Search

Summary Background Peanut and tree nut allergy is characterized by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Peanut allergy is more common than tree nut allergy, but many subjects develop hypersensitivity to both peanuts and tree nuts. Whether this is due to the presence of cross-reactive allergens remains unknown. Objective The aim of this study was to investigate the presence of allergenic cross-reactivity between peanut and tree nuts. Methods Western blotting and ELISA were performed using sera from subjects with or without peanut and tree nut allergy to assess immunoglobulin E (IgE) reactivity to peanut and tree nut extracts. Inhibition ELISA studies were conducted to assess the presence of allergenic cross-reactivity between peanut and tree nuts. Results Western blot and ELISA results showed IgE reactivity to peanut, almond, Brazil nut, hazelnut and cashew nut for peanut- and tree nut-allergic subject sera. Raw and roasted peanut and tree nut extracts showed similar IgE reactivities. Inhibition ELISA showed that pre-incubation of sera with almond, Brazil nut or hazelnut extracts resulted in a decrease in IgE binding to peanut extract, indicating allergenic cross-reactivity. Pre-incubation of sera with cashew nut extract did not cause any inhibition. Conclusion These results show that multiple peanut and tree nut sensitivities observed in allergic subjects may be due to cross-reactive B cell epitopes present in different peanut and tree nut allergens. The plant taxonomic classification of peanut and tree nuts does not appear to predict allergenic cross-reactivity.

Background: Subgroups of asthma patients have extremely severe respiratory symptoms that require chronic use of steroids for disease control. These patients are at risk of significant side-effects from chronic exposure to high doses of oral steroids. Intra­venous immunoglobulin (IVIG) has immunomodulatory properties as shown by its use in some immune disorders. A few trials have suggested a possible benefit in individuals with severe asthma. Aims: To evaluate the role of IVIG as an adjunctive therapy in steroid-dependent asthma, monitoring the outcomes of lung function and measured reduction in oral steroid requirement Method: Seven patients with severe steroid-dependent asthma were given IVIG at a dose of 1 g/kg each month for 6 months. Baseline pulmonary function tests and immunoglobulin levels were obtained. At the end of 6 months, the end-points observed were lung function and the degree of reduction in the dose of oral steroids. The number of hospital admissions during the 12 months following commencement of IVIG was compared with the preceding 12 months. Results: There was a significant reduction in daily prednisolone dose from 56 ± 31 mg to 39 ± 35 mg (P = 0.04, Wilcoxon rank sum test) and a decrease in the number of hospital admissions from 5.9 ± 2.9 to 3.6 ± 3.5 (P = 0.04). No significant improvement occurred in lung function. Conclusion: IVIG provides a potentially important adjunctive therapy in severe steroid-dependent asthma, reducing steroid requirement and decreasing hospital admissions, but not improving lung function. (Intern Med J 2003; 33: 341−344)

Allergen-specific immunotherapy is a clinically proven effective treatment for many allergic diseases, including asthma; however, it is not currently available for latex allergy because of the high risk of anaphylaxis. There is, therefore, a crucial need for an animal model of latex allergy in which to develop effective immunotherapy. Previous mouse models of latex allergy either did not characterize the allergic pulmonary immune response or used crude latex extracts, making it difficult to quantify the contribution of individual proteins and limiting their usefulness for developing specific immunotherapy. We immunized mice with recombinant Hev b 5, a defined major latex allergen, or latex glove protein extract, representing the range of occupationally encountered processed latex allergens. The immune response was compared with that seen in ovalbumin-immunized mice. Immunization with Hev b 5 or glove extract elicits hallmarks of allergic pulmonary Th2-type immune responses, comparable to those for ovalbumin, including (1) serum antigen-specific IgE, (2) an eosinophilic inflammatory infiltrate in the lung, (3) increased interleukin-5 in lung bronchoalveolar lavage fluid, and (4) mucus hypersecretion by epithelial cells in the lung airways. This mouse model will aid the development of potentially curative treatments for latex-sensitized individuals, including those with occupational asthma.

