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Background New preventive strategies against the development of allergic diseases focus on potentially immunomodulatory components, such as bacterial LPSs. Optimal time frames for initiating immunomodulation to receive a sufficient effect against allergen sensitization are still unclear. Objective Using a mouse model, we investigated the influence of prenatal LPS exposure on later allergen-mediated sensitization and airway inflammation in the offspring. Methods Pregnant BALB/c mice were repeatedly exposed to aerosolized LPS (LPS Escherichia coli ; 3× per week, day 7 of gestation time up to delivery). Some of the offspring were further exposed to aerosolized LPS before allergen sensitization with ovalbumin (OVA; administered intraperitoneally day 28 up to day 42) and OVA airway challenges (days 56-58). Positive control animals were placebo exposed to PBS instead of LPS, and negative control animals were first placebo exposed and later placebo sensitized with PBS instead of OVA. Results Compared with positive control animals, prenatal LPS exposure suppressed (1) allergen-specific sensitization (IgE production), (2) eosinophilic airway inflammation (reduced numbers of eosinophils in bronchoalveolar lavage fluids), and (3) in vivo airway reactivity in response to methacholine. These effects occurred only when prenatal was combined with further postnatal LPS exposure. Suppression of allergen-mediated inflammatory responses was associated with increased Toll-like receptor and T-bet expression by lung tissues and a shift toward predominantly T H 1 immune responses in spleen cells cultured with OVA in vitro . Conclusion Prenatal initiated and postnatal sustained LPS exposure increased endotoxin susceptibility and prevented later allergen sensitization in offspring through inhibition of T H 2 immune responses. Clinical implications Immunomodulation with bacterial compounds during gestation time might be an effective mode for first-step primary prevention against allergic diseases.

Exposure to environmental tobacco smoke (ETS) has been shown to increase symptoms of allergic bronchial asthma, but direct effects on the expression of inflammatory markers have not been demonstrated thus far.The aim of this study was to assess the correlation of ETS exposure with the expression of proinflammatory mediators in airway secretions, including IFN-gamma and IL-12, as well as IL-5 and IL-13, in allergic asthmatic schoolchildren and healthy control subjects.By using the nasopharyngeal aspiration technique, airway secretions were collected from 24 atopic children with asthma (age, 6-16 years) and 26 healthy control subjects, and the concentration of cytokines was measured with immunoenzymatic methods.IL-13 levels were highly increased in patients with asthma (P.005), and parental tobacco smoke resulted in a significant increase in airway IL-13 secretion in these children compared with that seen in nonexposed children and healthy control subjects (median, 860 pg/mL vs 242 pg/mL and 125 pg/mL, respectively). Furthermore, a positive correlation between IL-13 levels and serum IgE concentrations (r(s) = 0.55) was found in children with allergic asthma.These results indicate that ETS augments the expression and secretion of IL-13 in allergic asthma and that nasopharyngeal aspiration is a suitable method to assess cytokine measurements in airways in children. Measurements of IL-13 in secretions might be taken into account as a noninvasive marker of airway inflammation and to assess the detrimental effects of ETS.

Background Airway inflammation plays a critical role in the pathogenesis of asthma. In susceptible individuals, airway allergen exposure results in the recruitment of inflammatory cells into lung tissue, leading to a local inflammatory response. Central to the induction and regulation of this process are T lymphocytes. Objective Blocking of the newly discovered costimulatory T-cell molecule inducible costimulator (ICOS) with monoclonal antibodies was shown to ameliorate allergic airway inflammation in models of murine asthma. Although these observations indirectly support an association between ICOS and the development of allergic inflammation, the role of the ICOS + T cell in the pathogenesis of allergic airway disease remains unclear. Methods We used an adoptive transfer model to analyze further the role of antigen-specific ICOS + T cells during the effector phase of allergic airway inflammation. In vitro stimulated CD4 + T cells from mice transgenic for an ovalbumin-specific T-cell receptor (DO11.10) were sorted into ICOS-enriched and ICOS-depleted T-cell fractions and transferred into BALB/c recipient mice. Results Transfer of the ICOS-enriched T-cell population followed by allergen airway challenges induced pronounced infiltration of recipient T and B cells and local production of allergen-specific IgE by intrapulmonary plasma cells. In contrast, transfer of the ICOS-depleted T-cell fraction resulted in the recruitment of significantly lower numbers of B cells and no local IgE production. Conclusion These data indicate that expression of ICOS defines a subset of T effector cells that are required for B-cell infiltration and local IgE production in lung tissues on allergen airway exposure.

