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• W2029056637 abstract "Despite structural and functional differences between the initial sites of contact with allergens in the gastrointestinal and nasal tracts, few animal models have examined the influence of the mucosal routes of sensitization on host reactivity to food or environmental antigens. We compared the oral and nasal routes of peanut sensitization for the development of a mouse model of allergy. Mice were sensitized by administration of peanut proteins in the presence of cholera toxin as adjuvant. Antibody and cytokine responses were characterized, as well as airway reactivity to nasal challenge with peanut or unrelated antigens. Oral sensitization promoted higher levels of IgE, but lower IgG responses, than nasal sensitization. Both orally and nasally sensitized mice experienced airway hyperreactivity on nasal peanut challenge. The peanut challenge also induced lung eosinophilia and type 2 helper T-cell-type cytokines in orally sensitized mice. In contrast, peanut challenge in nasally sensitized mice promoted neutrophilia and higher levels of lung MAC-1+ I-Ab low cells and inflammatory cytokines. In addition, nasal but not oral, sensitization promoted lung inflammatory responses to unrelated antigens. In summary, both oral and nasal peanut sensitization prime mice for airway hyperreactivity, but the initial mucosal route of sensitization influences the nature of lung inflammatory responses to peanut and unrelated allergens. Despite structural and functional differences between the initial sites of contact with allergens in the gastrointestinal and nasal tracts, few animal models have examined the influence of the mucosal routes of sensitization on host reactivity to food or environmental antigens. We compared the oral and nasal routes of peanut sensitization for the development of a mouse model of allergy. Mice were sensitized by administration of peanut proteins in the presence of cholera toxin as adjuvant. Antibody and cytokine responses were characterized, as well as airway reactivity to nasal challenge with peanut or unrelated antigens. Oral sensitization promoted higher levels of IgE, but lower IgG responses, than nasal sensitization. Both orally and nasally sensitized mice experienced airway hyperreactivity on nasal peanut challenge. The peanut challenge also induced lung eosinophilia and type 2 helper T-cell-type cytokines in orally sensitized mice. In contrast, peanut challenge in nasally sensitized mice promoted neutrophilia and higher levels of lung MAC-1+ I-Ab low cells and inflammatory cytokines. In addition, nasal but not oral, sensitization promoted lung inflammatory responses to unrelated antigens. In summary, both oral and nasal peanut sensitization prime mice for airway hyperreactivity, but the initial mucosal route of sensitization influences the nature of lung inflammatory responses to peanut and unrelated allergens. The prevalence of peanut allergy has doubled in the last decade, and it now affects more than 3 million individuals in the United States.1Sicherer SH Munoz-Furlong A Sampson HA Prevalence of peanut and tree nut allergy in the United States determined by means of a random digit dial telephone survey: a 5-year follow-up study.J Allergy Clin Immunol. 2003; 112: 1203-1207Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar This health care problem is further enhanced by potential cross-reactive allergens. Thus, clinical symptoms were reported in peanut allergic patients who had ingested food of the same botanical family2Bernhisel-Broadbent J Sampson HA Cross-allergenicity in the legume botanical family in children with food hypersensitivity.J Allergy Clin Immunol. 1989; 83: 435-440Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 3Hefle SL Lemanske Jr, RF Bush RK Adverse reaction to lupine-fortified pasta.J Allergy Clin Immunol. 1994; 94: 167-172Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 4Moneret-Vautrin DA Guerin L Kanny G Flabbee J Fremont S Morisset M Cross-allergenicity of peanut and lupine: the risk of lupine allergy in patients allergic to peanuts.J Allergy Clin Immunol. 1999; 104: 883-888Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar or even taxonomically unrelated products.5Sicherer SH Burks AW Sampson HA Clinical features of acute allergic reactions to peanut and tree nuts in children.Pediatrics. 