New protective antigen of group A streptococci. (33/7228)

It is widely believed that the surface M protein of group A streptococci is the predominant surface protein of these organisms containing opsonic epitopes. In the present study, we identified a new surface protein, distinct from M protein, that evokes protective antibodies. A type 18 M-negative mutant was found to be both resistant to phagocytosis in human blood and virulent in mice. The wild-type strain, but not the M-negative mutant, was opsonized by antisera against purified recombinant M18 protein or a synthetic peptide copying the NH2-terminus of M18. However, antisera raised against a crude pepsin extract of the M-negative mutant opsonized both strains, indicating the presence of a protective antigen in addition to type 18 M protein. This antiserum was used to identify and purify a 24-kDa protein fragment (Spa, streptococcal protective antigen) that evoked antibodies that opsonized the M18 parent and the M-negative mutant. The results of passive mouse protection tests confirmed the presence of protective epitopes within Spa. The deduced amino acid sequence of a 636-bp 5' fragment of the spa18 gene showed no homology with sequences in GenBank. These studies reveal the presence of a new protective antigen of certain strains of group A streptococci that may prove to be an important component of vaccines to prevent streptococcal infections.  (+info)

Purification and some chemical properties of 30 kDa Ginkgo biloba glycoprotein, which reacts with antiserum against beta 1-->2 xylose-containing N-glycans. (34/7228)

From the seeds of Ginkgo biloba, a glycoprotein, which is a major component that reacts with an antiserum against beta 1-->2 xylose-containing N-glycans, has been purified and characterized. The N-terminal amino acid sequence of the purified glycoprotein was H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-L-L-F-G-Q-. The molecular mass was estimated to be 17 kDa and 16 kDa by SDS-PAGE under reducing conditions, however, the molecular mass of this glycoprotein in the native state was 30,762 by MALDI-TOF MS, suggesting that this glycoprotein consists of two subunits; one is glycosylated and the other is not. The structure of N-glycan linked to this glycoprotein (designated 30 kDa GBGP) was identified as Man3Fuc1Xyl1GlcNAc2, which is the predominant N-glycan linked to the storage glycoproteins in the same seeds (Kimura, Y et al. (1998) Biosci. Biotechnol. Biochem. 62, 253-261). From the peptic digest of the carboxymethylated glycosylated subunit, one glycopeptide was purified by RP-HPLC and the amino acid sequence was identified as H-K-A-N-N(Man3Fuc1Xyl1Glc-NAc2)-V-T-V-A-F, which corresponded to the N-terminal amino acid sequence.  (+info)

Non-proteolytic activation of human prorenin by anti-prorenin prosegment (pf#1: 1P-15P) antiserum. (35/7228)

Recombinant human prorenin was activated by incubation with anti-prorenin prosegment (L1PPTDTTTFKRIFLKR15P) antiserum at 4 degrees C. This activation was dependent on the concentration of the antiserum and incubation time. After the activation no molecular weight alteration of prorenin was observed by immunoblotting analysis. A peptide of L1PPTDTTTF8P as well as L1PPTDTTTFKRIFLKR15P potently interfered with the activation. Most of the activated prorenin bound to Protein A Sepharose CL 4B. The Km and Vmax values of the activated prorenin were 0.2 microM and 23.7 micrograms Ang I/ml/h, respectively, which were similar in level to those of mature renin obtained by trypsinization.  (+info)

Cell cycle-dependent regulation of FLIP levels and susceptibility to Fas-mediated apoptosis. (36/7228)

Activation-induced cell death of peripheral T cells results from the interaction between Fas and Fas ligand. Resting peripheral T cells are resistant to Fas-induced apoptosis and become susceptible only after their activation. We have investigated the molecular mechanism mediating the sensitization of resting peripheral T cells to Fas-mediated apoptosis following TCR stimulation. TCR activation decreases the steady state protein levels of FLIP (FLICE-like inhibitory protein), an inhibitor of the Fas signaling pathway. Reconstitution of intracellular FLIP levels by the addition of a soluble HIV transactivator protein-FLIP chimera completely restores resistance to Fas-mediated apoptosis in TCR primary T cells. Inhibition of IL-2 production by cyclosporin A, or inhibition of IL-2 signaling by rapamycin or anti-IL-2 neutralizing Abs prevents the decrease in FLIP levels and confers resistance to Fas-mediated apoptosis following T cell activation. Using cell cycle-blocking agents, we demonstrate that activated T cells arrested in G1 phase contain high levels of FLIP protein, whereas activated T cells arrested in S phase have decreased FLIP protein levels. These findings link regulation of FLIP protein levels with cell cycle progression and provide an explanation for the increase in TCR-induced apoptosis observed during the S phase of the cell cycle.  (+info)

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. (37/7228)