Background: Exposure to endotoxins, allergens, or both in early life might regulate the development of tolerance to allergens later in life. Objective: We investigated whether continuous exposure of infant mice to aerosolized endotoxin, allergen, or both inhibits subsequent allergen-induced immune and inflammatory responses. Methods: Infant BALB/c mice were pre-exposed to aerosolized endotoxin, ovalbumin (OVA), or both (3 times a week for the first 4 weeks of life) before systemic sensitization (days 1-14) and repeated airway challenge (days 28-30) with OVA. Results: Compared with that seen in negative control animals, systemic sensitization and airway allergen challenges induced high serum levels of allergen-specific IgE (0.7 ± 0.09 vs 0.02 ± 0.01 OD units), predominant T H 2-type cytokine production (IL-5 by splenic mononuclear cells in vitro, 1.2 ± 0.2 vs 0.04 ± 0.06 ng/mL), airway inflammation (bronchoalveolar lavage fluid leukocytes, 125 ± 15 vs 64 ± 7/μL; eosinophils, 28 ± 5 vs 1 ± 0/μL) and development of in vivo airway hyperreactivity (maximal enhanced pause, 11 ± 1.9 vs 4 ± 0.2). Pre-exposure with LPS before sensitization increased production of specific IgG2a (67 ± 10 vs 32 ± 5 U/mL) but failed to prevent T H 2-mediated immune responses. Pre-exposure with OVA or with OVA plus LPS completely suppressed allergen sensitization, airway inflammation, and development of in vivo airway hyperreactivity; values were similar to those of negative control animals. Inhibition was due to allergen-specific T-cell anergy indicated by omitted allergen-specific T H 2 and T H 1 immune responses. In addition, combined exposure to endotoxin and allergen induced a general shift toward an unspecific T H 1 immune response. Conclusion: Exposure with endotoxins before allergen sensitization is not able to induce unresponsiveness but might decrease the susceptibility for sensitization to a variety of common allergens. (J Allergy Clin Immunol 2003;112:389-96.)

Background: Binding of allergens with IgE to the IgE receptors on mast cells and basophils results in the release of inflammatory mediators as sulfidoleukotrienes (SLTs), triggering allergic cascades that result in allergic symptoms, such as asthma and rhinitis. Objective: We sought to investigate whether anti-IgE (Oma-lizumab), a humanized monoclonal anti-IgE antibody, in addition to specific immunotherapy (SIT) affects the leukotriene pathway. Methods: Ninety-two children (age range, 6-17 years) with sensitization to birch and grass pollens and with seasonal allergic rhinitis were included in a phase III, placebo- controlled, multicenter clinical study. All subjects were randomized to one of 4 treatment groups. Two groups subcutaneously received birch SIT and 2 groups received grass SIT for at least 14 weeks before the start of the birch pollen season. After 12 weeks of SIT titration, placebo or anti-IgE was added for 24 weeks. The primary clinical efficacy variable was symptom load (ie, the sum of daily symptom severity score and rescue medication score during pollen season). Blood samples taken at baseline and at the end of study treatment after the grass pollen season were used for separation of leukocytes in this substudy. After in vitro stimulation of the blood cells with grass and birch pollen allergens, SLT release (LTC4, LTD4, and LTE4) was quantified by using the ELISA technique. Results: Before the study treatment, SLT release to birch and grass pollen exposure did not differ significantly among the 4 groups. Under treatment with anti-IgE + SIT-grass (n = 23), a lower symptom load occurred during the pollen season compared to placebo + SIT-grass (n = 24, P = .012). The same applied to both groups receiving birch SIT (n = 23 and n=22, respectively; P = .03). At the end of treatment, the combination of anti-IgE plus grass SIT, as well as anti-IgE plus birch SIT, resulted in significantly lower SLT release after stimulation with the corresponding allergen (416 ng/L [5th-95th percentile, 1-1168] and 207 ng/L [1-860 ng/L], respectively) compared with placebo plus SIT (2490 ng/L [384-6587 ng/L], P = .001; 2489 ng/L [1-5670 ng/L], P = .001). In addition, treatment with anti-IgE was also followed by significantly lower SLT releases to the allergens unrelated to SIT (grass SIT: 300 ng/L [1-2432 ng/L] in response to birch allergen; birch SIT: 1478 ng/L [1-4593 ng/L] in response to grass pollen) in comparison with placebo (grass SIT: 1850 ng/L [1-5499 ng/L], P = .001; birch SIT: 2792 ng/L [154-5839 ng/L], P = .04]. Conclusion: Anti-IgE therapy reduces leukotriene release of peripheral leukocytes stimulated with allergen in children with allergic rhinitis undergoing allergen immunotherapy independent of the type of SIT allergen used. (J Allergy Clin Immunol 2002;110:728-35.)