1998; 102: e6Crossref PubMed Scopus (385) Google Scholar Allergic respiratory symptoms have also been described in peanut-allergic patients after inhalation of airborne peanut particles in school5Sicherer SH Burks AW Sampson HA Clinical features of acute allergic reactions to peanut and tree nuts in children.Pediatrics. 1998; 102: e6Crossref PubMed Scopus (385) Google Scholar or on airline flights.6Sicherer SH Furlong TJ DeSimone J Sampson HA Self-reported allergic reactions to peanut on commercial airliners.J Allergy Clin Immunol. 1999; 104: 186-189Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 7Rayman RB Peanut allergy in-flight.Aviat Space Environ Med. 2002; 73: 501-502PubMed Google Scholar In this regard, food allergens are now well recognized to play a significant role as aeroallergens in the etiology of asthmatic symptoms in individuals with food allergies.8Roberts G Golder N Lack G Bronchial challenges with aerosolized food in asthmatic, food-allergic children.Allergy. 2002; 57: 713-717Crossref PubMed Scopus (104) Google Scholar Sensitization to food allergens such as peanut generally occurs in the gastrointestinal (GI) tract. However, it could also occur as a consequence of direct or cross-sensitization by inhalational exposure to peanut or cross-reactive environmental antigens. For example, peanut allergy is frequently associated with pollen allergy,9Crespo JF Pascual C Vallecillo A Esteban MM Sensitization to inhalant allergens in children diagnosed with food hypersensitivity.Allergy Proc. 1995; 16: 89-92Crossref PubMed Scopus (6) Google Scholar, 10Eriksson NE Formgren H Svenonius E Food hypersensitivity in patients with pollen allergy.Allergy. 1982; 37: 437-443Crossref PubMed Scopus (319) Google Scholar, 11Ortolani C Pastorello EA Farioli L Ispano M Pravettoni V Berti C Incorvaia C Zanussi C IgE-mediated allergy from vegetable allergens.Ann Allergy. 1993; 71: 470-476PubMed Google Scholar, 12Enrique E Cistero-Bahima A Bartolome B Alonso R San Miguel-Moncin MM Bartra J Martinez A Platanus acerifolia pollinosis and food allergy.Allergy. 2002; 57: 351-356Crossref PubMed Scopus (78) Google Scholar and peanut allergens share sequence homologies with environmental antigens.13Vieths S Scheurer S Ballmer-Weber B Current understanding of cross-reactivity of food allergens and pollen.Ann NY Acad Sci. 2002; 964: 47-68Crossref PubMed Scopus (423) Google Scholar A study on children with a history of at least one acute allergic reaction showed that initial reactions to peanut occurred at 24 months of age, with the large majority resulting from a first oral exposure.5Sicherer SH Burks AW Sampson HA Clinical features of acute allergic reactions to peanut and tree nuts in children.Pediatrics. 1998; 102: e6Crossref PubMed Scopus (385) Google Scholar Because IgE-mediated allergic reactions require prior exposure to the allergen, one cannot rule out earlier sensitization through inhalation of airborne peanut particles. In addition, the presence of cross-reactive IgE to pollen and peanut antigens in pollen-allergic patients14van der Veen MJ van Ree R Aalberse RC Akkerdaas J Koppelman SJ Jansen HM van der Zee JS Poor biologic activity of cross-reactive IgE directed to carbohydrate determinants of glycoproteins.J Allergy Clin Immunol. 1997; 100: 327-334Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar and the reports that these individuals can develop positive skin tests to peanut15Asero R Mistrello G Roncarolo D Amato S Caldironi G Barocci F van Ree R Immunological cross-reactivity between lipid transfer proteins from botanically unrelated plant-derived foods: a clinical study.Allergy. 2002; 57: 900-906Crossref PubMed Scopus (173) Google Scholar, 16de Martino M Novembre E Cozza G de Marco A Bonazza P Vierucci A Sensitivity to tomato and peanut allergens in children monosensitized to grass pollen.Allergy. 1988; 43: 206-213Crossref PubMed Scopus (97) Google Scholar suggest that allergic symptoms to peanut may also be caused by respiratory sensitization with cross-reactive allergens. Structural and functional differences have been described between the gut-associated lymphoid tissues and the nasopharyngeal-associated lymphoid tissues17Kiyono H Fukuyama S NALT- versus Peyer's-patch-mediated mucosal immunity.Nat Rev Immunol. 