Background: Tree pollen contains many allergens showing cross-reactivity to proteins from pollen, seeds, and fruits of different plant species. Amongst Fagales, responsible for several allergenic responses, hazel provides the best material to study pollen as well as food allergens in one species. The aim of this study was to identify and characterize the physiological function of an allergen from hazel pollen and to determine possible cross-reactivity to proteins from hazelnut. Methods: Monoclonal antibodies (mAbs) against hazel pollen crude extract were produced. On the basis of IgE binding, demonstrated by sera from patients allergic to hazel pollen, one mAb indicating the best correlation has been selected, and the putative allergen was purified by preparative gel electrophoresis. Isoforms were investigated by two-dimensional PAGE, and for molecular identification a hazel pollen cDNA library was constructed. In situ localization of the allergen during pollen development was performed by immunofluorescence labelling. Results: Immunological staining of crude hazel pollen extract with specific IgE and mAb revealed a 70-kD protein. Immunoblot studies with mAb showed cross-reactive proteins of 70–72 kD in different plant tissues and species. After protein purification, the IgE-binding reactivity of the allergen has been reconfirmed, and two isoforms were detected. Molecular cloning identified the allergen as a luminal binding protein (BiP) of the Hsp70 family with 88–92% sequence identity in various plants. Further immunocytological studies indicated involvement of BiP during pollen development. Conclusions: Chaperons like BiP play an important role in protein synthesis and in the protection of cellular structures during stress-related processes. Because of their highly conserved protein sequences, we propose that such allergens could be responsible for at least a part of the allergenic cross-reactivity between proteins from different pollens and plant foods.

Summary Background Although allergy to latex is a well-characterized phenomenon, some hospitals continue to provide staff with powdered latex gloves as an option to low- or non-powdered gloves. Objective We aimed to measure the extent to which inhalation of latex particles could be reduced by the use of protective masks or by replacing powdered latex gloves with non-powdered latex gloves. Methods Twenty healthcare workers in a hospital setting wore nasal air samplers (NAS) and Institute of Occupational Medicine (IOM) samplers for four 20-min periods. Subjects wore powdered gloves, non-powdered gloves and no gloves during three sampling periods, and in the fourth, subjects applied an aerosol barrier face-mask or a particulate face-mask (N95) while wearing powdered gloves. All samples were stained for particles bearing Hev b 5 allergen by the Halogen assay. Results All subjects inhaled Hev b 5 bearing particles in all sampling periods. IOM samplers collected particles at 70% of the rate of NAS. The number of particles inhaled while wearing powdered gloves was 23.8-fold higher than when not wearing gloves and 9.7-fold higher than when wearing non-powdered latex gloves (P

Latex allergy is an important allergic disease for which safe and readily available immunotherapy is currently lacking. Despite advances in latex glove technology and reduction in allergen content, there remains a core of severely allergic health care workers (HCW), particularly with concominant food allergy, for whom allergen avoidance is insufficient. Current experience with immunotherapy using crude latex extracts has shown an unacceptable level of local and systemic side-effects. Latex allergens are extremely potent with a heightened capacity to cross-link effector cell-bound IgE and induce anaphylaxis. The predominant pattern of allergen reactivity among HCW is different from that among children with spina bifida, perhaps due to exposure to latex glove proteins, particularly via inhalation, rather than particle bound latex proteins present in urinary catheters. Recent studies using purified skin testing reagents have indicated that the most clinically important latex allergens amongst HCW are Hev b 5, 6 and 7. Elucidation of the molecular and cellular mechanisms of the immune response to these allergens is pivotal to facilitate the search for safer immunotherapy of latex allergy among HCW.

Summary Background Hev b 5 is a major latex allergen recognized predominantly by latex-allergic health care workers (HCWs). Recombinant Hev b 5 (rHev b 5) was previously expressed as a fusion protein with maltose binding protein (MBP), itself an immunogenic molecule; therefore non-fusion rHev b 5 is desirable. Moreover, standardized immunological assays for the detection of Hev b 5 are currently lacking and may have important implications for both allergen avoidance and diagnosis in latex allergy. Objectives To generate and use Hev b 5-specific mAbs to determine the relative abundance of Hev b 5 in different latex extracts, correlating this with the IgE reactivity of latex-allergic HCWs and to produce non-fusion rHev b 5. Methods For the production of mAbs, mice were immunized with rHev b 5/MBP fusion protein and mAbs selected with rHev b 5/MBP but not MBP reactivity. The mAb reactivity was compared with polyclonal IgE from latex-allergic HCWs using direct and inhibition ELISA and immunoblot assays. Recombinant Hev b 5 was expressed and purified in the pPROEX-HTa bacterial expression system. Results Four Hev b 5-specific mAbs were produced. Immunoblotting and ELISA using the mAbs indicate abundant Hev b 5 in high protein powdered latex glove extracts as compared with crude latex sap extracts. High quality surgical gloves with no detectable protein have no detectable Hev b 5. Inhibition ELISAs using serum IgE from latex-allergic HCWs and Hev b 5-specific mAbs gave strong correlation. Non-fusion recombinant Hev b 5 was successfully expressed and purified, showing reactivity with both the Hev b 5-specific mAbs and serum IgE of latex-allergic HCWs. Conclusion Hev b 5-specific mAbs and human IgE from latex-allergic HCWs demonstrate the greater content of Hev b 5 in high protein powdered glove extracts. This may explain the observed higher frequency of sensitization to this allergen in HCWs.

