Collagen-induced arthritis (CIA) is mediated by self-reactive CD4+ T cells that produce inflammatory cytokines. TGF-beta2-treated tolerogenic antigen-presenting cells (Tol-APCs) are known to induce tolerance in various autoimmune diseases. In this study, we investigated whether collagen-specific Tol-APCs could induce suppression of CIA. We observed that Tol-APCs could suppress the development and severity of CIA and delay the onset of CIA. Treatment of Tol-APCs reduced the number of IFN-gamma- and IL-17-producing CD4+ T cells and increased IL-4- and IL-5-producing CD4+ T cells upon collagen antigen stimulation in vitro. The suppression of CIA conferred by Tol-APCs correlated with their ability to selectively induce IL-10 production. We also observed that treatment of Tol-APCs inhibited not only cellular immune responses but also humoral immune responses in the process of CIA. Our results suggest that in vitro-generated Tol-APCs have potential therapeutic value for the treatment of rheumatoid arthritis as well as other autoimmune diseases.
Animals ; Antigen-Presenting Cells/*drug effects/*immunology ; Arthritis, Experimental/*immunology ; Chickens ; Collagen Type II/immunology ; Immune Tolerance/drug effects ; Mice ; Mice, Inbred BALB C ; Ovalbumin/immunology ; Th1 Cells/drug effects/immunology ; Th2 Cells/*drug effects/*immunology ; Transforming Growth Factor beta2/*pharmacology

In this study, we report the development of a new dual reporter vector system for the analysis of promoter activity. This system employs green fluorescence emitting protein, EGFP, as a reporter, and uses red fluorescence emitting protein, DsRed, as a transfection control in a single vector. The expression of those two proteins can be readily detected via flow cytometry in a single analysis, with no need for any further manipulation after transfection. As this system allows for the simultaneous detection of both the control and reporter proteins in the same cells, only transfected cells which express the control protein, DsRed, can be subjected to promoter activity analysis, via the gating out of all un-transfected cells. This results in a dramatic increase in the promoter activity detection sensitivity. This novel reporter vector system should prove to be a simple and efficient method for the analysis of promoter activity.
Enzyme Multiplied Immunoassay Technique ; Flow Cytometry ; Fluorescence ; Luminescent Proteins ; Proteins ; Transfection

TGF-beta-induced tolerogenic-antigen presenting cells (Tol-APCs) could induce suppression of autoimmune diseases such as collagen-induced arthritis (CIA) and allergic asthma. In contrast, many studies have shown that NKT cells are involved in the pathogenesis of Th1-mediated autoimmune joint inflammation and Th2-mediated allergic pulmonary inflammation. In this study, we investigated the effect of CD1d-restricted NKT cells in the Tol-APCs-mediated suppression of autoimmune disease using a murine CIA model. When CIA-induced mice were treated with Tol-APCs obtained from CD1d+/- or CD1d-/- mice, unlike CD1d+/- APCs, CD1d-/- Tol-APCs failed to suppress CIA. More specifically, CD1d-/- Tol-APCs failed to suppress the production of inflammatory cytokines and the induction of Th2 responses by antigen-specific CD4 T cells both in vitro and in vivo. Our results demonstrate that the presence of CD1d-restricted NKT cells is critical for the induction of Tol-APCs-mediated suppression of CIA.
Animals ; Antibodies/blood ; Antibody Formation/immunology ; Antibody Specificity/immunology ; Antigen-Presenting Cells/*immunology ; Antigens, CD1d/immunology ; Arthritis, Experimental/blood/*immunology/*prevention & control ; Collagen Type II/immunology ; Cytokines/blood ; Immune Tolerance/*immunology ; Inflammation Mediators/blood ; Mice ; Natural Killer T-Cells/*immunology ; Th1 Cells/immunology

Invariant natural killer T (iNKT) cells develop in the thymus upon recognition of CD1d expressed on developing thymocytes. Although CD4 and CD8 coreceptors are not directly involved in the interaction between CD1d and the T cell receptors (TCRs) of iNKT cells, a conspicuous lack of CD8+ iNKT cells in mice raised the question of whether CD8+ iNKT cells are excluded due to negative selection during their thymic development, or if there is no lineage commitment for the development of murine CD8+ iNKT cells. To address this question, we analyzed iNKT cell-specific TCR Valpha14+ transgenic mice, where the Valpha14 transgene forces the generation of iNKT cells. This allows detailed study of the iNKT cell repertoire. We were able to identify CD8+ iNKT cells which respond to the NKT cell-specific glycolipid ligand alpha-galactosylceramide. Unlike conventional iNKT cells, CD8+ iNKT cells produce predominantly IFN-gamma but not IL-4 upon antigen stimulation. We also confirmed the presence of CD8+ iNKT cells in wild type mice. Our results suggest that CD8+ NKT cells do exist in mice, although their population size is quite small. Their Th1-skewed phenotype might explain why the population size of this subtype needs to be controlled tightly.
Animals ; CD8-Positive T-Lymphocytes/*immunology/metabolism ; Galactosylceramides/immunology ; Interferon-gamma/immunology ; Interleukin-4/immunology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Natural Killer T-Cells/*immunology/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/*genetics ; Transgenes

OBJECTIVE: To emphasize the need for precise diagnosis of amyotrophic lateral sclerosis (ALS), a progressive and degenerative disease of upper and lower motor neurons that often present initially with weakness at the upper or lower extremities, and frequently misdiagnosed as myelopathy, radiculopathy, peripheral neuropathy or arthropathy that may ultimately lead to unnecessary treatments including surgical procedures. METHOD: We retrospectively reviewed medical records of 331 ALS patients who visited our hospital between 1998 and 2008. Symptoms at onset, progression of disease, radiologic findings, surgeries prior to diagnosis of ALS, outcome after surgery or conservative treatments, and electrodiagnostic study results were reviewed. RESULTS: Among the 331 patients with ALS, 34 (10.3%) had a history of surgical procedure and 37 (11.1%) underwent conservative treatment prior to diagnosis of ALS. 34 patients with a mean disease duration at diagnosis of 20.0+/-14.9 months, had surgery for symptoms that were later attributable to ALS. In 30 of the 34 patients, symptoms did not resolve after the intervention. 37 patients with a mean disease duration at diagnosis of 16.6+/-14.3 months, underwent conservative treatments such as physical therapy prior to diagnosis of ALS. Only in one patient (2.7%), symptoms improved after conservative treatment. CONCLUSION: In the absence of a single confirmatory study for the diagnosis of ALS, clinical findings may be misinterpreted, leading to an erroneous diagnosis. Therefore, closer and more careful follow-up is necessary for patients with limb weakness in the absence of sensory symptoms, or bulbar abnormalities such as dysarthria and dysphagia.
Amyotrophic Lateral Sclerosis ; Deglutition Disorders ; Dysarthria ; Extremities ; Follow-Up Studies ; Humans ; Lower Extremity ; Medical Records ; Motor Neurons ; Peripheral Nervous System Diseases ; Radiculopathy ; Retrospective Studies ; Spinal Cord Diseases