2004; 4: 699-710Crossref PubMed Scopus (589) Google Scholar that are the first sites of contact with ingested and inhaled antigens, respectively. But it remains unclear how priming through each site could influence subsequent allergic or inflammatory reactions. It is widely accepted that IgE and cytokines produced by Type 2 helper T (Th2) cells play a pivotal role in allergic manifestations.18Bochner BS Undem BJ Lichtenstein LM Immunological aspects of allergic asthma.Annu Rev Immunol. 1994; 12: 295-335Crossref PubMed Scopus (268) Google Scholar, 19Renauld JC New insights into the role of cytokines in asthma.J Clin Pathol. 2001; 54: 577-589Crossref PubMed Scopus (345) Google Scholar However, recent studies suggest that a larger number of parameters contribute to allergic responses. For example, in addition to IgE, antibodies (Abs) of the IgG isotype could exert a regulatory effect on allergic reactions;20Macedo-Soares MF Itami DM Lima C Perini A Faquim-Mauro EL Martins MA Macedo MS Lung eosinophilic inflammation and airway hyperreactivity are enhanced by murine anaphylactic, but not nonanaphylactic, IgG1 antibodies.J Allergy Clin Immunol. 2004; 114: 97-104Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar however, underlying mechanisms are still poorly understood.21Jenmalm MC Bjorksten B Macaubas C Holt BJ Smallacombe TB Holt PG Allergen-induced cytokine secretion in relation to atopic symptoms and immunoglobulin E and immunoglobulin G subclass antibody responses.Pediatr Allergy Immunol. 1999; 10: 168-177Crossref PubMed Scopus (38) Google Scholar Th1 cells that were believed to only protect against allergic reactions by attenuating the activity of Th2 cells22Huang TJ MacAry PA Eynott P Moussavi A Daniel KC Askenase PW Kemeny DM Chung KF Allergen-specific Th1 cells counteract efferent Th2 cell-dependent bronchial hyperresponsiveness and eosinophilic inflammation partly via IFN-gamma.J Immunol. 2001; 166: 207-217Crossref PubMed Scopus (121) Google Scholar now appear to also support Th2 cell-induced allergic asthma.23Hansen G Berry G DeKruyff RH Umetsu DT Allergen-specific Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation.J Clin Invest. 1999; 103: 175-183Crossref PubMed Scopus (583) Google Scholar, 24Randolph DA Carruthers CJ Szabo SJ Murphy KM Chaplin DD Modulation of airway inflammation by passive transfer of allergen-specific Th1 and Th2 cells in a mouse model of asthma.J Immunol. 1999; 162: 2375-2383Crossref PubMed Google Scholar, 25Randolph DA Stephens R Carruthers CJ Chaplin DD Cooperation between Th1 and Th2 cells in a murine model of eosinophilic airway inflammation.J Clin Invest. 1999; 104: 1021-1029Crossref PubMed Scopus (294) Google Scholar In addition, Th1 cells have been shown to recruit and activate neutrophils for subsequent airway hyperreactivity (AHR).26Takaoka A Tanaka Y Tsuji T Jinushi T Hoshino A Asakura Y Mita Y Watanabe K Nakaike S Togashi Y Koda T Matsushima K Nishimura T A critical role for mouse CXC chemokine(s) in pulmonary neutrophilia during Th type 1-dependent airway inflammation.J Immunol. 2001; 167: 2349-2353Crossref PubMed Scopus (54) Google Scholar The route of allergen sensitization may influence the pattern of Ab and T-cell responses and, therefore, the nature of potential adverse reactions. This increasing complexity of mechanisms underlying allergic and nonallergic inflammatory responses further limits our understanding of adverse effects that occur in individuals with allergies. Peanut allergy has been mostly investigated in animal models sensitized by the subcutaneous,27Teuber SS Del Val G Morigasaki S Jung HR Eisele PH Frick OL Buchanan BB The atopic dog as a model of peanut and tree nut food allergy.J Allergy Clin Immunol. 2002; 110: 921-927Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar the intraperitoneal,28Helm RM Furuta GT Stanley JS Ye J Cockrell G Connaughton C Simpson P Bannon GA Burks AW A neonatal swine model for peanut allergy.J Allergy Clin Immunol. 2002; 109: 136-142Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 29Pons L Ponnappan U Hall RA Simpson P Cockrell G West CM Sampson HA Helm RM Burks AW Soy immunotherapy for peanut-allergic mice: modulation of the peanut-allergic response.J Allergy Clin Immunol. 