Background: Bermuda grass pollen (BGP) is an increasingly important seasonal aeroallergen in Australia and other subtropical and temperate regions. BGP shares minimal allergenic cross-reactivity with pollens of rye grass or other Pooideae grasses often used for desensitization regimens in grass pollen allergy. Current allergen immunotherapy is seldom used in asthmatic patients due to IgE-mediated side effects. Since clinically effective immunotherapy is linked with altered allergen-specific T cell response, characterisation of human T cell reactivity to Cyn d 1, the major B cell allergen of BGP, should permit the design of effective and safe immunotherapy for BGP allergy. Methods: Short-term BGP-specific CD4+ T cell lines were established from peripheral blood of 14 BGP-sensitive patients before and after conventional 50% BGP and 50% 7-grass mix subcutaneous specific allergen immunotherapy (SIT). T cell diversity of antigen specificity and function was assessed by proliferation and cytokine production to BGP, Cyn d 1 and Cyn d 1 peptides. Results: Three highly immunogenic regions of Cyn d 1 were identified in 13/14 patients pre-SIT: Cyn d 1 (109–128), (181–209) and (217–241). The SIT regimen was clinically efficacious. Following SIT, decreased proliferation to BGP, Cyn d 1 and Cyn d 1 peptides was observed with a marked decrease in the IL-5:IFN-γ ratio. Conclusions: Cyn d 1 is a major T cell allergen of BGP. Decreased Cyn d 1-specific IL-5 dominant T cell responses were observed in association with clinically effective treatment with the 50% BGP and 50% 7-grass mix. Identified dominant T cell regions of Cyn d 1 should facilitate safer vaccine development for BGP-induced asthma in addition to rhinitis.

Allergen-specific immunotherapy (SIT) involves the administration of gradually increasing amounts of an allergen extract to reduce clinical symptoms of allergy. Well-controlled clinical trials have demonstrated the efficacy of SIT in the treatment of allergic diseases, including rhinoconjunctivitis and asthma, and best practice protocols have been established. Nevertheless, application of this potentially curative treatment is restricted, largely due to the risk of serious adverse events, especially in asthmatics. Although efficacy is high for venom-induced allergy, success rates for the more common aeroallergen-induced disease range from 60 to 80% depending on the allergen. The practice of SIT is currently being refined following major advances in our knowledge of basic immune mechanisms. In particular, new T cell-targeted strategies are being explored with the awareness of the pivotal role allergen-specific T cells play in initiating and regulating the immune response to allergens. Current SIT induces decreased IgE class switching and eosinophil activation by downregulating production of the T helper (Th) 2-type cytokines interleukin (IL)-4 and IL-5. Therefore, allergen preparations that have ablated IgE binding while retaining T cell reactivity should still be clinically effective but have substantially improved safety. These approaches include the use of small peptides based on dominant T cell epitopes of allergens and chemically modified or recombinant allergen molecules. Both approaches have already been tested, with promising results, in animal models; peptide immunotherapy has been shown effective in clinical trials. Defined hypoallergenic molecules or peptides offer ease of standardization in addition to efficacy and safety and will result in more widespread use of SIT in clinical practice. Elucidation of mechanisms for downregulating Th2-predominant responses to allergen by SIT will enable the development of laboratory assays for monitoring clinical efficacy.

