Autoimmune Peripheral Nervous System Hyperexcitability

A 25-year-old man was seen for assessment of progressive pain. He had a distant history of Guillain-Barré syndrome at age 8 years, at which time he had symmetrical proximal and distal weakness of the upper and lower extremities with loss of ambulation. No facial weakness, dysarthria, dysphagia, ptosis, diplopia, or respiratory weakness occurred. At his initial evaluation there was touch hypersensitivity of the muscles and skin. He had no weakness or cognitive involvement, although the pain made it difficult for him to concentrate. His creatine kinase value improved with hydration, but pain and muscle twitching persisted. On examination, he had diffuse extremity and truncal fasciculations and myokymia and reported pain in not only the areas of twitching but also other areas of his extremities and trunk. On neurophysiologic testing, fibular and tibial motor compound muscle action potentials were decreased in amplitude, with normal ulnar and median motor responses. Needle electromyography of muscles proximally and distally showed diffuse spontaneous firing of muscles ranging in frequency with waxing and waning characteristics. These findings were thought to be consistent with a primary hyperexcitable disorder of muscles with a superimposed old polyradiculoneuropathy and possibly a myopathy. Expanded autoimmune neuroimmunologic testing of serum identified immunoglobulin G-directed cerebellar molecular staining consistent with voltage-gated potassium channel autoantibodies. Radioimmunoprecipitation assay identified voltage-gated potassium channel-immunoglobulin Gs and led to reflex testing for contactin-associated protein 2-immunoglobulin G; autoantibodies were positive. Computed tomography of the chest with contrast was performed, and lymphadenopathy was identified. The patient was clinically diagnosed with contactin-associated protein 2 - immunoglobulin G–positive Isaacs syndrome. A trial of high-dose gabapentin was attempted, with only mild benefits. Next, intravenous immunoglobulin was initiated. Diabetes developed, and he was hospitalized requiring initiation of insulin. His condition is now managed variably with intravenous immunoglobulin and scheduled daily gabapentin. The immune system has long been recognized to help regulate pain via non- immunoglobulin G–mediated mechanisms. Specifically, cytokines decrease the nociceptive nerve fiber thresholds and are released after diverse tissue insults. This allows for speeded healing by increased blood flow and protection of the region by pain guarding mechanisms. It is now recognized that, in rare cases, immunoglobulin G-mediated autoimmunity can lead to otherwise idiopathic pain disorders.

A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

Cell-based assays (CBAs) and radioimmunoprecipitation assay (RIPA) are the most sensitive methods for identifying anti-acetylcholine receptor (AChR) antibody in myasthenia gravis (MG). But CBAs are limited in clinical practice by transient transfection. We established a stable cell line (KL525) expressing clustered AChR by infecting HEK 293T cells with dual lentiviral vectors expressing the genes encoding the human AChR α1, β1, δ, ϵ and the clustering protein rapsyn. We verified the stable expression of human clustered AChR by immunofluorescence, immunoblotting, and real-time PCR. Fluorescence-activated cell sorting (FACS) was used to detect anti-AChR antibodies in 103 MG patients and 58 healthy individuals. The positive results of MG patients reported by the KL525 was 80.6% (83/103), 29.1% higher than the 51.4% (53/103) of RIPA. 58 healthy individuals tested by both the KL525 CBA and RIPA were all negative. In summary, the stable expression of clustered AChR in our cell line makes it highly sensitive and advantageous for broad clinical application in CBAs.