2004; 114: 915-921Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar or the oral route30Bashir ME Andersen P Fuss IJ Shi HN Nagler-Anderson C An enteric helminth infection protects against an allergic response to dietary antigen.J Immunol. 2002; 169: 3284-3292Crossref PubMed Scopus (208) Google Scholar, 31Li XM Srivastava K Huleatt JW Bottomly K Burks AW Sampson HA Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model.J Immunol. 2003; 170: 3289-3295Crossref PubMed Scopus (141) Google Scholar, 32van Wijk F Hartgring S Koppelman SJ Pieters R Knippels LM Mixed antibody and T cell responses to peanut and the peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 in an oral sensitization model.Clin Exp Allergy. 2004; 34: 1422-1428Crossref PubMed Scopus (60) Google Scholar and challenged by the oral route.27Teuber SS Del Val G Morigasaki S Jung HR Eisele PH Frick OL Buchanan BB The atopic dog as a model of peanut and tree nut food allergy.J Allergy Clin Immunol. 2002; 110: 921-927Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 28Helm RM Furuta GT Stanley JS Ye J Cockrell G Connaughton C Simpson P Bannon GA Burks AW A neonatal swine model for peanut allergy.J Allergy Clin Immunol. 2002; 109: 136-142Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 30Bashir ME Andersen P Fuss IJ Shi HN Nagler-Anderson C An enteric helminth infection protects against an allergic response to dietary antigen.J Immunol. 2002; 169: 3284-3292Crossref PubMed Scopus (208) Google Scholar, 31Li XM Srivastava K Huleatt JW Bottomly K Burks AW Sampson HA Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model.J Immunol. 2003; 170: 3289-3295Crossref PubMed Scopus (141) Google Scholar The nasal route has been less extensively investigated. Furthermore, to our knowledge no study has compared inflammatory lung reactions to unrelated food or respiratory antigens in animal models sensitized by the oral and nasal routes. We compared Ab and T-cell responses induced by oral or nasal sensitization with whole-peanut protein extract (PPE) and cholera toxin (CT) as adjuvant. We then examined the influence of these responses on airway reactivity to nasal challenge with PPE or unrelated antigens. Our data show that the initial mucosal route of peanut sensitization affects the nature of the immune response and the lung reactivity to peanut but also to unrelated antigens. Female C57BL/6 mice were obtained from the Frederick Cancer Research Facility (National Cancer Institute, Frederick, MD). Mice were maintained in horizontal laminar flow cabinets and were free of microbial pathogens as determined by plasma Ab screening and tissue histopathology performed on sentinel mice. All mice received sterile food and water ad libitum. Studies were performed in accordance with institutional guidelines to avoid pain and distress. Whole-peanut protein extracts (PPE) were obtained as previously described by ammonium bicarbonate treatment of defatted peanut extracts.2Bernhisel-Broadbent J Sampson HA Cross-allergenicity in the legume botanical family in children with food hypersensitivity.J Allergy Clin Immunol. 1989; 83: 435-440Abstract Full Text PDF PubMed Scopus (241) Google Scholar Mice 8 to 12 weeks of age were sensitized on days 0 and 7 with whole PPE and CT as adjuvant. Anesthetized mice were nasally administered 100 μg of PPE and 1 μg of CT in a total volume of 10 μl with 5 μl placed into each nare. This volume of the nasal vaccine is retained in the nasal cavity after nasal administration to anesthetized mice.33Visweswaraiah A Novotny LA Hjemdahl-Monsen EJ Bakaletz LO Thanavala Y Tracking the tissue distribution of marker dye following intranasal delivery in mice and chinchillas: a multifactorial analysis of parameters affecting nasal retention.Vaccine. 2002; 20: 3209-3220Crossref PubMed Scopus (51) Google Scholar For sensitization by the oral route, mice were deprived of food for 2 hours and then orally treated with 250 μl of sodium bicarbonate as previously described.34Marinaro M Staats HF Hiroi T Jackson RJ Coste M Boyaka PN Okahashi N Yamamoto M Kiyono H Bluethmann H Fujihashi K McGhee JR Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4.J Immunol. 