MAB312RX Sigma-AldrichAnti-Neuron Cell Surface Antigen Antibody, clone A2B5-105 (7), Alexa Fluor® 488

ABSTRACT: Study Design. To examine the localization and expression of high-mobility group box-1 (HMGB-1) protein and its receptors after rat spinal cord injury.Objective. To elucidate the contribution of HMGB-1 and its receptors as potential candidates in a specific upstream pathway to the proinflammatory response leading to a cascade of secondary tissue damage after spinal cord injury.Summary of Background Data. HMGB-1 was recently characterized as a key cytokine with a potential role in nucleosome formation and regulation of gene transcription. No studies have investigated the role of HMGB-1 in spinal cord injury.Methods. Injured thoracic spinal cord from 62 rats aged 8-12 weeks and spinal cord from 20 control rats were examined. HMGB-1 was localized by immunofluorescence staining, costaining with cell markers, and by immunoelectron microscopy. The expression of HMGB-1 and its receptors, receptor for advanced glycation end products (RAGE), toll-like receptor (TLR)2, and TLR4, were also examined by immunohistochemistry.Results. HMGB-1 expression appeared earlier than that of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the spinal cord injury rats, with the HMGB-1 produced by both macrophages and neurons. HMGB-1 translocated from nucleus to cytoplasm in some neurons at an early stage after neural injury. Increased expression of HMGB-1, RAGE, and TLRs was observed after injury and interaction of HMGB-1 with RAGE or TLRs, particularly in macrophage, was confirmed at three days after injury.Conclusion. Our results demonstrated an earlier onset in the expression of HMGB-1 than TNF-α, IL-1β, and IL-6 after spinal cord injury. The release of HMGB-1 from neurons and macrophages is mediated through the HMGB-1/RAGE or TLR pathways. HMGB-1 seems to play at least some roles in the proinflammatory cascade originating the secondary damage after the initial spinal cord injury.

Rat insulinoma (RIN) cells, in comparison with adult islet cells, are relatively undifferentiated. They secrete low amounts of islet hormones, are unresponsive to glucose, and display pluripotency. A minority of RIN cells react with monoclonal antibodies A2B5 and 3G5 which recognize complex gangliosides on normal islet cells. In order to determine whether the expression of A2B5- or 3G5-reactive gangliosides is modulated during differentiation RIN cells were cultured with various concentrations of sodium butyrate (NaB), a known inducer of cellular differentiation. Expression of A2B5- and 3G5-reactive gangliosides was determined by indirect immunofluorescence and flow cytofluorimetry. NaB exposure resulted in a dose-dependent decrease in cell proliferation over 5 days of 1.5-, 2.9-, and 17.5-fold at 0.5, 1.0 and 3.0 mM, respectively, and a distinct change in cellular morphology. Cells exposed to NaB displayed prominent neurite-like projections. At 3 mM NaB, insulin secretion increased 7.9-fold and the percentage of cells expressing A2B5- and 3G5-reactive gangliosides increased by up to 4.4- and 5.5-fold, respectively. The expression of A2B5- or 3G5-reactive gangliosides per cell also increased, by 2.4- and 1.3-fold, respectively, at 3 mM NaB. These findings demonstrate that the expression of cell surface A2B5- and 3G5-reactive gangliosides is not static but increases with cell differentiation. NaB-treated RIN cells may serve as a model to study the role of gangliosides in the function and lineage relationships of islet cells.

Cultured rat dorsal root ganglion neurons expressed ganglioside GD3 when grown in the absence of non-neuronal cells. Among the non-neuronal cells, fibroblasts, but not Schwann cells, also stained for ganglioside GD3 during the first few days in culture. When neurons were combined with non-neuronal cells the intensity of the GD3 immunoreactive neuronal processes was diminished at sites contacted by Schwann cells. This contact-mediated effect was specific for ganglioside GD3 since no difference was seen with A2B5 or JONES antibodies, which recognize different gangliosides.