This study shows that the normal thymus produces immunoregulatory CD25+4+8- thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8- thymocytes, which constitute approximately 5% of steroid-resistant mature CD4+8- thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8- thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8- or CD4-8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8- thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25-4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8- thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8- thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous alpha-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8- thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.  (+info)

CD8+ T cells are a biologically relevant source of macrophage inflammatory protein-1 alpha in vivo. (38/7228)

Chemokines are small proteins that direct the migration of leukocytes to inflammatory foci. Many cell types, including macrophages, fibroblasts, endothelial cells, and lymphocytes, produce chemokines in vitro, but biologically relevant sources of chemokines in vivo have not been well characterized. To investigate the pertinent sources of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in vivo, we used MIP-1 alpha-deficient (MIP-1 alpha-/-) mice as donors and as recipients in adoptive transfer experiments after a lethal infection with Listeria monocytogenes (LM). Unexpectedly, we found that the production of MIP-1 alpha by CD8+ T cells was critical in this system, as the cells from MIP-1 alpha-/- mice primed with LM were significantly less effective in protecting naive mice against a lethal infection by LM than were the CD8+ T cells from wild-type (wt) mice. This requirement for donor T cell production of MIP-1 alpha was confirmed by the observation that wt donor T cells do not mediate protection when coadministered with an anti-MIP-1 alpha polyclonal antiserum. Production of MIP-1 alpha by the recipient mice was not required for protection, because wt and MIP-1 alpha-/- recipients were equally well protected by wt T cells. A 2- to 3-fold decrease in the number of transferred lymphocytes was seen in the spleens of mice receiving T cells from MIP-1 alpha-/- mice compared with those receiving wt T cells. In addition, CD8+ T cells from MIP-1 alpha-/- mice had a reduced ability to kill LM-infected target cells in vitro. These findings demonstrate that T cell production of MIP-1 alpha is required for clearance of an intracellular pathogen in vivo.  (+info)

Multiple roles for IL-12 in a model of acute septic peritonitis. (39/7228)

The present study addressed the role of IL-12 in a murine model of septic peritonitis, induced by cecal ligation and puncture (CLP). Although CLP surgery induced IL-12 production at 6 and 24 h after surgery, IL-12 immunoneutralization was clearly deleterious in this model: 54% of CLP mice receiving preimmune serum survived, whereas mice administered IL-12 antisera prior to CLP experienced a 25% survival rate. IL-12 immunoneutralization not only led to increased mortality, but also appeared to promote a shift away from IL-12 and IFN-gamma, in favor of IL-10. This cytokine shift corresponded to changes in bacterial load, as CLP mice receiving IL-12 antiserum yielded more CFUs from the peritoneal cavity at 24 h after CLP. To address the role of bacterial infection in IL-12 antiserum-induced mortality following CLP, antibiotics were administered for 4 days after surgery. Despite regular antibiotic administration, IL-12 immunoneutralization still reduced survival in CLP mice. Furthermore, histology of the ceca revealed that mice administered IL-12 antisera failed to show typical organization of the damaged cecum wall. Accordingly, Gram staining revealed bacteria within peritoneal fluids from these mice, while peritoneal fluids from CLP mice that received preimmune serum and antibiotics were free of bacteria. Altogether, these data suggested multiple important roles for IL-12 in the evolution of murine septic peritonitis.  (+info)

Neutralization of the CXC chemokine, macrophage inflammatory protein-2, attenuates bleomycin-induced pulmonary fibrosis. (40/7228)

Few studies have addressed the importance of vascular remodeling in the lung during the development of bleomycin-induced pulmonary fibrosis. For fibroplasia and deposition of extracellular matrix to occur, there must be a geometric increase in neovascularization. We hypothesized that net angiogenesis during the pathogenesis of fibroplasia and deposition of extracellular matrix during bleomycin-induced pulmonary fibrosis are dependent in part upon an overexpression of the angiogenic CXC chemokine, macrophage inflammatory protein-2 (MIP-2). To test this hypothesis, we measured MIP-2 by specific ELISA in whole lung homogenates in either bleomycin-treated or control CBA/J mice and correlated these levels with lung hydroxyproline. We found that lung tissue from mice treated with bleomycin, compared with that from saline-treated controls, demonstrated a significant increase in the presence of MIP-2 that was correlated to a greater angiogenic response and total lung hydroxyproline content. Neutralizing anti-MIP-2 Abs inhibited the angiogenic activity of day 16 bleomycin-treated lung specimens using an in vivo angiogenesis bioassay. Furthermore, when MIP-2 was depleted in vivo by passive immunization, bleomycin-induced pulmonary fibrosis was significantly reduced without a change in the presence of pulmonary neutrophils, fibroblast proliferation, or collagen gene expression. This was also paralleled by a reduction in angiogenesis. These results demonstrate that the angiogenic CXC chemokine, MIP-2, is an important factor that regulates angiogenesis/fibrosis in pulmonary fibrosis.  (+info)