Calcium Channel Autoantibodies Research Articles

A cell-based assay for the detection of pathogenic anti-voltage-gated calcium channel autoantibodies in immunoglobulin G from patients with type 1 diabetes

Recent studies have postulated the presence of functional autoantibodies (Abs) against L-type voltage gated calcium channels (VGCCs) in the serum of patients with type 1 diabetes, with various proposed physiological consequences, both islet cell associated and extra-glandular. Arguably, the most potentially damaging effect reported for these Abs is induction of apoptosis in pancreatic beta (β) cells, yet a convincing pathogenic mechanism remains to be demonstrated. In the current study, we report an assay of reactive oxygen species (ROS) stress induction in the rat insulinoma cell line Rin A12, as determined by 2′, 7′-Dichlorofluorescein diacetate (DCF-DA) fluorescence detection by flow cytometry. We demonstrate that incubation of Rin A12 cells with immunoglobulin G (IgG) containing anti-VGCC activity from patients with T1D mediates a significant increase in ROS, with subsequent induction of apoptosis, as determined by positivity for annexin V expression. Neither T1D patient-derived IgG lacking anti-VGCC activity or IgG from healthy donors altered ROS or annexin V expression, indicating the new assay is specific for the detection of functional anti-VGCC Abs. Subsequent screening of IgG samples derived from individual patients indicated a prevalence of approximately 75% in a cohort of 20 patients with T1D. The new cell-based assay provides, for the first time, experimental evidence supporting a plausible pathophysiological mechanism underlying anti-VGCC Ab-mediated apoptosis induction in β cells. Additionally, the assay is a considerable advance on previously published methods for detecting and characterising the functional activity of anti-VGCC Abs in patient-derived samples.

Frequency of Synaptic Autoantibody Accompaniments and Neurological Manifestations of Thymoma.

Thymoma is commonly recognized in association with paraneoplastic autoimmune myasthenia gravis (MG), an IgG-mediated impairment of synaptic transmission targeting the nicotinic acetylcholine receptor of muscle. Newly identified synaptic autoantibodies may expand the serological profile of thymoma. To investigate the frequency of potentially pathogenic neural synaptic autoantibodies in patients with thymoma. We retrospectively identified patients with histopathologically confirmed thymoma and serum available to test for synaptic autoantibodies (collected 1986-2014) at the Mayo Clinic Neuroimmunology Laboratory. We identified and classified 193 patients with thymoma into 4 groups: (1) lacking neurological autoimmunity (n = 43); (2) isolated MG (n = 98); (3) MG plus additional autoimmune neurological manifestations (n = 26); and (4) neurological autoimmunity other than MG (n = 26). Clinical presentation and serum profile of autoantibodies reactive with molecularly defined synaptic plasma membrane proteins of muscle, peripheral, and central nervous systems. Of the 193 patients with thymoma, mean patient age was 52 years and did not significantly differ by sex (106 women) or group. Myasthenia gravis was the most prevalent clinical manifestation (64%) followed by dysautonomia (16 patients [8%]) and encephalopathy (15 patients [8%]); 164 patients (85%) had at least 1 synaptic autoantibody, and 63 of these patients (38%) had at least 1 more. Muscle acetylcholine receptor was most frequent (78%), followed by ganglionic acetylcholine receptor (20%), voltage-gated Kv1 potassium channel-complex (13%), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (5%). Less frequent were aquaporin-4, voltage-gated Kv1 potassium channel-complex related proteins (leucine-rich glioma-inactivated 1 and contactin-associated protein-like 2), glycine receptor, and γ-aminobutyric acid-A receptor. Synaptic autoantibodies were significantly more frequent in patients with neurological autoimmunity than in those without and were most frequent in patients with neurological manifestations other than or in addition to MG. Synaptic autoantibodies, particularly those reactive with ion channels of the ligand-gated nicotinic acetylcholine receptor superfamily (namely α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, glycine, and γ-aminobutyric acid-A receptors), were prevalent in patients with thymoma. Autoantibodies of this extended spectrum may enhance autoimmune serological testing as an aid to preoperative thymoma diagnosis. Detection of currently known synaptic autoantibody specificities absent from this profile have potential algorithmic usefulness as negative predictors for thymoma (as recognized for neuronal voltage-gated calcium channel autoantibodies).

