Suppression of induction of experimental immune mediated blepharoconjunctivitis by tolerogenic conjugates of the antigen and monomethoxypolyethylene glycol.
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AIM: Covalent conjugates consisting of diverse antigens coupled to optimal numbers of monomethoxypolyethylene glycol (mPEG) molecules have been shown to suppress antigen specific antibody formation. In this study, the possibility was examined that the same conjugates might prevent experimental immune mediated blepharoconjunctivitis (EC, formerly EAC) which had been shown to be caused by CD4(+) T cells-that is, to cell mediated immunity. METHODS: 6-8 week old male Lewis rats were used. The test groups of rats received two intravenous injections, each of 300 microg, of a conjugate of ovalbumin mPEG (OVA(mPEG)(11)) in phosphate buffered saline (PBS), 14 and 28 days before the single immunisation with OVA in complete Freund's adjuvant. The rats were challenged 3 weeks later by eye drops containing OVA; 24 hours later they were sacrificed, and their eyes, blood, and lymph nodes were harvested for histological examination and determination of anti-OVA antibody titres and levels of cellular immunity. Two control groups received PBS or OVA in PBS before immunisation. Furthermore, the possibility that OVA(mPEG)(11) may have induced OVA specific suppressor cells was tested by establishing the effects of the co-transfer of splenocytes from OVA(mPEG)(11) treated rats with OVA primed lymph node cells on the manifestations of EC. RESULTS: Either PBS or OVA pretreated rats, which had not received OVA(mPEG)(11), developed high levels of antibodies and cell mediated immune responses to OVA, and application of eye drops led to blepharoconjunctivitis with massive cellular infiltration. In contrast, pretreatment with OVA(mPEG)(11) prevented cellular infiltration into the lids and conjunctivas, as well as the formation of detectable humoral and cellular immunity against OVA. Co-transfer of splenocytes from OVA(mPEG)(11) treated rats with OVA primed lymph node cells suppressed the cellular infiltration on application of OVA on the conjunctiva. CONCLUSIONS: These data indicate that intravenous injection of OVA(mPEG)(11) conjugates suppressed both humoral and cellular immunity by the effects of antigen specific suppressor cells, thus leading to the inhibition of development of EC. (+info)
Suppression of immediate and late responses to antigen by a non-anaphylactogenic anti-IgE antibody in a murine model of asthma.
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Eosinophils are recruited to the airways during allergic reactions, but animal models have shown that their mere presence is not sufficient for the development of bronchopulmonary hyperreactivity. Other factors, such as immunoglobulin (Ig)E, seem to be required. Using mice selected for the production of large amounts of IgE, the effects of antibody neutralization of IgE on antigen-induced lung recruitment of eosinophils and induction of bronchopulmonary hyperreactivity and of other indicators of inflammation were studied. A monoclonal non-anaphylactogenic rat anti-mouse IgE (mAb1-5), given within 24 h of the challenge with antigen, reduced tissue eosinophilia, the recruitment of IgE-bearing cells identified as basophils, mucous cell metaplasia, anaphylactic bronchoconstriction and bronchopulmonary hyperreactivity. mAb1-5 inhibited interleukin (IL)4 titres in the bronchoalveolar lavage fluid, but not those of I1-5. Inhibition by mAb1-5 may result from competitive displacement of immunoglobulin E from its different receptors, thus preventing cell stimulation. Moreover, the inhibition of the massive recruitment of immunoglobulin E-bearing basophils into the lungs within hours after challenge and of interleukin4 production by mAb1-5 may be important factors leading to the reduction of pulmonary eosinophilia and bronchopulmonary hyperreactivity. Thus, immunoglobulin (Ig)E and allergic IgE-bearing cells seem to play an essential role in the initial development of the late allergic airway responses. (+info)
IL-3 does not affect the allergic airway responses and leukotriene production after allergen challenge in rats.