Interference Due to Heterophilic Antibody, Biotin and Thyroid Hormone Autoantibody

Purpose: Immunoassay is susceptible to interference by other substances in the serum. The main substances interfering with thyroid function testing include heterophilic antibody, biotin, thyroid hormone autoantibody, and Macro-TSH. We reported a patient with fraudulently elevated FT4 and TSH and described various common experimental methods used to explore the existence of substances interfering with thyroid function testing.Methods and Results: FT4 and TSH were significantly lower when measured on the Architect platform (FT4: 7.09 pmol/L, TSH: 58.94 µIU/ml). Polyethylene glycol precipitation showed lower FT4 and TSH, suggesting the presence of a high molecular weight interfering substance. Heterophile blocking tube study showed heterophilic antibody interfered with TSH detection. 125I-hTSH binding study and radioimmunoprecipitation assay indicated that the patient didn’t contain anti-TSH autoantibody. The new generation of Elecsys immunoassay kit indicated that biotin interfered with TSH detection. Radioimmunoprecipitation assay showed that all four kinds of thyroid hormone autoantibodies were positive. After reviewing 24 literatures, we provided the diagnostic strategy for investigation of interferences with thyroid function immunoassays.Conclusion: We reported a case with falsely elevated TSH due to the combined action of heterophilic antibody and biotin and fraudulently elevated FT4 caused by thyroid hormone autoantibody. When there is a discrepancy between thyroid function and clinical manifestation, the presence of immunoassay interference with one or more indicators needs to be considered.

293 Autoimmune autonomic ganglionopathy: the NHNN experience

BackgroundAutoimmune Autonomic Ganglionopathy (AAG) is a rare, immune-mediated condition characterised by subacute pandysautonomia. 50% have auto-antibodies to the ganglionic nicotinic acetylcholine receptor (gAChR), affecting synaptic transmission at autonomic ganglia.MethodsWe describe 13 patients (5 female, median age 47) presenting with widespread autonomic failure, confirmed on cardiovascular, sudomotor and pupillometry testing at the National Hospital for Neurology and Neurosurgery (NHNN), and high gAChR antibody levels (>200 pm) measured by radioimmunoprecipitation assay at Oxford University.ResultsOf the 13 patients, 8 had other autoimmune conditions, 3 had antecedent infection and 3 were paraneoplastic. All had orthostatic hypotension, gastro-intestinal and urinary symptoms, 11 had documented pupillary abnormalities (9 mixed sympathetic and parasympathetic deficits, 2 subclinical sympathetic deficits), 11 had secretomotor dysfunction, 8 had generalised/partial anhidrosis, 8 had sexual dysfunction and 7 had evidence of small fibre dysfunction on neurophysiology.Ten received immunomodulatory treatment; 6 plasma exchange and 4 combination treatment including intravenous immunoglobulin, mycophenolate and rituximab. Earlier treatment was associated with greater clinical response.DiscussionPatients with AAG and high gAChR antibody levels have widespread dysfunction of the autonomic nervous system, which can respond dramatically to immunomodulation. Further research is needed to develop robust clinical biomarkers to guide treatment and monitor response.

A Family Cluster of Chagas Disease Detected through Selective Screening of Blood Donors: A Case Report and Brief Review

Chagas disease (CD) is a protozoan infection caused byTrypanosoma cruzi, which is transmitted by triatomine insect vectors in parts of Latin America. In a nonendemic country, such as Canada, spread can still occur via vertical transmission, and infected blood or organ donations. The Canadian Blood Services and Héma-Québec have both implemented selective screening of blood donors for CD based on risk factors. In 2011, Héma-Québec identified two seropositive ‘at-risk’ Chilean siblings who had donated blood in Montreal, Quebec. They were referred to the JD MacLean Centre for Tropical Diseases (Montreal, Quebec) for confirmatory testing (T cruziexcreted-secreted antigen ELISA, polymerase chain reaction and/or radioimmunoprecipitation assay) and follow-up. Screening of the rest of the family revealed two other seropositive family members (the mother and sister). While their geographical history in Chile suggests vectorial transmission, this family cluster of CD raises the possibility of vertical transmission. Congenital infection should always be considered among CD-positive mothers and pregnant women. With blood donor screening, Canadian physicians will increasingly see patients with CD and should know how to manage them appropriately. In addition to the case presentation, the authors review the transmission, screening and clinical management of CD in a nonendemic context.