Systematic evaluation is advocated for difficult asthma, but how best to deliver such care is unclear and outcome data are scarce.We describe our institution's structured approach to difficult asthma management and report on the outcomes of such an approach.Eighty-two consecutive patients with difficult asthma referred to our clinic from respiratory specialists were evaluated in 3 key areas: diagnostic confirmation, comorbidity detection, and inflammatory phenotyping. We then optimized treatment including relevant comorbidity interventions. The outpatient protocol was supported by comorbidity questionnaires, an electronic clinic template, and standardized panel discussion. Asthma outcomes were assessed at 6 months.Sixty-eight patients completed follow-up. Asthma diagnosis was refuted in 3 patients and the remaining 65 patients were included in the study analysis. There was no overall escalation of inhaled or oral corticosteroids. Patients had a median of 3 comorbidities, and a median of 3 comorbidity interventions. Control of chronic rhinosinusitis and dysfunctional breathing improved among patients with these diagnoses (22-item Sino-Nasal Outcome Test score from 47 ± 20 to 37 ± 22, P = .017; Nijmegen score from 32 ± 6 to 25 ± 9, P = .003). There were overall improvements in the Asthma Control Test score (from 14 ± 5 to 16 ± 6, P.001), the Asthma Quality of Life Questionnaire (from 4.29 ± 1.4 to 4.65 ± 1.5, P = .073), and the frequency of exacerbations over 6 months (from 2 [interquartile range, 0-4] to 0 [interquartile range, 0-2], P.001).In patients referred with difficult asthma from respiratory specialists, a structured approach coupled with targeted comorbidity interventions improved control of key comorbidities and enhanced asthma outcomes.

Background Activins A and B, members of the TGF-β superfamily, are produced as part of the physiological response to tissue damage and the resulting proinflammatory response. Given that lung allograft reperfusion results in an inflammatory response, it is likely that the activins and their binding protein follistatin will form part of the regulatory response. There is a need to document the response of these proteins to allograft reperfusion to determine if there is a role for the use of follistatin to control the biological actions of the activins because some of these are potentially damaging. Methods Serum from 48 consecutive patients undergoing lung transplantation (LTx) was collected at 2, 6, 12, and 26 weeks post-LTx. The serum levels of activin A and B and follistatin were measured by enzyme-linked immunosorbent assay and specific radioimmunoassays and compared with clinical events. Results Serum activin A and B levels were at the upper limit of the normal ranges at 2 weeks post-LTx decreasing thereafter to 12 weeks post-LTx (P < 0.05). In contrast, serum follistatin levels were unchanged between 2 and 12 weeks, with a late significant increase at 24 week post-LTx (P < 0.01). Patients with primary graft dysfunction had lower serum follistatin levels (7.7 vs 9.5 ng/mL; P = 0.04) and a higher activin A/follistatin ratio (13.1 vs 10.4; P = 0.02) at 2 weeks post-LTx. Conclusions Activin and follistatin levels vary with time form LTX and reflect a proinflammatory environment. Future studies will elucidate associations with chronic lung allograft dysfunction and the therapeutic potential of exogenous follistatin administration.

Crustacean allergy is a major cause of food-induced anaphylaxis. We showed previously that heating increases IgE reactivity of crustacean allergens. Here we investigate the effects of thermal processing of crustacean extracts on cellular immune reactivity. Raw and cooked black tiger prawn, banana prawn, mud crab and blue swimmer crab extracts were prepared and IgE reactivity assessed by ELISA. Mass spectrometry revealed a mix of several allergens in the raw mud crab extract but predominant heat-stable tropomyosin in the cooked extract. PBMC from crustacean-allergic and non-atopic control subjects were cultured with the crab and prawn extracts and proliferation of lymphocyte subsets was analysed by CFSE labelling and flow cytometry. Effector responses were assessed by intracellular IL-4 and IFN-γ, and regulatory T (CD4+CD25+CD127loFoxp3+) cell proportions in cultures were also compared by flow cytometry. For each crustacean species, the cooked extract had greater IgE reactivity than the raw (mud crab p

Food allergies are increasing worldwide, demonstrating a considerable public health concern. Shellfish allergy is one of the major food groups causing allergic sensitization among adults and children, affecting up to 2% of the general world population. Tropomyosin (TM) is the major allergen in shellfish and frequently used in the diagnosis of allergic sensitization and the detection of cross-contaminated food. To improve and establish better and more sensitive diagnostics for allergies and immunotherapeutics, large quantities of pure allergens are required. To establish a reproducible method for the generation of pure recombinant tropomyosin we utilized in this study different Escherichia coli strains (NM522, TOP10 and BL21(DE3)RIPL). In addition, isopropyl-β-d-thiogalactoside (IPTG) induction was compared with a novel auto-induction system to allow the generation of larger quantities of recombinant allergen. We demonstrated that the B-strain of E. coli is better for the expression of TM compared to the K-strain. Moreover, a higher yield could be achieved when using the auto-induction system, with up to 62 mg/l. High yield expressed recombinant TM from King prawn (KP) was compared to recombinant TM from Black tiger prawn (Pen m 1). We demonstrated that recombinant TM from KP and known isoallergen Pen m 1 have very similar molecular and immunological characteristics. Overall, we demonstrate that auto-induction can be used to express larger quantities of recombinant allergens for the development of diagnostic, to quantify allergens as well as immunotherapeutics employing isoallergens.