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T cell cytokines are important in asthma. Interleukin (IL)-3, an important growth factor for mast cells and eosinophils has been shown to be increased in the airways of asthmatic subjects, but its precise functions are uncertain. The aim of this study was to determine whether recombinant human (rh) IL-3 affected airway responses, inflammation and leukotriene production after antigen challenge in Brown Norway (BN) rats. Having established that rhIL-3 (>12.5 microg subcutaneously b.i.d. for 4 days) caused a doubling of mast cell numbers in the airways of BN rats, sensitized rats were pretreated with rhIL-3 (50 microg) or vehicle subcutaneously b.i.d. for 4 days. Ovalbumin (OA) challenge was performed and the early (EAR), and late (LAR) airway response and the associated biliary leukotriene (LT) excretion measured. The pulmonary cellularity was evaluated by means of lung digestion 8 h after challenge. IL-3 increased the number of eosinophils isolated from the lungs after antigen challenge (0.77+/-0.23 versus 0.38+/-0.12 x 10(6) cells, p=0.03). However, there were no effects on the numbers of neutrophils, lymphocytes and macrophages. Neither the EAR nor the LAR after OA challenge were altered by IL-3. Likewise biliary cysteinyl-LT excretion was similar in IL-3-treated animals and controls after challenge. In conclusion, interleukin-3 caused an increase in the numbers of mast cells and eosinophils around the airways without affecting the magnitude of either early or late airway responses or mediator release after antigen challenge. The present results suggest that airway inflammation can occur in rats without increasing the allergic asthmatic response. (+info)
Effect of respiratory syncytial virus on subsequent allergic sensitization to ovalbumin in guinea-pigs.
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Children with acute respiratory syncytial virus (RSV) bronchiolitis often develop recurrent wheezing, asthma and allergic sensitization, but the role of RSV in the pathogenesis of these sequelae is unclear. This study examined whether RSV infection potentiates subsequent allergic sensitization, airway hyperresponsiveness (AHR) and airway inflammation induced by repeated exposures to aerosolized ovalbumin (OA) in guinea-pigs. Guinea-pigs received either RSV or sham inoculum, followed by exposures to OA- or saline-containing aerosols to form the following groups: 1) noninfected, nonsensitized controls (sham/saline group); 2) RSV-infected, nonsensitized animals (RSV/ saline group); 3) noninfected, OA-sensitized animals (sham/OA group); 4) RSV infection and first OA exposure on the same day (RSV/OA group), and 5) RSV infection six days prior to first OA exposure (RSV6/OA group). Three days after the final aerosol exposure, circulating OA-specific immunoglobulin (Ig)G1 antibody titres and AHR to inhalation acetylcholine challenge were measured and morphometry performed to evaluate allergic inflammation of the airways. OA-exposed animals developed OA-specific IgG1 antibodies, AHR and airway eosinophilia (sham/OA, RSV/OA and RSV6/OA groups. RSV infection alone induced significant AHR and airway eosinophilia (RSV/saline group). RSV infection, and concomitant exposure to OA (RSV/OA group) enhanced OA-specific IgG1 antibodies, but not airway eosinophilia or AHR. Such increases were not observed in the RSV6/OA group. In conclusion, respiratory syncytial virus potentiates the production of ovalbumin-specific immunoglobulin G1 antibodies in guinea-pigs, but circulating titres of these antibodies do not reflect the extent of airway hyperresponsiveness or airway inflammation. In addition, respiratory syncytial virus infection alone can produce slight increases in airway hyperresponsiveness that are associated with increased numbers of eosinophils in the airways. (+info)
Primed T cells are more resistant to Fas-mediated activation-induced cell death than naive T cells.