PM400 Utility of calcium channel autoantibodies in predicting sudden cardiac death and all-cause death in chronic heart failure patients caused by DCM and ICM

PM400 Utility of calcium channel autoantibodies in predicting sudden cardiac death and all-cause death in chronic heart failure patients caused by DCM and ICM

Achalasia, chronic sensory neuropathy, and N-type calcium channel autoantibodies: beneficial response to IVIG.

Autoimmune gastrointestinal dysmotility (AGID) can result from paraneoplastic onconeuronal antibodies. Patients may present with regional hypomotility anywhere along the gastrointestinal tract. We report a case of a woman who developed an insidious sensory neuropathy and achalasia. She was found to have a high-titer of N-type voltage gated-calcium channel (VGCC) antibodies. She demonstrated clinical and electrophysiological improvement of her neuropathy, as well as improvement of her swallowing and gait, after treatment with intravenous immunoglobulins.

Serologic Profiles Aiding the Diagnosis of Autoimmune Gastrointestinal Dysmotility

Autoimmune gastrointestinal dysmotility is a limited autoimmune dysautonomia occurring idiopathically or in the context of an anatomically remote neoplasm, previously documented or unsuspected. Here we report 24 Mayo Clinic patients in whom the profile of serum autoantibodies aided this diagnosis. All patients were ascertained serologically in the course of service evaluation for autoantibodies consistent with neurologic autoimmunity. Review of their histories, motility studies, and laboratory findings revealed that all had presented with subacute gastrointestinal dysmotility. Recorded motility abnormalities included esophageal dysmotility 8 (6 had achalasia), delayed gastric emptying 12, slow small intestinal transit 7, slow colonic transit 4, and pelvic floor dyssynergia 4. Four patients underwent abdominal surgery; 2 commenced total parenteral nutrition. Plasma membrane cation channel autoantibodies were detected in 23 patients: neuronal voltage-gated calcium channel (5 N-type and 1 P/Q-type), acetylcholine receptor (11 ganglionic-type and 4 muscle-type), and neuronal voltage-gated potassium channel autoantibodies (4). Two patients had antineuronal nuclear autoantibodies, type 1. Approximately half of the patients had neural autoantibodies (including skeletal muscle striational and glutamic acid decarboxylase, 65kd isoform) or other antibody markers of organ-specific autoimmunity (thyroid or gastric parietal cell specificities). Neoplasia was diagnosed in 11 patients (9 recent, 2 remote): lung, breast and endometrial, gastrointestinal and thymoma. Moderate to dramatic improvement in gastrointestinal symptoms was reported after immunotherapy in 4 of 4 patients treated and after pyridostigmine treatment in 2 of 2 patients treated. Autoimmune serology aids the diagnosis of autoimmune gastrointestinal dysmotility, both paraneoplastic and idiopathic, and might guide management.