Background Allergic bronchopulmonary aspergillosis (ABPA) often presents with persistently uncontrolled asthma despite the use of corticosteroids and antifungal therapy. Omalizumab is a humanized anti-IgE monoclonal antibody currently used to treat severe asthma. Objective The aim was to assess the clinical and immunologic effects of omalizumab in ABPA in a randomized, placebo-controlled trial. Methods Patients with chronic ABPA were randomized to 4-month treatment with omalizumab (750 mg monthly) or placebo followed by a 3-month washout period in a cross-over design. The main endpoint was number of exacerbations. Other clinical endpoints included lung function, exhaled nitric oxide (FeNO), quality of life and symptoms. In vitro basophil activation to Aspergillus fumigatus extract and basophil FceR1 and surface-bound IgE levels were assessed by flow cytometry. Results Thirteen patients were recruited with mean total IgE 2314 ± 2125 IU/mL. Exacerbations occurred less frequently during the active treatment phase compared with the placebo period (2 vs 12 events, P = .048). Mean FeNO decreased from 30.5 to 17.1 ppb during omalizumab treatment ( P = .03). Basophil sensitivity to A. fumigatus and surface-bound IgE and FceR1 levels decreased significantly after omalizumab but not after placebo. Conclusion Omalizumab can be used safely to treat ABPA, despite high serum IgE levels. Clinical improvement was accompanied by decreased basophil reactivity to A. fumigatus and FceR1 and surface-bound IgE levels.

The innate response generated after initial allergen exposure is crucial for polarising adaptive immunity, but little is known about how it drives an atopic or type-2 immune response. The present study characterises the response of skin-draining afferent lymph in sheep following injection with peanut (PN) extract in the presence or absence of aluminium hydroxide (AlOH) adjuvant. Lymph was collected and innate cell populations characterised over an 84 h time period. The innate response to PN extract in afferent lymph displayed an early increase in neutrophils and monocytes without any changes in the dendritic cell (DC) population. PN antigen was transported by neutrophils and monocytes for the first 36 h, after which time DCs were the major antigen trafficking cells. AlOH adjuvant gradually increased antigen uptake by DCs at the later time points. Following lymphatic characterisation, sheep were sensitised with PN extract by three subcutaneous injections of PN in AlOH, and the level of PN-specific immunoglobulin E (IgE) was determined. Sheep with higher levels of steady-state DCs in afferent lymph showed increased monocytic recruitment in afferent lymph and reduced PN-specific IgE following sensitisation. In addition, DCs from afferent lymph that had ingested PN antigen increased the expression of monocyte chemoattractant mRNA. The results of this study show that the innate response to PN extract involves a dynamic change in cell populations in the afferent lymph over time. In addition, DCs may determine the strength of the initial inflammatory cell response, which in turn may determine the nature of the antigen-specific adaptive response.

The aim of this study was to investigate the molecular basis of human IgE–allergen interaction by screening a phage-displayed peptide library with an allergen-specific human IgE-mimicking monoclonal antibody (mAb). A mAb that reacted with major grass pollen allergens was successfully identified and shown to inhibit human IgE–allergen interaction. Biopanning of a phage-displayed random peptide library with this mAb yielded a 12 amino acid long mimotope. A synthetic peptide based on this 12-mer mimotope inhibited mAb and human IgE binding to grass pollen extracts. Our results indicate that such synthetic peptide mimotopes of allergens have potential as novel therapeutic agents.

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 microg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

The increase in recognition and understanding of asthma as an immune-mediated disease has provided greater insights into both the etiology and pathology of the disorder. Because infections have profound effects on the immune system, orchestrating changes in humoral and cell-mediated responses, they influence both the pathogenesis of asthma and its ongoing status. Data regarding respiratory tract and systemic infections have increased our understanding of both the underlying immune genesis of asthma and the acute inflammatory airway changes that occur during asthma. This review deals with both these aspects of respiratory infections.

A novel isoform of a major rye grass pollen allergen Lol p 5 was isolated from a cDNA expression library. The new isoform, Lol p 5C, shares 95% amino acid sequence identity with Lol p 5A. Both isoforms demonstrated shared antigenic activity but different allergenic activities. Recombinant Lol p 5C demonstrated 100% IgE reactivity in 22 rye grass pollen sensitive patients. In comparison, recombinant Lol p 5A showed IgE reactivity in less than 64% of the patients. Therefore, Lol p 5C represents a novel and highly IgE-reactive isoform allergen of rye grass pollen.

