Antifactor VIII antibody inhibiting allogeneic but not autologous factor VIII in patients with mild hemophilia A.
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Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF. (+info)
Evidence of partial protection against foot-and-mouth disease in cattle immunized with a recombinant adenovirus vector expressing the precursor polypeptide (P1) of foot-and-mouth disease virus capsid proteins.
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A recombinant live vector vaccine was produced by insertion of cDNA encoding the structural proteins (P1) of foot-and-mouth disease virus (FMDV) into a replication-competent human adenovirus type 5 vaccine strain (Ad5 wt). Groups of cattle (n = 3) were immunized twice, by the subcutaneous and/or intranasal routes, with either the Ad5 wt vaccine or with the recombinant FMDV Ad5-P1 vaccine. All animals were challenged by intranasal instillation of FMDV 4 weeks after the second immunizations. In the absence of a detectable antibody response to FMDV, significant protection against viral challenge was seen in all of the animals immunized twice by the subcutaneous route with the recombinant vaccine. The observed partial protection against clinical disease was not associated with a reduction in titre of persistent FMDV infections in the oropharynx of challenged cattle. (+info)
Expression and characterization of a DNase I-Fc fusion enzyme.
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Recombinant human deoxyribonuclease I (DNase I) is an important clinical agent that is inhaled into the airways where it degrades DNA to lower molecular weight fragments, thus reducing the viscoelasticity of sputum and improving the lung function of cystic fibrosis patients. To investigate DNases with potentially improved properties, we constructed a molecular fusion of human DNase I with the hinge and Fc region of human IgG1 heavy chain, creating a DNase I-Fc fusion protein. Infection of Sf9 insect cells with recombinant baculovirus resulted in the expression and secretion of the DNase I-Fc fusion protein. The fusion protein was purified from the culture medium using protein A affinity chromatography followed by desalting by gel filtration and was characterized by amino-terminal sequence, amino acid composition, and a variety of enzyme-linked immunosorbent assays (ELISA) and activity assays. The purified fusion contains DNase I, as determined by a DNase I ELISA and an actin-binding ELISA, and an intact antibody Fc region, which was quantified by an Fc ELISA, in a 2:1 stoichiometric ratio, respectively. The dimeric DNase I-Fc fusion was functionally active in enzymatic DNA digestion assays, albeit about 10-fold less than monomeric DNase I. Cleavage of the DNase I-Fc fusion by papain resulted in a specific activity comparable to the monomeric enzyme. Salt was inhibitory for wild type monomeric DNase I but actually enhanced the activity of the dimeric DNase I-Fc fusion. The DNase I-Fc fusion protein was also less Ca2+-dependent than DNase I itself. These results are consistent with a higher affinity of the dimeric fusion protein to DNA than monomeric DNase I. The engineered DNase I-Fc fusion protein described herein has properties that may have clinical benefits. (+info)
Modulation of the humoral immune response by antibody-mediated antigen targeting to complement receptors and Fc receptors.
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During an ongoing immune response, immune complexes, composed of Ag, complement factors, and Igs, are formed that can interact with complement receptors (CRs) and IgG Fc receptors (Fc gamma R). The role of CR1/2 and Fc gamma R in the regulation of the immune response was investigated using OVA that was chemically conjugated to whole IgG of the rat anti-mouse CR1/2 mAb 7G6. FACS analysis using the murine B cell lymphoma IIA1.6 confirmed that the 7G6-OVA conjugate recognized CR1/2. Incubating IIA1.6 cells with 7G6-OVA triggered tyrosine phosphorylation and Ag presentation to OVA-specific T cells in vitro. Immunizing mice with 7G6-OVA at a minimal dose of 1 microgram i.p. per mouse markedly enhanced the anti-OVA Ig response, which was primarily of the IgG1 isotype subclass. The 7G6-OVA did not enhance the anti-OVA response in CR1/2-deficient mice. OVA coupled to an isotype control Ab induced a considerably lower anti-OVA response compared with that induced by OVA alone, suggesting inhibition by interaction between the Fc part of the Ab and the inhibitory Fc gamma RIIb on B cells. This findings was supported by the observation that IIA1.6 cells which were incubated with 7G6-OVA lost the ability to present Ag upon transfection with Fc gamma RIIb. In sum, 7G6-conjugated OVA, resembling a natural immune complex, induces an enhanced anti-OVA immune response that involves at least CR1/2-mediated stimulation and that may be partially suppressed by Fc gamma RIIb. (+info)
Early membrane rupture events during neutrophil-mediated antibody-dependent tumor cell cytolysis.
