Bridging PCR and partially overlapping primers for novel allergen gene cloning and expression insert decoration.
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AIM: To obtain the entire gene open reading frame (ORF) and to construct the expression vectors for recombinant allergen production. METHODS: Gene fragments corresponding to the gene specific region and the cDNA ends of pollen allergens of short ragweed (Rg, Ambrosia artemisiifolia L.) were obtained by pan-degenerate primer-based PCR and rapid amplification of the cDNA ends (RACE), and the products were mixed to serve as the bridging PCR (BPCR) template. The full-length gene was then obtained. Partially overlapping primer-based PCR (POP-PCR) method was developed to overcome the other problem, i.e., the non-specific amplification of the ORF with routine long primers for expression insert decoration. Northern blot was conducted to confirm pollen sources of the gene. The full-length coding region was evaluated for its gene function by homologue search in GenBank database and Western blotting of the recombinant protein Amb a 8(D106) expressed in Escherichia coli pET-44 system. RESULTS: The full-length cDNA sequence of Amb a 8(D106) was obtained by using the above procedure and deduced to encode a 131 amino acid polypeptide. Multiple sequence alignment exhibited the gene D106 sharing a homology as high as 54-89% and 79-89% to profilin from pollen and food sources, respectively. The expression vector of the allergen gene D106 was successfully constructed by employing the combined method of BPCR and POP-PCR. Recombinant allergen rAmb a 8(D106) was then successfully generated. The allergenicity was hallmarked by immunoblotting with the allergic serum samples and its RNA source was confirmed by Northern blot. CONCLUSION: The combined procedure of POP-PCR and BPCR is a powerful method for full-length allergen gene retrieval and expression insert decoration, which would be useful for recombinant allergen production and subsequent diagnosis and immunotherapy of pollen and food allergy. (+info)
Accessory cell function of airway epithelial cells.
(10/104)
Accessory cell function of airway epithelial cells. We previously demonstrated that airway epithelial cells (AECs) have many features of accessory cells, including expression of class II molecules CD80 and CD86 and functional Fcgamma receptors. We have extended these studies to show that freshly isolated AECs have mRNA for cathepsins S, V, and H [proteases important in antigen (Ag) presentation], invariant chain, human leukocyte antigen (HLA)-DM-alpha and HLA-DM-beta, and CLIP, an invariant chain breakdown product. A physiologically relevant Ag, ragweed, was colocalized with HLA-DR in AECs, and its uptake was increased by granulocyte-macrophage colony-stimulating factor and IFN-gamma treatments, which had no effect on CD80 and CD86 expression. We demonstrate the presence of other costimulatory molecules, including B7h and B7-H1, on AECs and the increased expression of B7-H1 on AECs after treatment with granulocyte-macrophage colony-stimulating factor and IFN-gamma. Finally, we compared T cell proliferation after allostimulation with AECs and dendritic cells (DCs). The precursor frequency of peripheral blood T cells responding to AECs was 0.264% compared with 0.55% for DCs. DCs stimulated CD45RO(+), CD45RA(+), CCR7(+) and CCR7(-)CD4(+), and CD8(+) T cells, whereas AECs stimulated only CD45RO(+), CD45RA(-), CCR7(-), CD4(+), and CD8(+) T cells. There was no difference in cytokine production, type of memory T cells stimulated (effector vs. long-term memory), or apoptosis by T cells cocultured with AECs and DCs. The localization of AECs exposed to the external environment may make them important in the regulation of local immune responses. (+info)
Superoxide dismutase failed to attenuate allergen-induced nasal congestion in ragweed-sensitized dogs.
(11/104)
We hypothesized that augmentation of antioxidant defenses with exogenous superoxide dismutase (SOD), an enzyme that provides an initial defense against oxidative injury, would attenuate allergen-induced nasal congestion in the canine model of allergic rhinitis. Nasal congestion was evaluated by the measurements of nasal resistance and the volume of the nasal passage. In five nonsensitized dogs, 30,000 U of SOD from bovine erythrocytes delivered by aerosol to the nasal passages before histamine challenge reduced the histamine-induced nasal congestion. At 30 min postchallenge, nasal resistance was 1.14 +/- 0.2 cmH2O.l(-1).min(-1) in the saline pretreatment study vs. 0.36 +/- 0.02 cmH2O.l(-1).min(-1) in the SOD pretreatment study (P < 0.05), and volume of nasal passage was 10.9 +/- 0.5 cm3 vs. 17.4 +/- 1.3 cm3 (P < 0.05), respectively. In five sensitized dogs, however, neither an analogous pretreatment with SOD nor intranasal aerosolized pretreatment with 30,000 U of SOD conjugated to polyethylene glycol attenuated ragweed-induced nasal congestion. Also, systemic application of SOD did not attenuate responses to challenges with histamine and ragweed in nonsensitized and sensitized dogs, respectively. The antioxidant-induced attenuation of nasal congestion in nonsensitized dogs confirms validity of the model and indicates the involvement of free radical-mediated damage in the genesis of the histamine-induced congestion. In sensitized dogs, the data do not support the hypothesis that oxidative stress is a clinically significant component of acute ragweed-induced nasal congestion. The data do not support the use of SOD for acute protection against allergic rhinitis. (+info)
Involvement of mast cells in adenosine-mediated bronchoconstriction and inflammation in an allergic mouse model.