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Memory T cells respond in several functionally different ways from naive T cells and thus function as efficient effector cells. In this study we showed that primed T cells were more resistant to Fas-mediated activation-induced cell death (AICD) than naive T cells using OVA-specific TCR transgenic DO10 mice and Fas-deficient DO10 lpr/lpr mice. We found that apoptosis was efficiently induced in activated naive T cells at 48 and 72 h after Ag restimulation (OVA peptide; 0.3 and 3 microM), whereas apoptosis was not significantly increased in activated primed T cells at 24-72 h after Ag restimulation. We further showed that the resistance to AICD in primed T cells was due to the decreased sensitivity to apoptosis induced by Fas-mediated signals, but TCR-mediated signaling equally activated both naive and primed T cells to induce Fas and Fas ligand expressions. Furthermore, we demonstrated that primed T cells expressed higher levels of Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein (FLIP), an inhibitor of Fas-mediated apoptosis, at 24-48 h after Ag restimulation than naive T cells. In addition, Bcl-2 expression was equally observed between activated naive and primed T cells after Ag restimulation. Thus, these results indicate that naive T cells are sensitive to Fas-mediated AICD and are easily deleted by Ag restimulation, while primed/memory T cells express higher levels of FLIP after Ag restimulation, are resistant to Fas-mediated AICD, and thus function as efficient effector cells for a longer period. (+info)
26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate.
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Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein-antigens in the cytoplasm. Initially, antigens are degraded by the 26 S proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2K(b)-restricted peptide epitope, derived from ovalbumin SIINFEKL (termed ODC-ova). Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODC-ova by the 26 S proteasome resulted in the generation of the K(b)-ligand. Mass spectrometry analysis indicated that in addition to SIINFEKL, the N-terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC-ova degradation. The overall yield of SIINFEKL was approximately 5% of the amount of ODC-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope. (+info)
Histidine-rich glycoprotein regulates the binding of monomeric IgG and immune complexes to monocytes.
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Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein which we have shown previously inhibits the formation of insoluble immune complexes (IC). In this study we examined the ability of HRG to regulate the binding of monomeric IgG and IC to monocytes. Initial studies demonstrated that HRG interacts with FcgammaRI on the monocytic cell line THP1 and blocks the binding of monomeric IgG to these cells. However, despite totally blocking the binding of monomeric IgG to FcgammaRI, pre-incubation of THP1 cells with HRG had no effect on the binding of IC to these cells. In contrast, depending on the HRG:IgG molar ratio, pre-incubation of monomeric IgG with HRG resulted in either enhanced or reduced IgG binding to FcgammaRI. Similarly, under certain highly defined conditions, incorporation of HRG in IgG-containing IC potentiated the binding of IC to THP1 cells. The key conditions involved incorporating approximately equimolar concentrations of HRG and IgG in the IC, the IC being formed at a near equivalence antigen:antibody ratio and usually physiological concentration (20 microM) of Zn(2+) being present. Collectively these observations indicate that HRG is an important regulator of IC uptake by monocytes. Thus HRG can interact with FcgammaRI on monocytes and block monomeric IgG binding, whereas when incorporated in IgG containing IC, HRG can enhance the uptake of IC by monocytes, probably via its heparan sulfate binding domain. (+info)
Chicken ovalbumin is synthesized and secreted by Escherichia coli.
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By recombinant DNA methods, the chicken ovalbumin structural gene has been fused to Escherichia coli lac transcriptional and translational control regions. When a plasmid containing the hybrid gene was introduced into E. coli, a protein identified as ovalbumin by immunoreactivity and sodium dodecyl sulfate/polyacrylamide gel electrophoresis was synthesized. The chicken ovalbumin made in bacteria was full length (43,000 daltons) and constituted 1.5% of the cellular protein. In addition, the microbially synthesized ovalbumin was secreted through the cell membrane into the periplasmic space of E. coli. The ability of the E. coli secretory apparatus to recognize chicken ovalbumin, which is normally synthesized and secreted in hen oviducts, suggests that common features exist in the secretion-recognition mechanisms found in these two organisms. The bacterial synthesis of significant amounts of chicken ovalbumin demonstrates that the E. coli cellular machinery may be utilized to synthesize a higher eukaryotic protein which is relatively stable in the bacterial intracellular environment. (+info)