Autoimmune Gastrointestinal Dysmotility Treated Successfully With Pyridostigmine

Autoimmune gastrointestinal dysmotility (AGID) is a limited form of autoimmune autonomic neuropathy occurring idiopathically or in a paraneoplastic context. This disorder is considered rare, but is underrecognized as a cause for GI dysmotilities of varying anatomic extent, severity, and duration. We describe the diagnosis and management of an instructive case. A 60-year-old (nondiabetic) woman presented with a 15-year history of severe isolated gastroparesis. Paraneoplastic autoantibody evaluation aided the diagnosis of AGID. This included indirect immunofluorescence (neuronal nuclear and cytoplasmic antibodies), radioimmunoprecipitation assays (neuronal and muscle plasma membrane cation channel antibodies), and enzyme-linked immunosorbent assay (muscle striational antibodies). Serologic testing revealed both ganglionic neuronal acetylcholine receptor and N-type voltage-gated calcium channel autoantibodies. This profile was consistent with AGID and, despite the long history, raised the possibility of lung, breast, or ovarian carcinoma or thymoma. An underlying neoplasm was excluded by appropriate investigations. In a 1-month trial of oral pyridostigmine therapy, the patient's GI symptoms improved and her weight stabilized. Pyridostigmine was continued at a low dose, and was supplemented by tegaserod. Autoimmune serology is a valuable adjunct to the diagnosis and guide to management of patients with AGID. The favorable response to acetylcholinesterase inhibitors, despite a 15-year history, suggests an immunopharmacologic rather than an inflammatory cytotoxic pathology. Immunomodulatory therapy may not always be required. Of numerous autoantibodies currently recognized as biomarkers of AGID, the ganglionic acetylcholine receptor autoantibody is the only proven pathophysiologic effector. Certain neuronal nuclear and cytoplasmic autoantibodies are highly predictive of an underlying malignancy.

Voltage-operated calcium channel heterogeneity in pancreatic beta cells: physiopathological implications.

Voltage-operated calcium channels play crucial roles in stimulus-secretion coupling in pancreatic beta cells. A growing body of evidence indicates that these channels in beta cells are heterogeneous. In particular, not all the high-threshold calcium channels expressed belong to the best known L-type. In rat insulinoma cells, for example, L, N, and P/Q-type channels are present, while in human beta cells L-type and P/Q-type dominate. Where present, N-type and P/Q-type channels participate, alongside with the dominant L-type, in the control of sugar- or depolarization-induced hormone release. Distinct biophysical properties and selective modulation of the channel subtypes are likely to play important physiological roles. T-type channels are involved in beta cell apoptosis, while calcium channel autoantibodies recognizing high-threshold channels in beta cells, have been described both in neurological and diabetic patients. Subtype-selective calcium channel drugs have the potential for being beneficial in beta cell pathological states.

Accelerated autoantibody clearance by IVIG therapy

Recently, it has been postulated that the beneficial effect of intravenous immunoglobulins (IVIG) in antibody-mediated autoimmune disorders is based on an accelerated catabolism of autoantibodies due to saturation of FcRn. In the present study, we determined the magnitude and time course of this effect in a computer simulation to see whether this mechanism may indeed explain clinical observations. A pharmacokinetic model for IgG clearance was established in which concentration changes were calculated in small time steps. To validate the model we performed in vivo experiments with mice. A computational simulation accurately predicted the magnitude of the observed in vivo effect of IVIG administration on endogenous IgG levels. For human patients our model predicted a gradual decrease in autoantibody levels after IVIG administration (2 g/kg) with a maximum reduction of about 25% after 3–4 weeks and a duration of several months. This corresponds well to the effects of IVIG therapy observed in two patient studies: one on calcium-channel autoantibodies by Bain et al. (Neurology 1996; 47: 678–683) and another on ANCA by Jayne et al. (J Autoimmunity 1993; 6: 207–219). In conclusion, accelerated IgG clearance can explain a modest decrease in autoantibody levels observed in several patient studies. However, in some other clinical studies larger or more rapid effects have been observed, which can not be explained by accelerated clearance, suggesting that IVIG can also reduce autoantibody levels via other mechanisms such as down-regulation of antibody production or neutralization by anti-idiotypic antibodies.

