Death signals from the B cell antigen receptor target mitochondria, activating necrotic and apoptotic death cascades in a murine B cell line, WEHI-231.
(25/2060)
B cell antigen receptor (BCR)-mediated cell death has been proposed as a mechanism for purging the immune repertoire of anti-self specificities during B cell differentiation in bone marrow. Mitochondrial alterations and activation of caspases are required for certain aspects of apoptotic cell death, but how the mitochondria and caspases contribute to BCR-mediated cell death is not well understood. In the present study, we used the mouse WEHI-231 B cell line to demonstrate that mitochondrial alterations and activation of caspases are indeed participants in BCR-mediated cell death. The peptide inhibitor of caspases, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), blocked cleavage of poly(ADP-ribose) polymerase and various manifestation of nuclear apoptosis such as nuclear fragmentation, hypodiploidy and DNA fragmentation, indicating that signals from the BCR induced the activation of caspases. In addition, z-VAD-fmk delayed apoptosis-associated changes in cellular reduction-oxidation potentials as determined by hypergeneration of superoxide anion, as well as exposure of phosphatidylserine residues in the outer plasma membrane. By contrast, although z-VAD-fmk retarded cytolysis, it was incapable of preventing disruption of the plasma membrane even under the same condition in which it completely blocked nuclear apoptosis. Mitochondrial membrane potential loss was also not blocked by z-VAD-fmk. Bongkrekic acid, a specific inhibitor of mitochondrial permeability transition pores, suppressed not only the mitochondrial membrane potential but also the change of plasma membrane permeability. Overexpression of Bcl-xL prevented mitochondrial dysfunction, nuclear apoptosis and membrane permeability cell death triggered by BCR signal transduction. These observations indicate that death signals from BCR may first cause mitochondrial alterations followed by activation of both necrotic and apoptotic cascades. (+info)
Preparation and application of anti-idiotypic antibody against anti-gibberellin A4 antibody.
(26/2060)
A monoclonal anti-idiotypic antibody was raised against anti-gibberellin A4 (GA4) antibody, which recognizes biologically active gibberellins such as GA1 and GA4 specifically. Amino acid sequences of variable regions of both anti-GA4 and anti-idiotypic antibodies were analyzed. By using the property of the anti-idiotypic antibody to compete with GA1/4 in binding to the anti-GA4 antibody, we successfully applied the anti-idiotypic antibody to ELISA as a tracer for measuring GA1/4. The single-chain Fv (scFv) gene of the anti-idiotypic antibody was constructed, and scFv expressed in E. coli showed binding activity to anti-GA4 antibody. These results suggest the possible application of anti-idiotypic antibody as a handy and stable source of an enzymatic tracer for ELISA by production of fusion protein of the scFv and an appropriate enzyme. (+info)
Anti-Sa antibody in Chinese rheumatoid arthritis.
(27/2060)
OBJECTIVE: To test anti-Sa antibody in different autoimmune connective tissue diseases and analyze the relationship between Sa antibody and clinical manifestations and laboratory tests in rheumatoid arthritis. METHOD: Sa antigen was extracted from human placenta. Anti-Sa antibody was tested in 40 normal people and 478 connective tissue disease (CTD) patients using Western Blotting (WB). RESULTS: Sa antigen was a protein with molecular weights of 50 kD and 55 kD. Anti-Sa antibody was positive in 31.9% (61/191) rheumatoid arthritis (RA), 3.0% (2/67) Sjogren's syndrome (SS), 4.3% (2/46) systemic lupus erythmatosus (SLE) and 0% (0/66) Behcet's disease, 0% (0/60) polymyositis/dermatomyositis (PM/DM), 0% (0/66) other CTD and 0% (0/40) normal controls. Anti-Sa antibody was different from other auto-antibodies in RA. In rheumatoid arthritis its sensitivity, specificity, positive prediction rate, negative prediction rate were 31.9%, 98.6%, 93.8% and 68.5% respectively. Anti-Sa antibody positive patients were significantly different from anti-Sa antibody negative patients in moming stiffness, ESR, ANA and X-ray grade. CONCLUSION: Anti-Sa antibody was a new auto-antibody for the diagnosis of RA. Anti-Sa antibody positive patients seem to have more serious inflammation and more advanced disease process. (+info)
Inhibitors of PI 3-kinase and MEK kinase differentially affect mediator secretion from immunologically activated human basophils.
(28/2060)
Effects of inhibitors of PI 3-kinase and MEK kinases were investigated on histamine, leukotriene C4(LTC4), and cytokine release from human basophils stimulated with anti-IgE. The PI 3-kinase antagonists wortmannin (> 10 nM) and LY 294002 (>1 microM) strongly inhibited anti-IgE-induced release of all mediators by 40-100%. This was contrasted by the effects of the MEK kinase inhibitor PD 098059, which weakly inhibited histamine, interleukin (IL)-4, and IL-13 release but was substantially more efficacious at blocking LTC4 production (>70% at 10 microM). Previous studies have shown that arachidonic acid synthesis is controlled by MEK kinases. We observed that wortmannin, LY 294002, and PD 098059 reduce basophil ERK-1,2 activation, thus implying that, with regard to arachidonic acid metabolism, MEK kinases are a downstream target for PI-3-kinase. Our results demonstrate a universal regulatory role played by PI 3-kinases in basophil mediator production and release, whereas MEK kinase signaling is largely limited to controlling arachidonic acid metabolism. (+info)
Molecular and cellular basis of the altered immune response against arsonate in irradiated A/J mice autologously reconstituted.