Background: T cells are pivotal in the elicitation of allergic diseases. Analogues of T-cell epitope peptides with a modification at a T-cell receptor (TCR) contact site can alter selected T-cell effector functions. Thus the ability to modulate allergen-specific T-cell responses towards T H1 -like by stimulation with peptide analogues may downregulate allergic inflammation. Objectives: The purpose of this study was to characterize the minimal epitope recognized by cloned T cells of a dominant Lol p 5 epitope, p105-116, and identify the critical residues involved in TCR and MHC contact. Methods: Using peptides with progressive truncation of N- and C-terminal residues in T-cell proliferation assays, we identified the core epitope recognized by cloned CD4 + T cells. An additional series of peptides with single amino acid substitutions were used in T-cell proliferation and live-cell MHC binding assays. Taken together, these results allowed identification of MHC binding and TCR contact residues of p105-116. Results: The core epitope of p105-116 was identified as residues 107-114. Within this core epitope, 3 residues were found to be important for MHC binding, positions 107, 110, and 112, whereas those at positions 108, 109, 110, 111, and 113 were putative TCR contact residues. Conclusions: The identification of the TCR and MHC contact residues of a dominant Lol p 5 T-cell epitope and analogues of this peptide capable of modulating T-cell responses will allow the evaluation of these peptides' potential as immunotherapeutic agents for rye grass pollen allergic disease. (J Allergy Clin Immunol 1999;103:255-61.)

Background. Although allergen immunotherapy is effective for allergic rhinitis, its role in treating asthma is unclear. Methods. We examined the efficacy of immunotherapy for asthma exacerbated by seasonal ragweed exposure. During an observation phase, adults with asthma who were sensitive to ragweed kept daily diaries and recorded peak expiratory flow rates between July and October. Those who reported seasonal asthma symptoms and medication use as well as decreased peak expiratory flow were randomly assigned to receive placebo or ragweed-extract immunotherapy in doses that increased weekly for an additional two years. Results. During the observation phase, the mean (SE) peak expiratory flow rate measured in the morning during the three weeks representing the height of the pollination season was 454 (20) litres per minute in the immunotherapy group and 444 (16) litres per minute in the placebo group. Of the 77 patients who began the treatment phase, 64 completed one year of the study treatment and 53 completed two years. During the two treatment years, the mean peak expiratory flow rate was higher in the immunotherapy group (489 (16) litres per minute vs. 453 (17) in the placebo group (p= 0.06) during the first year, and 480 (12) litres per minute vs. 461 (13) in the placebo group (p = 0.03) during the second). Medication use was higher in the immunotherapy group than in the placebo group during observation and lower during the first treatment year (p = 0.01) but did not differ in the two groups during the second year (p=0.7). Asthma symptom scores were similar in the two groups (p = 0.08 in year 1 and p = 0.3 in year 2). The immunotherapy group had reduced hayfever symptoms, skin test sensitivity to ragweed, and sensitivity to bronchial challenges and increased IgG antibodies to ragweed as compared with the placebo group; there was no longer a seasonal increase in IgE antibodies to ragweed allergen in the immunotherapy group after two years of treatment. Reduced medication costs were counterbalanced by the costs of immunotherapy. Conclusions. Although immunotherapy for adults with asthma exacerbated by seasonal ragweed exposure had positive effects on objective measures of asthma and allergy, the clinical effects were limited and many were not sustained for two years.

There is increasing interest in the use of engineered particles for biomedical applications, although questions exist about their proinflammatory properties and potential adverse health effects. Lung macrophages and dendritic cells (DC) are key regulators of pulmonary immunity, but little is known about their uptake of different sized particles or the nature of the induced immunological imprint. We investigated comparatively the immunological imprints of inert nontoxic polystyrene nanoparticles 50 nm in diameter (PS50G) and 500 nm in diameter (PS500G). Following intratracheal instillation into naive mice, PS50G were preferentially taken up by alveolar and nonalveolar macrophages, B cells, and CD11b+ and CD103+ DC in the lung, but exclusively by DC in the draining lymph node (LN). Negligible particle uptake occurred in the draining LN 2 h postinstillation, indicating that particle translocation does not occur via lymphatic drainage. PS50G but not PS500G significantly increased airway levels of mediators that drive DC migration/maturation and DC costimulatory molecule expression. Both particles decreased frequencies of stimulatory CD11b+MHC class IIhi allergen-laden DC in the draining LN, with PS50G having the more pronounced effect. These distinctive particle imprints differentially modulated induction of acute allergic airway inflammation, with PS50G but not PS500G significantly inhibiting adaptive allergen-specific immunity. Our data show that nanoparticles are taken up preferentially by lung APC stimulate cytokine/chemokine production and pulmonary DC maturation and translocate to the lung-draining LN via cell-associated transport. Collectively, these distinctive particle imprints differentially modulate development of subsequent lung immune responses. These findings support the development of lung-specific particulate vaccines, drug delivery systems, and immunomodulators.

