BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic synovial inflammation which leads to joint destruction. Gene therapy of RA targets the players of inflammation or articular destruction. However, viral vectors have safety problems and side effects, while non-viral vectors suffer from inefficient gene transfer and fast loss of gene expression. To overcome the limits of non-vial vectors, an EBV-based plasmid which is known to exert prolonged high level gene expression can be used. METHODS: pEBVGFP, pEBVIL-10, and pEBVvIL-10 were constructed by cloning GFP, IL-10, and vIL-10 genes into an EBV-based plasmid, respectively. The pGFP was used as a control plasmid. Each constructs were lipofected into HIG-82 rabbit synoviocytes. The expression of GFP was monitored by FACS and confocal microscopy. IL-10 and vIL-10 expressions were measured by ELISA. RESULTS: GFP expression 2 days after transfection was achieved in 33.2% of cells. GFP-expressing cells transfected with pGFP decreased rapidly from 4 days after transfection and disappeared completely by 11 days. Cells transfected with pEBVGFP began to decrease slowly from 4 days. But GFP expression was detected for over 35 days. In addition, HIG-82 cells transfected with pEBVIL-10 (44.6+/-1.5 ng/ml) or pEBVvIL-10 (51.0+/-5.7 ng/ml) secreted these cytokines at high levels. High level cytokine production by hygromycin selection was maintained at least for up to 26 days after transfection. CONCLUSION: These results suggest that the EBV-based plasmid has a potential to improve non-viral gene transfer system and may be applicable to treat RA without the drawbacks of viral vectors.
Arthritis, Rheumatoid ; Clone Cells ; Cloning, Organism ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Gene Expression* ; Genetic Therapy ; Inflammation ; Interleukin-10 ; Joints ; Microscopy, Confocal ; Plasmids* ; Transfection

BACKGROUND: Hu syndrome, a neurological disorder, is characterized by the remote effect of small cell lung cancer on the neural degeneration. The suspicious effectors for this disease are anti-Hu autoantibodies or Hu-related CD8+ T lymphocytes. Interestingly, the same effectors have been suggested to act against tumor growth and this phenomenon may represent natural tumor immunity. For these diagnostic and therapeutic reasons, the demand for antibodies against Hu protein is rapidly growing. METHODS: Polyclonal and monoclonal antibodies were generated using recombinant HuR protein. Western blot analyses were performed to check the specificity of generated antibodies using various recombinant proteins and cell lysates. Extracellular stimuli for HuR expression had been searched and HuR-associated proteins were isolated from polysome lysates and then separated in a 2-dimensional gel. RESULTS: Polyclonal and monoclonal antibodies against HuR protein were generated and these antibodies showed HuR specificity. Antibodies were also useful to detect and immunoprecipitate endogenous HuR protein in Jurkat and BJAB. This report also revealed that TNF-alphatreatment in BJAB up-regulated HuR expression. Lastly, protein profile in HuR-associated mRNA- protein complexes was mapped by 2-dimensional gel electrophoresis. CONCLUSION: This study reported that new antibodies against HuR protein were successfully generated. Currently, project to develop a diagnostic kit is in process. Also, this report showed that TNF-alphaup-regulated HuR expression in BJAB and protein profile associated with HuR protein was mapped.
Antibodies ; Antibodies, Monoclonal ; Autoantibodies ; Blotting, Western ; Electrophoresis ; ELAV Proteins ; ELAV-Like Protein 1 ; Nervous System Diseases ; Ovarian Neoplasms* ; Polyribosomes ; Recombinant Proteins ; Sensitivity and Specificity ; Small Cell Lung Carcinoma ; T-Lymphocytes ; Tumor Necrosis Factor-alpha

BACKGROUND: Human seminal plasma (HSP)-induced hypersensitivity is one of the serious complications with sexual intercourse. The clinical manifestations of HSP-induced hypersensitivity may be related to the release of vasoactive mediators from mast cell induced by HSP. It has recently been reported that HSP modulates immune systems and induces mast cell degranulation and histamine release from rat peritoneal mast cells (RPMC). Ketotifen and disodium cromoglycate (DSCG), anti-asthmatic and anti-allergic drugs, have a role of mast cell stabilization and inhibit mast cell-induced leukocyte rolling and adhesion. But the inhibitory agents of HSP-induced mast cell activation are unknown. This study was performed to investigate the effects of DSCG and ketotifen on the HSP-induced mast cell activation. METHODS: For this, influences of DSCG and ketotifen on the human seminal plasma-induced degranulation, histamine release and morphological changes of RPMC were observed. RESULTS: The mast cell degranulation and histamine release of RPMC by HSP were induced in a dose-dependent fashion. The HSP-induced cytomorphological changes such as swelling, intracellular vacoules, and interrupted cell boundary were significantly inhibited by pretreatment with DSCG or ketotifen. DSCG and Ketotifen inhibited the HSP-induced degranulation and histamine release from RPMC. CONCLUSION: From the above results, it is suggested that DSCG and ketotifen have a inhibitory effect of the HSP-induced mast cell activation. DSCG and ketotifen may be used for treatment of HSP-induced hypersensitivity.
Animals ; Coitus ; Cromolyn Sodium* ; Histamine ; Histamine Release ; Humans* ; Hypersensitivity ; Immune System ; Ketotifen* ; Leukocyte Rolling ; Mast Cells* ; Rats ; Semen