Background: Systemic administration of IL-12 can prevent airway hyperresponsiveness (AHR) in mice after sensitization and repeated allergen challenge. However, systemic IL-12 has been associated with severe adverse effects. Objective: We determined whether IL-12 administration to the airways in a dose sufficiently low so as not to result in systemic effects can modify allergic inflammation and AHR after allergen challenge. Methods: Mice were sensitized to ovalbumin by intraperitoneal injection and challenged with ovalbumin aerosol on 3 consecutive days. During the period of challenge, IL-12 was administered intranasally following 2 regimens, designated high (500 ng) or low (50 ng). We monitored airway responsiveness to inhaled methacholine by barometric body plethysmography, lung inflammatory cells, local cytokine production, and, to assess systemic effects of IL-12 treatment, spleen weights and numbers of eosinophils in the bone marrow. Results: Allergen challenge resulted in increases in airway responsiveness and in numbers of lung eosinophils. These increases were prevented by both high- and low-dose IL-12. Additionally, IL-12 administration resulted in enhanced local interferon-γ production and prevented the increases in local IL-4 and IL-5 production after airway challenge. A high dose, but not a low dose, of IL-l2 resulted in increased spleen weights and prevented the increase in numbers of bone marrow eosinophils after allergen challenge. Conclusion: These data indicate that local administration of IL-12 can prevent AHR and reduce lung eosinophilia after allergen challenge in sensitized mice without eliciting systemic adverse effects. IL-12 exerts these effects by inducing local T H1 -type responses in the airways in a setting that is normally dominated by T H2 -type responses. (J Allergy Clin Immunol 1998;102:86-93.)

Background Results from phase III clinical trials in adults and phase II clinical trials in children and adolescents demonstrate that tiotropium is an effective treatment when added to inhaled corticosteroid (ICS) maintenance therapy. Objective We sought to assess the efficacy and safety of once-daily tiotropium Respimat added to ICSs with or without a leukotriene receptor antagonist in a phase III trial in adolescent patients with moderate symptomatic asthma. Methods In this 48-week, double-blind, placebo-controlled, parallel-group study, 398 patients aged 12 to 17 years were randomized to receive 5 μg (2 puffs of 2.5 μg) or 2.5 μg (2 puffs of 1.25 μg) of once-daily tiotropium or placebo (2 puffs) administered through the Respimat device every evening, each as add-on treatment to ICS background therapy, with or without a leukotriene receptor antagonist; long-acting β 2 -agonist therapy was not permitted during the study. Results Improvement in peak FEV 1 within 3 hours after dosing at 24 weeks (primary end point) was statistically significant with both tiotropium doses compared with placebo: 5 μg of tiotropium, 174 mL (95% CI, 76-272 mL); 2.5 μg of tiotropium, 134 mL (95% CI, 34-234 mL). Significant improvements in trough FEV 1 at week 24 (a secondary end point) were observed with the 5-μg dose only. Trends for improvement in asthma control and health-related quality of life over the 48-week treatment period were observed. Conclusions Once-daily tiotropium significantly improved lung function and was safe and well tolerated when added to at least ICS maintenance therapy in adolescent patients with moderate symptomatic asthma. Larger responses were observed with the 5-μg tiotropium dose.