(12/104)
In allergen-induced asthma, activation of lung mast cells leads to bronchial constriction, increased mucus secretion, and an increase in the localization of inflammatory cells to the airways. The purpose of this study was to explore the role of mast cells in adenosine-mediated airway reactivity and inflammation using the mast cell degranulating agent, compound 48/80 (C48/80). Mice were sensitized and challenged with ragweed (or 0.9% saline) followed by C48/80 administration twice a day in increasing doses for 5 days. Dose-responsiveness to the nonspecific adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) was established, and lung lavage was performed 24 h later for cell differential analysis to evaluate inflammation. At a dose of 375 microg/ml (aerosolized NECA), C48/80 pretreatment resulted in a significant attenuation in airway reactivity when compared with sensitized control mice (330.07 versus 581.57%, respectively). Lung lavage from the C48/80 treated mice showed a decrease in eosinophils (17.7 versus 60.9%, respectively) and an increase in macrophages when compared with the sensitized control group (76.4 versus 30.8%, respectively). These results support the conclusion that mast cell degranulation plays an important role in adenosine receptor-mediated airway hyperresponsiveness and inflammation. (+info)
Mismatched antigen prepares gamma delta T cells for suppression of airway hyperresponsiveness.
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Gammadelta T cells suppress airway hyperresponsiveness (AHR) induced in allergen-challenged mice but it is not clear whether the suppression is allergen specific. The AHR-suppressive cells express TCR-Vgamma4. To test whether the suppressive function must be induced, we adoptively transferred purified Vgamma4(+) cells into gammadelta T cell-deficient and OVA-sensitized and -challenged recipients (B6.TCR-Vgamma4(-/-)/6(-/-)) and measured the effect on AHR. Vgamma4(+) gammadelta T cells isolated from naive donors were not AHR-suppressive, but Vgamma4(+) cells from OVA-stimulated donors suppressed AHR. Suppressive Vgamma4(+) cells could be isolated from lung and spleen. Their induction in the spleen required sensitization and challenge. In the lung, their function was induced by airway challenge alone. Induction of the suppressors was associated with their activation but it did not alter their ability to accumulate in the lung. Vgamma4(+) gammadelta T cells preferentially express Vdelta4 and -5 but their AHR-suppressive function was not dependent on these Vdeltas. Donor sensitization and challenge not only with OVA but also with two unrelated allergens (ragweed and BSA) induced Vgamma4(+) cells capable of suppressing AHR in the OVA-hyperresponsive recipients, but the process of sensitization and challenge alone (adjuvant and saline only) was not sufficient to induce suppressor function, and LPS as a component of the allergen was not essential. We conclude that AHR-suppressive Vgamma4(+) gammadelta T cells require induction. They are induced by allergen stimulation, but AHR suppression by these cells does not require their restimulation with the same allergen. (+info)
Induction and inhibition of the Th2 phenotype spread: implications for childhood asthma.
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The interactions between genetic and environmental factors play a major role in the development of childhood asthma. We hypothesized that a pre-existing Th2/asthmatic response can promote Th2 responses to newly encountered Ags (i.e., phenotype spread). To test this hypothesis, we developed a mouse model in which the requirements for the induction and inhibition of phenotype spread to a clinically relevant neo-allergen (i.e., ragweed) were investigated. Our results indicate that 1) phenotype spread to the neo-allergen can be induced only within the first 8 h after a bronchial challenge with the first Ag (OVA); 2) Th2 differentiation of naive CD4(+) T cells occurs in bronchial lymph nodes; 3) trafficking of naive CD4(+) T cells to local lymph nodes and IL-4 produced by OVA-activated Th2 cells play essential roles in the differentiation of naive CD4(+) T cells to Th2 cells; and 4) suppression of the production of chemokines involved in the homing of naive CD4(+) T and Th2 cells to bronchial lymph nodes by a TLR9 agonist inhibited phenotype spread and abrogated the consequent development of experimental asthma. These findings provide a mechanistic insight into Th2 phenotype spread and offer an animal model for testing relevant immunomodulatory interventions. (+info)
Cutting edge: atopy promotes Th2 responses to alloantigens and increases the incidence and tempo of corneal allograft rejection.
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A large body of evidence suggests that corneal allograft rejection is mediated by a type 1 Th cell response and that deviation toward type 2 immunity favors graft survival. However, clinical observations indicate that patients with severe ocular allergies have increased risk of corneal allograft rejection. We used a mouse model of atopic conjunctivitis to evaluate the effects of Th2 immune deviation on corneal allograft survival and possible mechanisms of graft rejection. Our results reveal the following novel findings: 1) atopic conjunctivitis promotes systemic Th2 immune responses to corneal graft donor alloantigens; 2) corneal allografts in atopic host eyes have an increased incidence and swifter tempo of rejection; 3) increased rejection is associated with alterations in systemic T cell-mediated responses to donor alloantigens; and 4) corneal allograft rejection in atopic hosts does not require the direct involvement of infiltrating eosinophils. (+info)
Variation in ragweed (Ambrosia artemisiifolia L.) pollen concentration in central Croatia, 2002-2003.
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The aim of the study was to determine the onset, duration and termination of the ragweed pollen season; intradiurnal, daily and monthly pollen variation, and possible differences in the occurrence and concentration of ragweed pollen according to sampling sites between 2002 and 2003. The study was conducted at three sampling sites in central Croatia over two pollen seasons (2002 and 2003) using the volumetric method of sampling (Hirst type sampler). In 2003, the ragweed pollen season was by 43 % longer and the percentage proportion of ragweed pollen by 3.4 % greater in comparison with 2002. The total ragweed pollen count and number of days with ragweed pollen concentration greater than 30 pollen grains per m (3) air showed a declining tendency from East to West in both seasons. The intradiurnal peak concentration occurred between 10.00-14.00. The air concentration of ragweed pollen decreased with temperature decline and precipitation. Results of the study provided useful information to individuals allergic to ragweed pollen thereby allowing them to adjust their outdoor activities to avoid contact with the allergen. (+info)