BACKGROUND: Telomerase, a ribonucleoprotein enzyme capable of synthesizing telomeric repeats, attracts attention for its possible role in determining the replicative capacity of normal somatic cells, transformed cells, and cells of the germline lineage. Differently from normal somatic cells with no telomerase activity, normal lymphocytes has been reported to have telomerase activity comparable to that found in transformed cells during development and activation, which substantiate a role in supporting the capacity of lymphocytes for extensive clonal expansion. METHODS: Here, in order to define the telomerase regulation in murine T lymphocytes, telomerase activity in cloned murine CD8+ T cells and naive CD8+ T cells isolated from C57BL/6 mice was examined. Next, the regulatory mechanism of telomerase activity at transcriptional and post- translational levels was investigated by determining the expression level of the TERT protein, a key component for telomerase activity. RESULTS: It was demonstrated that telomerase activity was expressed in an inactivated state as well as in an activated state in the murine CD8+ T lymphocytes by using TRAP assay. The increase of telomerase activity was partially dependent on the net increase of TERT expression. Also, telomerase activity was decreased after treatment with protein kinase inhibitors, indicating that telomerase activation was prevented by inhibition of phosphorylation. CONCLUSION: Therefore, these results suggest that telomerase activity is constitutively expressed in the murine resting T lymphocytes and controlled by both transcriptional regulation and post-translational modifications.
Animals ; Clone Cells ; Lymphocytes ; Mice ; Phosphorylation ; Protein Kinase Inhibitors ; Protein Processing, Post-Translational ; Ribonucleoproteins ; T-Lymphocytes* ; Telomerase*

BACKGROUND: Anti-cardiolipin antibody (Anti-CL Ab) is one of the various anti-phospholipid antibodies (Anti-PL Abs) and found in the plasma of patients with systemic lupus erythematosus (SLE), atherosclerosis, and other infectious diseases. While anti-PL Abs found in the sera of patients with infectious diseases bind directly to CL, binding of anti-PL Abs to CL circulating in the sera of patients with autoimmune diseases is mediated by beta2-glycoprotein 1 (beta2-GP1). The purpose of this study is to investigate the effect of beta2-GP1 on the antigen binding assay of anti-CL Abs present in the sera of patients with atherosclerosis, which has been known as one of autoimmune diseases. METHODS: ELISA was performed with sera containing anti-CL Abs from three patients with atherosclerosis in the presence or absence of beta2-GP1 or FBS. RESULTS: Reactivity of anti-CL Abs to CL was increased in the presence of beta2-GP1 or FBS in a dose dependent manner. CONCLUSION: beta2-GP1 or FBS could be used as co-factor in CL ELISA with anti-CL Abs present in the sera of patients with atherosclerosis. It is suggested that anti-CL Abs found in atherosclerosis patients are similar in terms of antigen binding property to those circulating in the patients with autoimmune diseases, not to infectious diseases.
Antibodies* ; Atherosclerosis ; Autoimmune Diseases ; Cardiolipins* ; Communicable Diseases ; Enzyme-Linked Immunosorbent Assay ; Humans ; Lupus Erythematosus, Systemic ; Plasma

BACKGROUND: B-1 cells differ from conventional B-2 cells both phenotypically and functionally. The aim of this study was to investigate the difference between peritoneal B-1 cells and splenic B-2 cells in proliferation. METHODS: We obtained sorted B-1 cells from peritoneal fluid and B-2 cells from spleens of mice. During the culture of these cells, immunoglobulin secreted into the culture supernatants was evaluated by enzyme- linked immunosorbent assay. Entering of S phase in response to LPS-stimuli was measured by proliferative assay. RESULTS: Spontaneous Immunoglobulin M production occurred in peritoneal B-1 cells but not in splenic B-2 cells. LPS stimulated peritoneal B-1 cells secreted IgM at day 1, but splenic B-2 cells at day 2. In thymidine incorporation, peritoneal B-1 cells entered actively S phase after 24hours LPS-stimulation but splenic B-2 cells entered actively S phase after 48 hours. CONCLUSION: IgM secretion and S phase entering occurred early in peritoneal B-1 cells compared to splenic B-2 cells.
Animals ; Ascitic Fluid ; Immunoglobulin M ; Immunoglobulins ; Mice ; S Phase ; Spleen ; Thymidine

