Introduction The detection of onconeural antibodies has been very useful in helping to define the paraneoplastic etiology of a given neurological syndrome. neurological syndromes, Lung malignancy, Autoantibody 1. Introduction The detection of onconeural antibodies has been very useful in helping to define the paraneoplastic etiology of a given neurological syndrome. Onconeural antibodies are detected in many laboratories by screening of sera on frozen sections of rat or mouse cerebellum (Moll et al., 1995; Vincent et al., 1998). Positive immunoreactivities are usually confirmed by immunoblot of recombinant proteins or neuronal homogenates. Sera from patients with small-cell lung Myricitrin (Myricitrine) carcinoma (SCLC), the most common tumor associated with paraneoplastic neurological syndromes (PNS), sometimes harbor antibodies against neural antigens that have not previously been recognized as associated with PNS (Gure et al., 2000). When this happens, it is important first to establish whether the antibody is related to the presence of a specific type of malignancy, Myricitrin (Myricitrine) and second to investigate whether the antibody is usually associated with a particular PNS. Even if the antibody is not directly related to the immune response that causes a PNS, it may indicate the presence of an underlying malignancy undiagnosed at the time of the antibody determination. Here we describe a new antibody specificity, and Rabbit Polyclonal to SIN3B show that it is a marker for SCLC. This antibody may help in the diagnosis of SCLC-related PNS, particularly the LambertCEaton myasthenic syndrome (LEMS). 2. Methods 2.1. Patients We retrieved from your archives of one of the investigators (FG) those samples which had shown immunoreactivity restricted to the nuclei of the Bergmann glia (Yamada and Watanabe, 2002) of the Purkinje cell layer and to subpopulations of glial cell nuclei in the white matter during routine immunohistochemistry of rat cerebellum to detect onconeural antibodies, as previously explained (Fig. 1) (Graus et al., 1997). The reactivity was defined as anti-glial nuclear antibody (AGNA). Open in a separate windows Fig. 1 (A) Frozen section of paraformaldehyde-fixed rat cerebellum immunoreacted with AGNA positive serum. There is intense labeling of the nuclei of Bergmann glia in the Purkinje cell layer. (B) Higher magnifications of the same section incubated with AGNA. (C) Double labeling study showing that AGNA reacts with the nuclei (reddish) of Bergmann glia, which are not labeled with NeuN a neuronal nucleus-specific monoclonal antibody (green). Note that NeuN labels the nuclei of granular cells and other cerebellar neurons except Purkinje cells, as previously explained (Mullen et al., 1992). Bar=38 m (A), 18 m (B), and 24 m (C). Sections counterstained with hematoxylin in (A) and (B). To define the clinical associations with AGNA, we analyzed the serum of 113 patients without PNS and SCLC, 122 with other malignancy types, and 19 with idiopathic LEMS, defined by the absence of malignancy after at least 3 years of follow-up (ONeill et al., 1988). To ascertain the frequency of AGNA among different PNS associated with SCLC, we examined the presence of AGNA immunoreactivity in 30 patients with paraneoplastic LEMS, 27 with paraneoplastic cerebellar degeneration (PCD), 10 with sensory neuropathy, 10 with opsoclonus, and 8 with limbic encephalitis. All these patients had definite SCLC, except six who experienced X-ray evidence only of lung malignancy. None of the sera harbor anti-Hu, anti-Ri, anti-Zic4 (Bataller et al., 2004b) or other associated antibodies that could obscure the presence of AGNA immunoreactivity. 2.2. AGNA immunohistochemistry AGNA immunoreactivity was analyzed by immunohistochemistry (serum screening dilution 1:500; biotinylated IgG from AGNA positive serum at 50 g/ml) using an avidinCbiotin technique on paraformaldehyde-fixed frozen rat tissues or acetone-fixed human Myricitrin (Myricitrine) cerebellum as explained previously (Graus et al., 1997). Myricitrin (Myricitrine) To show if AGNA of different patients recognized comparable epitopes, rat cerebellar sections were pre-incubated with undiluted normal serum, an anti-Hu-positive serum, or AGNA-positive serum for 3 h followed by a biotinylated IgG obtained from a positive AGNA serum and developed with a standard avidinCbiotin technique as explained (Bernal et al., 2003). To study if there was AGNA immunoreactivity.