Background Threshold levels for peanut allergy determined by using oral challenges are important for the food industry with regard to allergen labeling. Moreover, the utility of biological markers in predicting threshold levels is uncertain. Objective We sought to use a modified oral food challenge regimen that might determine threshold levels for peanut allergy mimicking a more real-life exposure and to correlate the eliciting dose (ED) and severity of clinical reaction in children with peanut allergy with B-cell, T-cell, and effector cell markers. Methods A modified food challenge procedure with doses scheduled 2 hours apart was used in 63 children with peanut allergy. All children received a maximum of 8 semi-log increasing titration steps of roasted peanuts ranging from 3 to 4500 mg of peanut protein until objective allergic reactions occurred. Severity of symptoms was graded from I to V. Biological markers were measured before challenge. Results Forty-five of 63 patients showed objective symptoms after greater than 30 minutes, with a median latency of clinical reaction of 55 minutes. By using a log-normal dose-distribution model, the ED5 was calculated to be 1.95 mg of peanut protein. The ED was significantly and inversely correlated with peanut- and Ara h 2–specific IgE levels, skin prick test responses, basophil activation, and T H 2 cytokine production by PBMCs. Symptom severity did not correlate with any of the markers or the ED. Conclusion This modified food challenge procedure might better reflect threshold levels for peanut allergy than the standard procedure because most of the patients reacted at a time interval of greater than 30 minutes. By using this model, threshold levels, but not severity, could be correlated with biological markers.

Background Under inflammatory conditions, T cell–dependent (TD) protein antigens induce proinflammatory T- and B-cell responses. In contrast, tolerance induction by TD antigens without costimulation triggers the development of regulatory T cells. Under both conditions, IgG antibodies are generated, but whether they have different immunoregulatory functions remains elusive. Objective It was shown recently that proinflammatory or anti-inflammatory effector functions of IgG molecules are determined by different Fc N-linked glycosylation patterns. We sought to examine the Fc glycosylation and anti-inflammatory quality of IgG molecules formed on TD tolerance induction. Methods We administered chicken ovalbumin (OVA) with or without costimulus to mice and analyzed OVA-reactive IgG Fc glycosylation. The anti-inflammatory function of differentially glycosylated anti-OVA IgGs was further investigated in studies with dendritic cell cultures and in an in vivo model of allergic airway disease. Additionally, we analyzed the Fc glycosylation pattern of birch pollen–reactive serum IgGs after successful allergen-specific immunotherapy in patients. Results Stimulation with TD antigens under inflammatory conditions induces plasma cells expressing low levels of α2,6-sialyltransferase and producing desialylated IgGs. In contrast, plasma cells induced on tolerance induction did not downregulate α2,6-sialyltransferase expression and secreted immunosuppressive sialylated IgGs that were sufficient to block antigen-specific T- and B-cell responses, dendritic cell maturation, and allergic airway inflammation. Importantly, successful specific immunotherapy in allergic patients also induced sialylated allergen-specific IgGs. Conclusions Our data show a novel antigen-specific immunoregulatory mechanism mediated by anti-inflammatory sialylated IgGs that are formed on TD tolerance induction. These findings might help to develop novel antigen-specific therapies for the treatment of allergy and autoimmunity.

