Live neurons similarly incubated with normal human IgG and antiCHu IgG served as controls

Live neurons similarly incubated with normal human IgG and antiCHu IgG served as controls. findings indicate that in a young woman with acute psychiatric symptoms, seizures, and central hypoventilation, a paraneoplastic immune-mediated syndrome should be considered. Recognition of this disorder is important because despite the severity of the symptoms, patients usually recover. The location and function of the isolated antigen suggest that the disorder is directly mediated by antibodies. Paraneoplastic limbic encephalitis (LE) often associates with brainstem dysfunction and predominantly affects older individuals with lung cancer.1 A review of 137 patients with LE showed that young individuals with germ-cell tumors of the testis or ovarian teratoma (OT) were more frequently affected than patients of any age with more prevalent tumors such as cancer of the breast, prostate, or colon.2 Subsequent studies defined the Nrf2-IN-1 LE associated with germ-cell tumors as a syndrome with dominant limbic, diencephalic and upper brainstem dysfunction, and the Ma proteins as the main autoantigens.3 Because germ-cell tumors often contain teratoma elements, we reasoned that a similar disorder may occur in women with OT. This led us to investigate the neurological and immunological features of four women with OT and encephalitis examined by us, and to review the clinical features of similar cases in the literature.4-12 None of our four patients had antibodies to Ma proteins, but we were impressed by the similarity and severity of the neurological symptoms, which often resembled an acute psychotic episode, malingering, or drug abuse. These patients often had cerebrospinal fluid (CSF) inflammatory abnormalities and the neurological syndrome improved after tumor resection, immunotherapy, or both. On the basis of these observations, we postulated that teratoma-associated encephalitis is an immune-mediated disorder and that if antibodies are involved they are not detected by conventional testing. We report the clinical features of this disorder along with the associated antibodies and preliminary characterization of one of the antigens. Patients and Methods Four patients were examined Nrf2-IN-1 by the authors, and sera or CSF was obtained at Rabbit polyclonal to ATS2 symptom presentation (three cases) or recurrence (one case) and Nrf2-IN-1 kept frozen at ?80C until use. A brief description of Patient 1 has been reported previously (Case 4 in Ances and colleagues12); this patient and Patient 2 are fully reported here (see online supplementary information). The clinical features of Patients 3 and 4 have been previously reported.9-11 Immunohistochemistry and Immunocompetition Assays Rats were anesthetized and euthanized by decapitation and the brain removed and processed as reported.12 Frozen 7m-thick sections were directly mounted on slides and the patients’ sera (diluted 1:250) or CSF (1:10) were tested using the avidin-biotin-peroxidase technique.12 To determine whether patients’ antibodies targeted the same epitopes, we used immunocompetition assays between IgG biotinylated from one patient’s serum and whole serum of other patients.13 Distribution of Immunolabeling in Hippocampal Neuronal Cultures Rat hippocampal neuronal cultures were prepared as reported.14 Neurons were grown on coverslides, fixed with paraformaldehyde (PFA), and serially incubated with patients’ sera (1:250) for 1 hour and fluorescein-labeled goat antiChuman IgG for 30 minutes. After washing, slides were incubated with biotinylated IgG from control patients with voltage-gated potassium channels (VGKCs) or normal individuals or mouse antibodies to the VGKC Kv1.2 (1:50; Upstate Biotechnology, Lake Placid, NY) or ARF6 (1:25; Chemicon International, Temecula, Nrf2-IN-1 CA). The reactivity of biotinylated human IgG was developed with avidin-rhodamine (1:2000; Vector, Burlingame, CA) and the reactivity of mouse antibodies was developed with Alexa Fluor rhodamine-labeled goat antiCmouse IgG (1:2000; Molecular Probes, Eugene, OR). Expression of Antigens in Live Hippocampal Neurons To determine whether the target antigens were accessible in live neurons, we added patients’ antibodies to the neuronal cell cultures for 30 minutes. Afterward, the media was removed and the neurons were gently washed in phosphate-buffered saline and fixed in PFA, and the bound antibodies were revealed with Alexa Fluor fluorescein-labeled antiChuman IgG (1:2,000). Live neurons similarly incubated with normal human IgG and antiCHu IgG served as controls. All human IgGs were used at concentration 0.37g/ml obtained from patients’ CSF. Isolation of cDNA Clones and Affinity Purification of Antibodies A uni-ZAP-XR rat hippocampal library (Stratagene, La Jolla, CA) was probed with sera from Patients 1, 2, and 3, as reported.15 Positive clones were purified with several rounds of antibody screening and subcloned.

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