Neuronal calcium channel autoantibodies coexisting with type 1 Purkinje cell cytoplasmic autoantibodies (PCA-1 or "anti-Yo")

Neuronal calcium channel autoantibodies coexisting with type 1 Purkinje cell cytoplasmic autoantibodies (PCA-1 or "anti-Yo")

New approaches to the treatment of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative motor neuron disease characterized by muscle weakness, wasting, spasticity, and weight loss, and eventually by respiratory failure. There is a degeneration and loss of motor neurons in the spinal cord, brainatem, and cerebral cortex. Death typically occurs within 3 to 4 years of the onset of the disease, although some patients may live considerably longer. [1,2] In the past few years there has been a substantial amount of research into the pathophysiology and treatment of ALS. Although a wide variety of therapeutic approaches have been attempted, until recently there was no drug that had an effect on the natural course of the disease. Riluzole, a glutamate antagonist, is the first such agent and the first drug approved for ALS treatment. Two placebo-controlled trials have demonstrated that the drug prolongs survival in patients with ALS. [3,4] Other investigational therapeutic approaches to the treatment of ALS include the use of neurotrophic factors, antioxidants, and other glutamate inhibitors. This article reviews the agents now available and those under investigation for the treatment of ALS, and discusses potential future approaches to therapy. A number of hypotheses concerning the pathogenesis of ALS have been proposed. These include: oxidative stress mediated by free radicals; neural toxicity (excitotoxicity) caused by the excessive stimulation of glutamate receptors by excitatory amino acids (e.g., glutamate, aspartate); a deficiency of neuronal growth factors; and autoimmunity (calcium channel autoantibodies). [5] There is growing evidence that oxidative stress is important in the initiation of the disease. [6] This hypothesis is supported by the discovery of mutations in the gene encoding for Cu/Zn superoxide dismutase (SOD1) in patients with familial ALS. [7] Glutamate is the principal excitatory neurotransmitter in the brain, and there is evidence that glutamate excitotoxicity is an important factor in the propagation of …

Incidence of serum anti-P/Q-type and anti-N-type calcium channel autoantibodies in the Lambert-Eaton myasthenic syndrome

The Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease in which autoantibodies are directed against voltage-gated calcium channels (VGCCs) at presynaptic nerve terminals. We first demonstrated the presence of P/Q-type and N-type VGCCs in digitonin extracts prepared from human and rabbit cerebellum using the specific ligands 125I-ω-conotoxin MVIIC ( 125I-ω-CmTx) and 125I-ω-conotoxin GVIA ( 125I-ω-CgTx), respectively. We then tested sera from 72 LEMS patients' 25 with proven small cell lung cancer (SCLC) and 66 healthy or other neurological, SCLC or autoimmune disease controls in an immunoprecipitation assay using 125I-ω-CmTx-labelled (P/Q-type) VGCCs in human cerebellar extract. Sixty-six of 72 LEMS serum samples (91.7%) were positive for the presence of VGCC antibodies, as defined as a titre greater than 3 standard deviations above the mean for the healthy controls ( n = 22). Rabbit cerebellar extract as antigen gave similar results ( r = 0.94, P < 0.001, n = 30). By contrast, only 24 72 (33%) LEMS sera were positive in the assay for anti-N-type VGCC antibodies using 125I-ω-CgTx. All these 24 were also positive in the 125I-ω-CmTx assay. All healthy and disease control sera were negative in both assays. The anti-P/Q-type VGCC antibody titres did not correlate with an electrophysiological index of disease severity across individuals; however, longitudinal studies in a LEMS patient with SCLC receiving chemotherapy, and in a non-SCLC LEMS patient receiving immunosuppressive therapy showed an inverse relation between antibody titre and disease severity. These results support the view that anti-P/Q-type VGCC antibodies are implicated in the motor disorder in LEMS, and show that the ω-CmTx radioimmunoassay is a highly specific and sensitive means of detecting them.

Heterogeneity of calcium channel autoantibodies detected using a small-cell lung cancer line derived from a Lambert-Eaton myasthenic syndrome patient.