1995; 155: 4621-4629Crossref PubMed Google Scholar Oral sensitization consisted of intragrastric administration of 1 mg of PPE plus 15 μg of CT in 250 μl of phosphate-buffered saline (PBS). Other doses of CT (5 μg nasal or 60 μg oral) and PPE (25, 50, or 200 μg nasal or 2 mg oral) were tested in separate experiments. Some experiments included mice given ovalbumin (OVA) (Sigma Chemical, Saint Louis, MO) as antigen instead of PPE. In these experiments, mice were then either nasally administered 100 μg of OVA plus 1 μg of CT or given 1 mg of OVA plus 15 μg of CT by the oral route. Plasma samples were collected 1 week after each sensitization, on days 7 and 14, for analysis of peanut-specific Ab responses. Mice nasally or orally sensitized to peanut were nasally challenged on days 15 and 16 with 200 μg of PPE in a total volume of 100 μl. More specifically, anesthetized mice were given 25 μl of PPE per nare, four times at 2- to 3-minute intervals. For analysis of lung responses to unrelated proteins, mice were challenged with 200 μg of OVA or 40 μg of Dermatophagoides farinae (Der f) protein extract (Greer Laboratories, Lenoir, NC) instead of PPE. Plasma levels of peanut-specific Abs were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well microplates (Falcon) were coated with 50 μg/ml PPE in PBS and incubated overnight at 4°C. After blocking with PBS-1% bovine serum albumin, serial dilutions of plasma samples were added and incubated overnight at 4°C. Peanut-specific IgG Abs were detected using 0.3 μg/ml of horseradish peroxidase (HRP)-labeled goat anti-mouse γ-heavy chain-specific Abs (Southern Biotechnology Associates, Birmingham, AL). Biotin-conjugated rat anti-mouse γl (clone A85-1), γ2a (clone R19-15), γ2b (clone R12-3), or γ3 (clone R40-82) heavy chain mAbs (BD PharMingen, San Diego, CA) were used at 0.5 μg/ml; and streptavidin-HRP (BD PharMingen) was diluted at 1:2000 for the detection of peanut-specific IgG subclasses. The colorimetric reaction was developed by the addition of 2,2′-azino-bis(3)-ethylbenzylthiazoline-6-sulfonic acid substrate (Sigma) and H2O2. Endpoint titers were expressed as the log2 of plasma dilution giving an optical density at 415 nm of ≥0.1 above those obtained with control plasma. To determine the potential of plasma of peanut-sensitized mice to react with irrelevant protein antigens, plasma samples were added to ELISA plates coated with OVA (1 mg/ml) or Der f protein extract (10 μg/ml). The removal of IgG has been shown to improve the detection of IgE Abs.35Lehrer SB Reish R Fernandes J Gaudry P Dai G Reese G Enhancement of murine IgE antibody detection by IgG removal.J Immunol Methods. 2004; 284: 1-6Crossref PubMed Scopus (31) Google Scholar Thus, dilutions of plasma samples were first depleted of IgG by overnight incubation at 4°C in protein G-coated 96-well plates (Reacti-Bind plates; Pierce, Rockford, IL). Total and antigen-specific IgE levels were then analyzed by ELISA. For detection of antigen-specific IgE Abs, IgG-depleted samples were added to ELISA plates coated with PPE (50 μg/ml, 100 μl/well). The IgE were detected with 0.5 μg/ml biotin-conjugated rat anti-mouse IgE (clone R35-118; BD PharMingen) followed by streptavidin-HRP (1:2000). The levels of total IgE Abs were determined using capture and detection antibodies, as well as IgE standard, from the BD OptEIA Set mouse IgE kit (BD Biosciences, San Diego, CA). Enhanced pause (Penh), an index that reflects changes in amplitude of pressure wave form and expiratory time, was measured 6 hours after the last nasal peanut challenge in mice placed in a barometric plethysmograph according to a previously described method.36Hamelmann E Schwarze J Takeda K Oshiba A Larsen GL Irvin CG Gelfand EW Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography.Am J Respir Crit Care Med. 1997; 156: 766-775Crossref PubMed Scopus (1151) Google Scholar Doses of metacholine (0, 10, and 20 mg/ml) were administered by nebulization. For each dose, Penh were measured every minute over 7 minutes. Controls included sham-sensitized and sham-challenged mice. Lungs were fixed in 10% buffered formaldehyde, paraffin-embedded, and cut into sections of 5 μm thickness. The sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin for the evaluation of inflammation. The presence of eosinophils in tissue sections was determined by the cyanide-resistant peroxidase activity as previously described.37Strath M Warren DJ Sanderson CJ Detection of eosinophils using an eosinophil peroxidase assay: its use as an assay for eosinophil differentiation factors.J Immunol Methods. 