Monitoring ADAMTS13 Autoantibody Titer Using a Novel Quantitative Radioimmunoprecipitation Assay In Patients With Acquired Thrombotic Thrombocytopenic Purpura

Background ADAMTS13-binding immunoglobulin G (IgG)-type autoantibodies are present in patients with acquired thrombotic thrombocytopenic purpura (TTP), thereby causing severe deficiency of plasma ADAMTS13 activity. Some specific autoantibodies directly inhibit the enzymatic activity by interfering with the access of its substrate, von Willebrand factor (VWF), particularly in the spacer domain, although others bind to almost all of the domains in the molecule, possibly leading to the acceleration of the clearance from blood stream. Not only the inhibitor titer, but the importance of ADAMTS13 autoantibody titer was highlighted by many previous clinical studies, associating with the prognosis such as recurrence rate. Objective We targeted to establish a novel high-sensitive assay to measure ADAMTS13-binding IgG autoantibody titer using three kinds of radioisotope-labeled antigens, ADAMTS13 whole molecule, MDTCS and T2-8/CUB, and aimed to analyze the association between the autoantibody titer and the clinical characteristics of TTP patients. Materials and Methods Human Cell-Free Protein Expression System (Takara #3281, Shiga, Japan) was used to synthesize radioisotope-labeled antigens. ADAMTS13 cDNA corresponding to whole molecule (A13), metalloprotease to spacer domains (MDTCS) and TSP1-2 to CUB2 domains (T2-8/CUB) were cloned into an expression vector pT7-IRES and mixed with Cell Lysate containing T7 RNA polymerase, methionine-free amino acids, ATP, translation enhancement factor and 35S-methionine. The correct synthesis and molecular size of the radiolabeled antigens were checked with SDS-PAGE. To assess the utility as a quantitative assay, each of the antigens was mixed with mouse anti-ADAMTS13 monoclonal antibodies, whose epitopes were determined in our previous study (Thromb Res. 2012; 130(3):e79-83), and the immune complex was precipitated with protein G beads, washed and measured in a liquid scintillation counter. Plasma samples from acquired TTP patients were tested to quantify the autoantibody titers using radiolabeled A13, MDTCS and T2-8/CUB antigens, respectively. As a control, plasma samples from healthy subjects with no histories of autoimmune disease were also tested. Results Each of the radiolabeled antigens was detected as a single band at the correct molecular weight size and successfully immnoprecipitated with several mouse anti-ADAMTS13 monoclonal antibodies, indicating the intact molecular conformation of the synthesized proteins using the cell-free protein synthesis system. Moreover, the appropriate dose-dependent escalation curves in accordance with the addition of the monoclonal antibodies were observed, thereby confirming the utility of the assay as a quantitative analysis. We tested TTP patient plasma at onset (n=5) and were able to detect ADAMTS13 autoantibody titers with each of the radiolabeled A13, MDTCS and T2-8/CUB antigens. We next applied this assay for monitoring ADAMTS13 autoantibody titer in a clinical course of TTP patient. The patient developed the first episode of TTP at the age of 2 month and treated with steroid pulse and plasma exchange therapy for six consecutive days. Remission was once achieved but 6 months later from the onset, the second episode of TTP occurred and the patient was treated with 11 plasma exchange and rituximab at the dose of 375 mg/m2 once a week for 4 weeks. Plasma samples at onset, after the first 6 consecutive plasma exchange and after rituximab administration were examined about autoantibody titer using this assay. Interestingly, the titers remained high even after the plasma exchange but declined clearly after the rituximab treatment, whereas no reduction of total IgG level was observed. These findings suggest that the autoantibody titration using this assay might be useful to assess the effect of treatment and associate with the prognosis related to the recurrence. Conclusion We developed a novel quantitative radioimmunoprecipitation assay to measure ADAMTS13 autoantibody titer related to three antigens, ADAMTS13 whole molecule, MDTCS and TSP2-8/CUB. This assay may serve not only as a diagnostic test but as a monitoring index to evaluate the prognosis of TTP. Disclosures: No relevant conflicts of interest to declare.