We investigated the heterogeneity of anti-voltage-gated calcium channel (VGCC) antibodies in the Lambert-Eaton myasthenic syndrome (LEMS) using a small-cell lung carcinoma line (MB) derived from an LEMS patient. Four of 13 LEMS patients had raised titers of anti-125I-omega-conotoxin-labeled (N-type) VGCCs, measured by radioimmunoassay using line MB as the source of antigen. Antagonists for L-type (nitrendipine and nifedipine) and N-type (omega-conotoxin) VGCCs inhibited K(+)-stimulated (voltage-dependent) Ca2+ flux into this line--by 22% for L-type and 2% for N-type at maximum concentration. Inhibition by the LEMS IgGs, by contrast, ranged from 46 to 78% at a concentration of 2 mg/ml. These differing effects on Ca2+ flux inhibition by LEMS IgGs on the one hand and by L- and N-type channel antagonists on the other, taken together with the observation that many of the sera failed to react with omega-conotoxin-labeled (N-type) channels in the immunoprecipitation assay, suggest that in many LEMS patients the autoantibodies target other VGCC subtypes besides L- or N-types, and that these are important in inducing the myasthenic disorder.

Calcium channel autoantibodies in myasthenic syndrome and small cell lung cancer.

Lambert-Eaton myasthenic syndrome (LEMS) is one of the neurologic paraneoplastic syndromes often found in patients with lung cancer. It is characterized by a generalized deficit of neurotransmitter release. Patients with small cell lung cancer (SCLC) in particular may develop LEMS, and SCLC is very often detected in patients affected by LEMS. LEMS is an autoimmune disease, and autoantibodies that interfere with neurotransmitter release by binding to presynaptic voltage-operated calcium channels (VOCCs) have been found in sera of patients with LEMS. Both human neuronal and SCLC cell lines express omega-conotoxin-sensitive VOCCs, and autoantibodies from patients affected by LEMS can precipitate these channels. We have now screened a large population of patients and control subjects in order to define the specificity and sensitivity of the anti-VOCC antibody assay. We have tested sera from 52 patients with LEMS with and without SCLC; 32 sera from patients with SCLC without LEMS, 31 from patients with non-SCLC, 34 from patients with inflammatory lung diseases, 17 from patients with other neurologic disorders, and 48 from healthy control subjects. We have found that a positive result with this radioimmunoassay is highly specific for LEMS, with or without SCLC, when the antibody titer is higher than 14.21 pM. Anti-VOCC antibodies have also been found in about 40% of patients with SCLC without LEMS, but they were absent in all the other populations tested. We can conclude that this serologic assay is a very useful aid in the diagnosis of LEMS, and it might be useful also for the early diagnosis of SCLC.

Calcium channel autoantibodies in the Lambert-Eaton myasthenic syndrome.

We have tested 36 patients with the Lambert-Eaton myasthenic syndrome for serum antibodies to voltage-gated calcium channels by using an immunoprecipitation assay with [125I] omega-conotoxin-labeled voltage-gated calcium channels extracted from a human neuroblastoma cell line, SKN-SH. Forty-four percent of these patients had significant levels of antibody (30-1,466 pM) compared with healthy control individuals (less than 15 pM). The incidence of positive sera in patients without associated small cell lung carcinoma (61%) was greater than in those patients with small cell lung carcinoma (28%). Results correlated strongly with results obtained using voltage-gated calcium channels extracted from the small cell lung carcinoma line, MAR5. Anti-voltage-gated calcium channel antibody titers did not correlate with disease severity across individuals, but longitudinal studies in 2 patients receiving immunosuppressive therapy showed a clear inverse relation between antibody titer and an electromyographic index of disease severity. The incidence of positive sera among patients with other neurological disorders was not significant, but 8 of 12 patients with rheumatoid arthritis or systemic lupus erythematosus had raised titers (30-82 pM). We conclude that the antibodies detected in this assay are heterogeneous and that some of them are likely to be implicated in this disorder of neuromuscular transmission. The assay should prove useful as an additional diagnostic aid in patients with Lambert-Eaton myasthenic syndrome.