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Although cell-mediated cytolysis is a fundamental immune effector response, its mechanism remains poorly understood at the cellular level. In this report, we image for the first time transient ruptures, as inferred by cytoplasmic marker release, in tumor cell membranes during Ab-dependent cellular cytolysis. The cytosol of IgG-opsonized YAC tumor cells was labeled with tetra-methylrhodamine diacetate followed by the formation of tumor cell-neutrophil conjugates. We hypothesized that tumor cell cytolysis proceeds via a series of discrete membrane rupture/resealing events that contribute to marker release. To test this hypothesis, we occluded the fluorescence image of the labeled tumor cells by passing an opaque disk into a field-conjugated plane between the light source and the sample. Multiple small bursts of fluorescent label release from tumor cells could be detected using a photomultiplier tube. Similarly, multiple fluorescent plumes were observed at various sites around the perimeter of a target. These findings support a multihit model of target cytolysis and suggest that cytolytic release is not focused at specific sites. Cytolytic bursts were generally observed at 20-s intervals, which match the previously described reduced nicotinamide-adenine dinucleotide phosphate and superoxide release oscillation periods for neutrophils; we speculate that metabolic oscillations of the effector cell drive the membrane damage of the target. (+info)
Induction of Ig somatic hypermutation and class switching in a human monoclonal IgM+ IgD+ B cell line in vitro: definition of the requirements and modalities of hypermutation.
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Partly because of the lack of a suitable in vitro model, the trigger(s) and the mechanism(s) of somatic hypermutation in Ig genes are largely unknown. We have analyzed the hypermutation potential of human CL-01 lymphocytes, our monoclonal model of germinal center B cell differentiation. These cells are surface IgM+ IgD+ and, in the absence of T cells, switch to IgG, IgA, and IgE in response to CD40:CD40 ligand engagement and exposure to appropriate cytokines. We show here that CL-01 cells can be induced to effectively mutate the expressed VHDJH-C mu, VHDJH-C delta, VHDJH-C gamma, VHDJH-C alpha, VHDJH-C epsilon, and V lambda J lambda-C lambda transcripts before and after Ig class switching in a stepwise fashion. In these cells, induction of somatic mutations required cross-linking of the surface receptor for Ag and T cell contact through CD40:CD40 ligand and CD80: CD28 coengagement. The induced mutations showed intrinsic features of Ig V(D)J hypermutation in that they comprised 110 base substitutions (97 in the heavy chain and 13 in the lambda-chain) and only 2 deletions and targeted V(D)J, virtually sparing CH and C lambda. These mutations were more abundant in secondary VHDJH-C gamma than primary VHDJH-C mu transcripts and in V(D)J-C than V lambda J lambda-C lambda transcripts. These mutations were also associated with coding DNA strand polarity and showed an overall rate of 2.42 x 10(-4) base changes/cell division in VHDJH-CH transcripts. Transitions were favored over transversions, and G nucleotides were preferentially targeted, mainly in the context of AG dinucleotides. Thus, in CL-01 cells, Ig somatic hypermutation is readily inducible by stimuli different from those required for class switching and displays discrete base substitution modalities. (+info)
Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws.
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Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatment with viral IL-10 significantly suppressed the development of Abs to autologous mouse type II collagen. Thus, the treatment of a single joint by local delivery of the vIL-10 gene may protect multiple joints of the same individual while avoiding deleterious side effects often associated with systemic therapy. (+info)
Regulatory effects of endogenous protease inhibitors in acute lung inflammatory injury.
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Inflammatory lung injury is probably regulated by the balance between proteases and protease inhibitors together with oxidants and antioxidants, and proinflammatory and anti-inflammatory cytokines. Rat tissue inhibitor of metalloprotease-2 (TIMP-2) and secreted leukoprotease inhibitor (SLPI) were cloned, expressed, and shown to be up-regulated at the levels of mRNA and protein during lung inflammation in rats induced by deposition of IgG immune complexes. Using immunoaffinity techniques, endogenous TIMP-2 in the inflamed lung was shown to exist as a complex with 72- and 92-kDa metalloproteinases (MMP-2 and MMP-9). In inflamed lung both TIMP-2 and SLPI appeared to exist as enzyme inhibitor complexes. Lung expression of both TIMP-2 and SLPI appeared to involve endothelial and epithelial cells as well as macrophages. To assess how these endogenous inhibitors might affect the lung inflammatory response, animals were treated with polyclonal rabbit Abs to rat TIMP-2 or SLPI. This intervention resulted in significant intensification of lung injury (as revealed by extravascular leak of albumin) and substantially increased neutrophil accumulation, as determined by cell content in bronchoalveolar lavage (BAL) fluids. These events were correlated with increased levels of C5a-related chemotactic activity in BAL fluids, while BAL levels of TNF-alpha and chemokines were not affected by treatment with anti-TIMP-2 or anti-SLPI. The data suggest that endogenous TIMP-2 and SLPI dynamically regulate the intensity of lung inflammatory injury, doing so at least in part by affecting the generation of the inflammatory mediator, C5a. (+info)