Summary Background Selected cytokines produced by allergen specific CD4+ T cells from atopic individuals contribute to both the specific and non-specific effector mechanisms of the allergic itnmune response. The chemokine family of cytokines and tumour necrosis factor (TNF)-α are leucocyte regulatory and proinflanimatory molecules. The chemokines include interleukin (IL)-8 and the RANTES/SIS cytokines. Objective There has been no systematic survey of chemokine production in T-cell subtypes. Because of their wide range of biological properties, it might be expected that they would be closely regulated by T cells. This paper illustrates one way (through the characterization of T-cell clones) these questions might be addressed. Methods Northern blot analysis was used to quantitate steady state transcription of selected cytokine genes and enzyme linked imtiiunosorbent assay (ELISA) was used to quantitate soluble product. Results mRNA expression of the chemokines (IL-8, HuMIP-1α and HuMIP-1β) and TNFα is upregulated in TH2-like cloned house dust mite reactive human CD4+ T cells under conditions of activation and during the induction phase of anergy. Although the development of anergy superinduces mRNA for both IL-8 and TNFα. protein production is low compared with that released during activation. In contrast. RANTES, a chemoattractant for CD4+/CD45RO+ memory T cells, eosinophils and basophils, is constitutively expressed at the RNA level by the T cells and not modulated by signals of activation and anergy induction. The production of IL-2, IL-4 and IL-5 mRNA and proteins during the induction of anergy peaks at 2h after stimulation, whereas the kinetics following activation of the T cells is delayed in comparison. Conclusion These data show that the induction of the anergic state coincides with post-transcriptional regulation of selected cytokine genes. Further study of these phenomena will impact on our understanding of the mechanisms of induction of anergy and the regulation of allergic immune responses in desensitization.

SummaryBackground Peanut allergy causes severe type 1 hypersensitivity reactions and conventional immunotherapy against peanut allergy is associated with a high risk of anaphylaxis. Objective Our current study reports proof of concept experiments on the safety of a stably denatured variant of the major peanut allergen Ara h 2 for immunotherapy. We determined the impact of structure loss of Ara h 2 on its IgE binding and basophil degranulation capacity, T cell reactivity as well as anaphylactic potential. Methods The secondary structure of untreated and reduced/alkylated Ara h 2 variants was determined by circular dichroism spectroscopy. We addressed human patient IgE binding to Ara h 2 by ELISA and Western blot experiments. RBL-SX38 cells were used to test the degranulation induced by untreated and reduced/alkylated Ara h 2. We assessed the anaphylactic potential of Ara h 2 variants by challenge of sensitized BALB/c mice. T cell reactivity was investigated using human Ara h 2-specific T cell lines and splenocytes isolated from sensitized mice. Results Reduction/alkylation of Ara h 2 caused a decrease in IgE binding capacity, basophil degranulation and anaphylactic potential in vivo. However, the human T cell response to reduced/alkylated and untreated Ara h 2 was comparable. Mouse splenocytes showed higher metabolic activity upon stimulation with reduced/alkylated Ara h 2 and released similar IL-4, IL-13 and IFNγ levels upon treatment with either Ara h 2 variant. Conclusions and Clinical Relevance Reduced/alkylated Ara h 2 might be a safer alternative than native Ara h 2 for immunotherapeutic treatment of peanut allergic patients.