1985; 83: 209-215Crossref PubMed Scopus (258) Google Scholar Briefly, lung sections were incubated for 1 minute at room temperature in 10 mmol/L KCN, pH 6.5. Slides were then rinsed in PBS and incubated for 15 minutes with the peroxidase substrate 3,3′-diaminobenzine (Vector Laboratories, Burlingame, CA). After washes in PBS, tissue sections were counterstained with hematoxylin. The eosinophils, which express a cyanide-resistant peroxidase activity, appeared as containing dark brown granules, and their frequency was estimated by microscopic observation at ×200 magnification. The neutrophils, which do not express a cyanide-resistant peroxidase, were segregated based on their characteristic morphology. For quantification of lung inflammation, the slides were coded, and peribronchial and perivascular inflammation was scored in a blinded fashion by two independent investigators. A value of 1 was given when slides showed no sign of inflammation. Slides were graded from 2 to 4 when bronchi were surrounded by a thin layer of inflammatory cells (2, few bronchi; 3, more bronchi; and 4, most bronchi). They were graded from 5 to 7 according to the number of bronchi that were surrounded by a thick layer of inflammatory cells (5, few bronchi; 6, more bronchi; and 7, most bronchi). Finally, slides were graded 8 or 9 when inflammation spread into the interstitial area (8, severe; and 9, extreme). Whole-lung tissue was dissociated by digestion with 1 mg/ml collagenase type V (Sigma) in RPMI-1640 (Cellgro Mediatech, Washington, DC), supplemented with 10 mmol/L HEPES, 2 mmol/L l-glutamine, 5 × 10−5 mol/L 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin (supplemented RPMI) to obtain single cell preparations. Mononuclear cells were collected at the 20 to 75% interface of discontinuous Percoll gradient and stained with anti-CD3 (clone 145-2C11), anti-CD4 (clone GK1.5), anti-B220 (clone RA3-6B2), anti-CD11c (clone HL3), anti-MAC-1 (clone M1/70), or anti-MHC class II Abs (I-Ab, clone AF6-120.1) (BD PharMingen). After washes and fixation, samples were analyzed by flow cytometry. Whole-lung tissue was dissociated by digestion with collagenase as described above. Mononuclear cells were collected and washed in supplemented RPMI. The CD4+ T cells were purified using the automated magnetic cell sorting (autoMACS) according to the protocol provided by the manufacturer (Miltenyi Biotech, Auburn, CA). Briefly, single cell suspensions were incubated with a biotinylated anti-CD4 mAb (BD PharMingen) for 30 minutes at 4°C and washed in PBS containing 2 mmol/L EDTA and 0.5% bovine serum albumin. Streptavidin-conjugated MicroBeads (Miltenyi Biotech) were then added to cells. After a 30-minute incubation at 4°C, cells were washed, and CD4+ T cells were purified by positive selection using autoMACS. Lung tissue was dissociated as described above; mononuclear cells were collected and washed in supplemented RPMI; and RNA was isolated using STAT-60 (Tel-Test, Friendswood, TX). The reverse transcription was performed with superscript II reverse transcriptase, dNTPs, and poly(dT) oligos. The real-time PCR (Lightcycler; Roche, Indianapolis, IN) was performed with primers generated with Oligo software (Plymouth, MN) and the SYBR green detection system according to the manufacturer. Results are expressed as crossing point (CP), defined as the cycle at which the fluorescence rises appreciably above the background fluorescence as determined by the Second Derivative Maximum Method (Roche Molecular Biochemicals LightCycler Software). The formula 20 − (CPcytokine − CPβ-actin) was used to represent the logarithm of the relative mRNA levels of a given cytokine. This formula allows the normalization of all results against β-actin levels to correct for differences in cDNA concentration between the starting templates. Differences of crossing points above two cycles were considered significant. Bronchoalveolar lavage fluids (BALF) were obtained via cannulation of