BACKGROUND: CD27 is recently known as a memory B cell marker and is mainly expressed in activated T cells, some B cell population and NK cells. CD27 is a member of tumor necrosis factor receptor family. Like CD40 molecule, CD27 has (P/S/T/A) X(Q/E)E motif for interacting with TNF receptor-associated factors (TRAFs), and TRAF2 and TRAF5 bindings to CD27 in 293T cells were reported. METHODS: To investigate the CD27 signaling effect in B cells, human CD40 extracellular domain containing mouse CD27 cytoplamic domain construct (hCD40-mCD27) was transfected into mouse B cell line CH12.LX and M12.4.1. RESULTS: Through the stimulation of hCD40-mCD27 molecule via anti-human CD40 antibody or CD154 ligation, expression of CD11a, CD23, CD54, CD70 and CD80 were increased and secretion of IgM was induced, which were comparable to the effect of CD40 stimulation. TRAF2 and TRAF3 were recruited into lipid-enriched membrane raft and were bound to CD27 in M12.4.1 cells. CD27 stimulation, however, did not increase TRAF2 or TRAF3 degradation. CONCLUSION: In contrast to CD40 signaling pathway, TRAF2 and TRAF3 degradation was not observed after CD27 stimulation and it might contribute to prolonged B cell activation through CD27 signaling.
Animals ; B-Lymphocytes* ; Cell Line ; Humans ; Immunoglobulin M ; Killer Cells, Natural ; Ligation ; Membranes ; Memory ; Mice* ; Necrosis* ; Receptors, Tumor Necrosis Factor ; T-Lymphocytes ; TNF Receptor-Associated Factor 2 ; TNF Receptor-Associated Factor 3 ; TNF Receptor-Associated Factor 5 ; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins

In recent years, adjuvants have received much attention because of the development of purified subunit and synthetic vaccines which are poor immunogens and require adjuvants to evoke the immune response. Therefore, immunologic adjuvants have been developed and testing for most of this century. During the last years much progress has been made on development, isolation and chemical synthesis of alternative adjuvants such as derivatives of muramyl dipeptide, monophosphoryl lipid A, liposomes, QS-21, MF-59 and immunostimulating complexes (ISCOMS). Biodegradable polymer microspheres are being evaluated for targeting antigens on mucosal surfaces and for controlled release of vaccines with an aim to reduce the number of doses required for primary immunization. The most common adjuvants for human use today are aluminum hydroxide and aluminum phosphate. Calcium phosphate and oil emulsions have been also used in human vaccination. The biggest issue with the use of adjuvants for human vaccines is the toxicity and adverse side effects of most of the adjuvant formulations. Other problems with the development of adjuvants include restricted adjuvanticity of certain formulations to a few antigens, use of aluminum adjuvants as reference adjuvant preparations under suboptimal conditions, non-availability of reliable animal models, use of non-standard assays and biological differences between animal models and humans leading to the failure of promising formulations to show adjuvanticity in clinical trials. The availability of hundreds of different adjuvants has prompted a need for identifying rational standards for selection of adjuvant formulations based on safety and sound immunological principles for human vaccines. The aim of the present review is to put the recent findings into a broader perspective to facilitate the application of these adjuvants in general and experimental vaccinology.
Acetylmuramyl-Alanyl-Isoglutamine ; Adjuvants, Immunologic ; Aluminum ; Aluminum Hydroxide ; Calcium ; Emulsions ; Humans ; Immunization ; ISCOMs ; Lipid A ; Liposomes ; Microspheres ; Models, Animal ; Polymers ; Vaccination ; Vaccines ; Vaccines, Synthetic

A pandemic influenza A (H1N1) virus strain was isolated from a pig farm in Korea in December 2009. The strain was propagated in and isolated from both the Madin-Darby canine kidney cell line and embryonated eggs. The partial and complete sequences of the strain were identical to those of A/California/04/2009, with >99% sequence similarity in the HA, NA, M, NS, NP, PA, PB1, and PB2 genes. The isolated strain was inactivated and used to prepare a swine influenza vaccine. This trial vaccine, containing the new isolate that has high sequence similarity with the pandemic influenza A (H1N1) virus, resulted in seroconversion in Guinea pigs and piglets. This strain could therefore be a potential vaccine candidate for swine influenza control in commercial farms.
Agriculture ; Animals ; Cell Line ; Eggs ; Guinea Pigs ; Influenza Vaccines ; Influenza, Human* ; Kidney ; Korea* ; Orthomyxoviridae* ; Ovum ; Pandemics* ; Seroconversion ; Swine

In this study, we compared two different tumor cell vaccines for their induction of anti-tumor immunity; one was a tumor cell clone expressing a membrane-bound form of IL-12 p35 subunit (mbIL-12 p35 tumor clone), and the other was a tumor clone expressing heterodimeric IL-12 as a single chain (mb-scIL-12 tumor clone). The stimulatory effect of mb-scIL-12 on the proliferation of ConA-activated splenocytes was higher than that of mbIL-12 p35 in vitro. However, the stimulatory effect of mbIL-12 p35 was equivalent to that of recombinant soluble IL-12 (3 ng/ml). Interestingly, both tumor clones (mbIL-12 p35 and mb-scIL-12) showed similar tumorigenicity and induction of systemic anti-tumor immunity in vivo, suggesting that tumor cell expression of the membrane-bound p35 subunit is sufficient to induce anti-tumor immunity in our tumor vaccine model.
Clone Cells ; In Vitro Techniques ; Interleukin-12* ; Vaccines