Cytotoxic T lymphocyte-mediated cell death in paraneoplastic sensory neuronopathy with anti-Hu antibody
Introduction
Paraneoplastic sensory neuronopathy (PSN) is a rare syndrome starting with dysesthetic pain and numbness in the distal extremities, then spreading through all four limbs and trunk causing severe sensory ataxia. More than 60% of PSN patients have small-cell lung cancer (SCLC) which usually is discovered after neurological symptoms appear. Many patients with PSN associated with SCLC harbor an antibody in their sera called anti-Hu, or ANNA-1, which mainly labels neuronal nuclei immunohistochemically and binds to several bands at about 35–42 kDa on Western blots of neuronal tissue homogenates [3]. This antibody is a good marker for diagnosing PSN and underlying lung cancer, but little evidence have been presented that this antibody directly induces neuronal cell loss. In contrast, patients with PSN show pleocytosis in their cerebrospinal fluid (CSF), and the hallmarks of their pathological features are inflammatory lymphocyte infiltration and loss of neurons in the dorsal root ganglia and other areas of the central nervous system, indicative of T cell-mediated neuronal damage. As there have been no reports verifying the role of T cells in PSN, we studied cytotoxic T cell (CTL) activity in patients with PSN associated with SCLC.
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Materials and methods
Recombinant HuD protein was constructed following the DNA sequence reported by Szabo et al. [12]. Briefly, a primer pair for the PCR was designed that corresponded to nucleotide positions 148–169 for the 5′ site and 448–467 for the 3′ site. The PCR product was ligated to the pET-16b plasmid vector carrying a His Tag sequence, transformed to E. coli BL21(DE3) cells and grown in rich broth media. Recombinant HuD protein production was induced with isopropyl-β-d-thiogalactopyranoside (IPTG). Cells
Results
Recombinant HuD protein production was recognized on SDS-PAGE at the expected molecular size, and anti-Hu antibody reacted specifically with that band. Immunohistochemistry clearly showed that the injected HuD protein was present in the cytoplasms of the fibroblasts (Fig. 2). Fibroblast viability was favorable (more than 90% of the cells were alive) for up to 4 h after the injection of bovine serum albumin or plasmid vector or HuD protein. The disappearance rate (results of quadruple
Discussion
Specific autoantibodies such an ‘anti Yo, Hu, Ri’, react with both tumor and nervous tissue, have been detected in some patients with paraneoplastic neurological syndromes. Patients who have antibodies tend to have a better cancer prognosis, the tumor being small, less metastatic and more slowly growing [2]. These patients tend to have rapidly progressive neurological symptoms and high titer autoantibodies in their sera and CSF. This suggests that these autoantibodies have an important role in
Acknowledgements
This work was supported by Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan.
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