the exposed trachea, by infusion of 0.6 ml of supplemented RPMI through a 22-gauge catheter into the lungs, followed by aspiration of this fluid into a syringe. A volume of 0.4 ml of fluid was consistently recovered. Aliquots were centrifuged, and supernatants were collected and stored at −70°C until analyzed. Cell pellets were subjected to cytospin, and the slides were stained with Giemsa (Sigma). Cytokines were measured in the supernatants of BALFs by ELISA. Nunc MaxiSorp Immunoplates (Nunc, Napersville, IL) were coated with anti-mouse tumor necrosis factor-α (TNF-α) (clone MP6-XT22), interferon (IFN)-γ (clone R4-6A2), interleukin-4 (IL-4) (clone BVD4-1D11), IL-5 (clone TRFK5), IL-6 (clone MP5-20F3), or IL-10 (clone JES5-2A5) mAbs (BD PharMingen) or IL-13 (R&D Systems, Minneapolis, MN) in 0.1 mol/L sodium bicarbonate buffer (pH 9.5) and incubated overnight at 4°C. After blocking with PBS-3% bovine serum albumin, cytokine standards and serial dilutions of supernatants of BALFs were added in duplicates. The plates were incubated with biotinylated anti-mouse TNF-α (clone MP6-XT3), IFN-γ (clone XMG-1.2), IL-4 (clone BVD6-24G2), IL-5 (clone TRFK4), IL-6 (clone MP5-32C11), IL-10 (clone JES5-16E3) (BD PharMingen), or IL-13 mAbs (R&D Systems), followed by HRP-labeled goat anti-biotin Ab (Vector Laboratories). The colorimetric reaction was developed with the addition of 2,2′-azino-bis(3)-ethylbenzylthiazoline-6-sulfonic acid substrate and H2O2. Standard curves were generated using murine rIFN-γ, rIL-5, rIL-6, rIL-10 (Genzyme, Cambridge, MA), rIL-4 (Endogen Corp., Boston, MA), rTNF-α, and rIL-13 (R&D Systems). The ELISAs were capable of detecting 3 pg/ml IL-4; 5 pg/ml IL-6; 10 pg/ml IL-5; 20 pg/ml IFN-γ, TNF-α, and IL-10; and 30 pg/ml IL-13. A quantikine ELISA kit (R&D Systems) was used for detection of IL-1β. The results are reported as the mean ± 1 SD. Statistical significance (P < 0.05) was determined by Student's t-test and by the Mann-Whitney U-test of unpaired samples. The results were analyzed using the InStat statistical software (San Diego, CA) for Apple computers. Although mucosal surfaces of the GI and respiratory tracts are considered the primary sites of sensitization to food antigens, it remains unclear how peanut priming through each site could influence subsequent allergic or inflammatory reactions. We first compared the plasma levels of peanut-specific Ab responses in mice that received whole PPE and CT as adjuvant by the oral and the nasal route. Both mucosal routes of sensitization promoted peanut-specific plasma IgG Abs, but higher levels of IgG responses were measured in mice sensitized by the nasal route (Figure 1). In addition, nasal and oral sensitization also induced different patterns of peanut-specific IgG subclass responses with a lower IgG1-to-IgG2a ratio in nasally sensitized mice (1.3 ± 0.1 vs. 1.8 ± 0.3, P < 0.01) (Figure 1). In contrast with plasma IgG responses, higher levels of peanut-specific IgE Ab responses were measured in orally sensitized mice (Figure 1). The difference in the levels of IgE responses between mice sensitized by the oral and nasal routes was maintained when lower or higher doses of antigen or adjuvant were used (data not shown). In addition, no difference was seen in antigen-specific IgE Ab responses between mice that were given OVA (1 mg orally or 100 mg nasally) instead of PPE (data not shown). Taken together, these results suggested that oral sensitization with PPE favors IgE Ab responses, whereas nasal sensitization more effectively primes for IgG Abs. The Penh values of mice orally or nasally sensitized with PPE were measured 6 hours after nasal challenge to determine whether these routes of mucosal sensitization primed for different AHR res" @default.
• W2029056637 created "2016-06-24" @default.
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• W2029056637 date "2005-12-01" @default.
• W2029056637 modified "2023-10-01" @default.
• W2029056637 title "Oral and Nasal Sensitization Promote Distinct Immune Responses and Lung Reactivity in a Mouse Model of Peanut Allergy" @default.
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• W2029056637 doi "https://doi.org/10.1016/s0002-9440(10)61246-1" @default.
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