Background DC play an important role in directing allergen-specific naive T-cells towards a Th1, Th2 or Treg cytokine profile. The path of T cell differentiation may be due to various maturation stages and activation stimuli influencing DC/T-cell interaction. In this context, IL-10 has been shown to exhibit immune regulatory capacities and suppress both DC and T cell functions. Aim To further delineate effects of different culture conditions and activation stimuli on IL-10 production by DC and the consequences on DC/T-cell interactions. Methods BMDC were generated from hemopoietic progenitors by culture with GM-CSF or with GM-CSF plus IL-4 for 6 days and than stimulated with LPS on day 6. BMDC were phenotypically characterized by FACS analysis, and the cytokine production was measured by ELISA. DC were co-cultured with allergen-specific naive T-cells. Results IL-10 production of BMDC generated with GM-CSF was strongly enhanced, compared to BMDC generated with GM-CSF plus IL-4. Co-culture of DC exhibiting increased IL-10 production with naive T-cells induced significantly less production of Th2-cytokines, like IL-5 and IL-13, whereas production of the Th1 cytokine IFN-γ as well as IL-10 was increased, compared to co-culture with DC with low IL-10 production. Vice versa, co-culture of naive T cells with BMDC characterized by low IL-10 production resulted in enhanced IL-5 and IL-13. Conclusion These data demonstrate that IL-10 production by DC may be a critical element in the regulation of T cell activation and differentiation towards an allergen-promoting phenotype. This notion may be exploited for future strategies for the prevention of allergic diseases.

Rationale IgE plays a central role in the pathogenesis of allergic asthma. It is detectable in serum as well as in bronchoalveolar lavage and correlates with disease severity. To further define the requirements for induction of local IgE production in lung, we utilized an adoptive transfer model of allergic airway inflammation. Methods and results Purified T cells from DO11.10 mice, carrying a T-cell receptor (TCR) specific for Ovalbumin (OVA), were stimulated in vitro with OVA 323–339 -peptide and transferred into naive Balb/c recipients. Repeated intra-nasal challenges of the recipients with OVA provoked the formation of intrapulmonary B-cell clusters preferentially located between bronchi and blood vessels. Immunohistologic characterization of these clusters showed inducible co-stimulator (ICOS) + T cells scattered within the B-cell infiltrates in close proximity to IgG1 and IgE producing plasma cells. To analyze whether the expression of ICOS was required for the induction of pulmonary B-cell infiltration and local IgE production, in vitro stimulated DO11.10 T cells were separated by magnetic cell sorting into ICOS-enriched and ICOS-depleted T-cell fractions, containing equal numbers of activated TCR transgenic T cells. Transfer of ICOS-enriched T-cells provoked markedly pronounced B-cell infiltrates with IgE and IgG1 producing plasma cells, while hardly any infiltrates and no IgE or IgG1 production was observed after transfer of the ICOS-depleted fraction. Conclusion These data suggest that allergen-specific ICOS + T-cells are involved in the development of peripheral T-/B-cell interaction in lung tissue and facilitate local IgE-production following allergen airway challenge.

Background The “hygiene hypothesis” postulates that a lack of immune stimulation early in life due to increasingly “sterile” living conditions in western countries augments the development of Th2-skewed immune responses to environmental allergens. Accordingly, immune modulating intervention in this period of life may be most favorable for preventive strategies against the development of allergic diseases. Aim Aim of the current study was to test the effects of oral administration of endotoxin in a rat model of food allergy. Methods Brown Norway rats were sensitized ip with ovalbumin (OVA) combined with pertussis and Al(OH)2 as adjuvans on days 35, 40 and 45. On days 60 and 61, rats were locally challenged with OVA by gavage feeding. Different amounts of endotoxin or PBS were administered orally, first once daily (days 7–21 post partum), than every third day up to an age of 42 days. Results Detection of increased allergen specific Ig serum levels and proliferative responses of spleen MNC confirmed successful systemic sensitization to OVA, but did not reveal significant differences between endotoxin and PBS-treated rats. In contrast, increased numbers of total lymphocytes and activated T-cells with enhanced in vitro IFN-γ and IL-10 cytokine production were detected in mesenteric lymph nodes (MLN) of endotoxin-, compared to PBS-treated rats. These observations were strongly dependent on the amount of administered endotoxin and only occurred with dosages above 0,2 ng/kg/day. Conclusion These data suggest that local administration of endotoxin in the intestine exhibits immune modulating effects with increased Th1 and T regulatory responses in a dose-dependent manner.