Summary Background The adhesion molecule LFA-1 contributes to the activation response of peripheral blood human CD4+ T cells. Less is known of its contribution to stimulation of long-term CD4+ T cell lines and clones or of its potential to co-stimulate CD4+ T cells of different functional phenotype. Objective This study was therefore performed to investigate co-stimulatory properties of the LFA-1 (CD11a/CD 18) complex in the activation of human CD4+ T cell lines and clones of TH-0. TH-1 and TH-2 subsets. Methods Co-stimulatory activity was measured by cross-linking antibodies to CD 11a or CD18 with anti-CD3 antibodies to plastic and then measuring the proliferative response of CD4+ T cells to these antibodies. Results A house duct mite allergen-specific CD4+ T cell line (TH-2) demonstrated much greater dependence on both C'DI la and CD IK than a mycobacterial antigen-specific CD4+ T cell line (TH-1). Co-stimulatory activity through LFA-1 was also provided to a house dust mite-specific CD4+ T cell clone (DE-9; TH-2) but not to an influenza haemagglutinin-specific CD4+ T cell clone (HA 1.7: TH-0). In contrast, soluble antibodies to CD 18 inhibited proliferativc responses of both DE-9 and HA1.7 to an immunogenic challenge of antigen and to stimulation by unti-CD3 antibodies. However, the allergen-specific T cells were more susceptible to inhibition. Signal transduction was also observed from the T-cell receptor to LFA-1. Ligation of the T-cell receptor modulated the phenotypic expression of LFA-1 and ICAM-1 on both HA1-7 and DE-9). Phenotypic modulation was observed as a result of both activation and the induction of non-responsiveness. Conclusion These experiments indicate that CD4+ T cells of TH-2 functional phenotype may have a greater requirement for the co-stimulatory activity of LFA-1 than CD4+ T cells of TH-0 or TH-1 phenotypes.

Background: Increasing production and consumption of mollusc is associated with the rise in prevalence of mollusc allergy worldwide, currently ranging from 0.15% to 1.3% of the general population. However, the elucidation of mollusc allergens for better diagnostics still lags behind other seafood groups such as fish and crustacean. Genomic data have been utilized previously for improved identification of non-food allergens by performing similarity searching using the BLAST program. Based on the published genome of the Pacific oyster (Crassostrea gigas) we aimed to identify the complete potential oyster allergen repertoire using ioinformatics analysis, and to investigate identified protein allergenicity using a combination of immuno-chemical methods and proteomic analysis. Results: Ninety-five potential allergenic proteins of the Pacific oyster were discovered using in silico analyses. These proteins were of same protein family and had more than 50% amino acid identity with their homologous allergens. The allergenicity of these proteins was characterized using a combination of immunoassay and transcriptome-derived proteomics analyses. However The 2D-immunoblotting results showed only twenty two IgE-reactive spots in the raw extract of the Pacific oyster, and six spots in the heated extract. The identity of these IgE-reactive proteins was investigated by mass spectrometry. Sixteen allergens were identified, some with two or more isoforms. Conclusions: The combination of genomics coupled to proteomics and IgE-reactivity profiling is a powerful method for the identification of novel allergens from food sources. Using this combination approach we were able to expand the current knowledge on IgE-reactivity to various proteins of the Pacific oyster. These newly identified allergens and knowledge of their gene sequences will facilitate the development of improved component resolved diagnosis and future immunotherapy approach for oyster allergy.

Background The diagnosis of allergic bronchopulmonary aspergillosis (ABPA) in asthma is often made in patients with total serum IgE levels greater than 1,000 IU/mL in conjunction with evidence of Aspergillus sensitization. The specificity of total serum IgE for the diagnosis of ABPA is low even when combined with serum Aspergillus specific IgE. Objective To determine the prevalence of ABPA and to identify alternative clinical predictors for ABPA among asthmatic patients with a total serum IgE level greater than 1,000 IU/ml. Methods This study was conducted in a tertiary hospital in Melbourne, Australia, with a large asthma and allergy service. Patients with asthma and total serum IgE levels greater than 1,000 IU/ml from January 1, 2005, through December 31, 2014, were included. Patients were considered to have concomitant allergic conditions if they had atopic eczema, allergic rhinitis, or both. The diagnosis of ABPA was based on the managing physician's documented diagnosis and referenced to criteria proposed by the International Society for Human and Fungal Mycology. Results The prevalence of ABPA in our cohort was 15.8%. Older age, elevated total serum IgE level, reduced lung function, and the absence of other concomitant allergic conditions increased the risk of ABPA. After multivariate logistic regression, patients without concomitant allergic conditions had an odds ratio of 4.4 (95% confidence interval, 1.9–10.1; P = .001) for ABPA when compared with patients with allergic conditions. Conclusion The absence of atopic eczema and allergic rhinitis in these patients increases the likelihood of ABPA. Eliciting an accurate allergy history may be a useful bedside clinical tool when considering the diagnosis of ABPA.