(29/2060)
The humoral immune response to arsonate (Ars) in normal A/J mice is dominated in the late primary and particularly in the secondary response by a recurrent and dominant idiotype (CRIA) which is encoded by a single canonical combination of the variable gene segments: VHidcr11-DFL16.1-JH2 and Vkappa10-Jkappa1. Accumulation of somatic mutations within cells expressing this canonical combination or some less frequent Ig rearrangements results in the generation of high-affinity antibodies. By contrast, in partially shielded and irradiated A/J mice (autologous reconstitution) immunized with Ars-keyhole limpet hemocyanin (KLH), both the dominance of the CRIA idiotype and the affinity maturation are lost, whereas the anti-Ars antibody titer is not affected. To understand these alterations, we have analyzed a collection of 27 different anti-Ars hybridomas from nine partially shielded and irradiated A/J mice that had been immunized twice with Ars-KLH. Sequence analysis of the productively rearranged heavy chain variable region genes from those hybridomas revealed that (i) the canonical V(D)J combination was rare, (ii) the pattern of V(D)J gene usage rather corresponded to a primary repertoire with multiple gene combinations and (iii) the frequency of somatic mutations was low when compared to a normal secondary response to Ars. In addition, immunohistological analysis has shown a delay of 2 weeks in the appearance of full blown splenic germinal centers in autoreconstituting mice, as compared to controls. Such a model could be useful to understand the immunological defects found in patients transplanted with bone marrow. (+info)
Humoral response suppression observed with CD23 transgenics.
(30/2060)
CD23, also known as the low affinity IgE receptor (FcepsilonRII), has been hypothesized to have a role in IgE regulation. A new CD23 transgenic mouse was generated using the MHC class I promoter and IgH enhancer to further test the hypothesis that CD23 plays a role in the down-regulation of IgE. Study of three founder lines by FACS showed overexpression to varying extents on both B and T lymphocytes. No alterations in lymphocyte populations was observed. All three founder lines exhibited strong suppression of IgE in response to DNP-keyhole limpet hemocyanin/alum and Nippostrongylus brasiliensis infection compared with that in parental or littermate controls. The founder line exhibiting the highest level of suppression also was less susceptible to Ag-induced systemic anaphylactic shock. Overall, the data support the concept that enhancing CD23 levels can be used to suppress IgE-mediated disease. The mechanism involves decreased IgE synthesis, because the serum half-life of IgE was not altered in transgenics, and enzyme-linked immunospot analysis demonstrated lower IgE-producing cells stimulated by injection of anti-IgD. Transgenics also exhibited significantly decreased IgG1 responses and exhibited lower levels of all Ig isotypes, although this was more variable in different founder lines. (+info)
Long survival of patients with small cell lung cancer after adjuvant treatment with the anti-idiotypic antibody BEC2 plus Bacillus Calmette-Guerin.
(31/2060)
Despite active therapies for small cell lung cancer (SCLC), most patients relapse and die of the disease. The present study evaluates immunization using the anti-idiotypic antibody BEC2, which mimics the ganglioside GD3 expressed on the surface of most SCLC tumors, combined with Bacillus Calmette-Guerin (BCG) as an immune adjuvant. We hypothesized that active immunization could alter the natural history of the disease. Fifteen patients who had completed standard therapy for SCLC received a series of five intradermal immunizations consisting of 2.5 mg of BEC2 plus BCG over a 10-week period. Blood was collected for serological analysis, and outcome was monitored. All patients developed anti-BEC2 antibodies, despite having received chemotherapy with or without thoracic radiation. We detected anti-GD3 antibodies in five patients, including those with the longest relapse-free survival. The median relapse-free survival for patients with extensive stage disease is 11 months and has not been reached for patients with limited stage disease (>47 months), with only one of seven patients having relapsed after a median follow-up of 47 months. Immunization of patients with SCLC after standard therapy using BEC2 plus BCG can induce anti-GD3 antibodies and is safe. The survival and relapse-free survival in this group of patients are substantially better than those observed in a prior group of similar patients. A Phase III trial is being conducted to evaluate BEC2 plus BCG as adjuvant therapy after chemotherapy and irradiation. (+info)
Evaluation of four methods for detection of immunoglobulin M antibodies to dengue virus.
(32/2060)
Dengue has become hyperendemic in many islands of the Caribbean region. The performance in a diagnostic laboratory of four commercial assays for detection of immunoglobulin M (IgM) antibodies was evaluated. Sera from 62 patients with dengue virus infection were studied. These included 18 patients from whom dengue virus type 2 was isolated in a 1997 outbreak (specimens collected a mean of 14 days after onset of symptoms), 8 patients with dengue hemorrhagic fever (mean time after onset, 11 days), and 36 patients in whom dengue was previously confirmed by serology (mean time after onset, 10 days). Thirty serum specimens from blood donors in a country where dengue is not endemic were used as negative controls. The methods evaluated were two IgM-capture enzyme-linked immunosorbent assays (ELISA) (MRL Diagnostics, Cypress, Calif., and PanBio, Queensland, Australia), a dot ELISA dipstick assay (Integrated Diagnostics, Baltimore, Md.), and a rapid immunochromatographic assay for dengue IgG and IgM (PanBio IC). IgG antibodies were also detected by an ELISA method (MRL Diagnostics). The sensitivities of the four assays were as follows: MRL Diagnostics IgM ELISA, 98.4%; PanBio IgM ELISA, 85.5%; Integrated Diagnostics IgM dot ELISA, 96. 8%; and PanBio IC, 83.9%. The specificities of all tests were 100%. Evidence of secondary dengue was found in all patients with dengue hemorrhagic fever and in 83% of the remaining patients. The MRL Diagnostics IgM ELISA appears to be more sensitive than the PanBio IgM ELISA, and this may be significant when IgM titers are low, particularly in patients with secondary dengue infections. The dot ELISA dipstick assay is equally sensitive and may be more appropriate for use in laboratories with lower workloads. (+info)