Nonallergic Subjects Research Articles

Allergic patients have more numerous and prolonged respiratory infections than nonallergic subjects

Allergic disorders are characterized by type 2 helper T cell (Th2)-polarization, thus physiological type 1 helper T cell (Th1)-dependent mechanisms involved in fighting respiratory infections (RI) may be defective. It has previously been reported that allergic children have more numerous and severe RI than nonallergic ones. The aim of the study was to evaluate the number and duration of RI in adult allergic and nonallergic subjects. Six hundred and twenty-four subjects were studied; 202 of them were allergic (i.e. suffering from allergic rhinitis). The number of RI as well as the duration of the disease were recorded for 2 years. Allergic subjects showed a significantly higher rate of RI episodes [adjusted incidence rate ratio (IRR) = 2.16, 95% confidence interval (CI) 1.94-2.41, P < 0.001] than subjects without allergy. The number of mild RI episodes was slightly higher in allergic subjects (IRR = 1.68, 95% CI 1.50-1.89, P < 0.001), while the number of severe episodes was markedly higher (IRR = 15.71, 95% CI 10.35-23.84, P < 0.001) when compared with nonallergic subjects. Moreover, allergic patients showed a longer total duration of RI than nonallergic subjects, with a mean difference of 17.4 days (95% CI 15.5-19.4, P < 0.001). This study provides evidence that adult allergic patients have more numerous and prolonged RI than nonallergic subjects.

Eosinophilic infiltration in the nasal mucosa of rhinitis patients: is it affected by the presence of asthma or the allergic status of the patients?

Asthma and rhinitis often coexist, and there is evidence to suggest that they have similar histopathologic features. To examine whether the inflammatory infiltration in the nasal mucosa in rhinitis is affected by the presence of asthma and allergy. Nasal mucosa biopsy samples were collected from 44 individuals: 18 with rhinitis and asthma (9 allergic and 9 nonallergic), 16 with rhinitis and no asthma (8 allergic and 8 nonallergic), and 10 nonallergic control subjects. The alkaline phosphatase-anti-alkaline phosphatase method was applied to 6-microm-thick cryostat sections using monoclonal antibodies against T cells (CD4 and CD8) and eosinophils (EG2). Slides were counted blindly, and results are expressed as cells per high-power field. Eosinophil counts were higher in the nasal mucosa of rhinitic patients vs controls. No differences in cellular infiltration were detected between rhinitic patients with and without asthma or between allergic and nonallergic patients. A trend toward higher CD4+ T-cell counts in the nasal mucosa of rhinitic patients was observed, whereas no differences were noted in CD8+ T-cell infiltration among the groups. Inflammatory infiltration, characterized by the presence of eosinophils and CD4+ T cells, was similar in the nasal mucosa in noninfectious rhinitis irrespective of the presence of asthma or the allergic status of the patient.

T‐cell activation in occupational asthma and rhinitis

Allergic asthma and rhinitis are described as associated with a Th2 activation. However, recent works indicate that a Th1 activation can also be associated with these diseases, concomitantly to a defect in regulatory T (Treg) cell activation. Occupational asthma (OA) and occupational rhinitis (OR) are peculiar cases of these diseases in which the T-cell activation profile is largely unknown. To characterize T-cell activation induced after a specific inhalation test (SIT) in OA and OR. A total of 21 subjects with OA, 10 subjects with OR, 10 exposed nonallergic (ENA) subjects, and 14 healthy volunteers were included. The SIT with the incriminated substance was performed in patients and ENA subjects. Blood and induced sputum were obtained before and after SIT. T cells were analysed for CD69, CD25, IL-13, and IFN-gamma expression by flow cytometry. IL-4 and IFN-gamma were assayed by enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants. Treg cells were identified as CD4(+)CD25(+high)CD45RO(+)CD69(-) T cells in peripheral blood. Baseline IFN-gamma production was decreased in OA and OR compared with controls. The SIT induced an increase in both Th1 and Th2 cells in blood and sputum from OA. In this group, the proportion of peripheral Treg cells decreased after SIT. Similar results were found in the CD8(+) population. ELISA assays were concordant with flow cytometry. In OR, an attenuated activation profile was found, with an increase in the proportion of IL-13-producing T cells after SIT. By contrast, in ENA subjects, SIT induced Th2 activation, with an increase in Treg cells and a decrease in Th1 cells. Our results demonstrate a gradient of T-cell activation from a tolerating profile in ENA subjects to an inflammatory profile in OA, with an intermediate stage in OR.

Allergic Subjects Have More Numerous Respiratory Infections and Severe Gastrointestinal Infections Than Non-Allergic Subjects: Preliminary Results

Allergic disorders are characterized by Th2-polarization, thus physiological Th1-dependent mechanisms for fighting infectious diseases (ID) may be defective. This study aims at evaluating the number and duration of respiratory (RI) and gastrointestinal diseases (GI) in allergic and non-allergic subjects in a particular community: the crew of an operative warship consisting of 189 persons (171 males and 18 females). The study period lasted for 5 months during an operative mission. The number and duration (days) of RI and GI were evaluated. Allergic subjects experienced more numerous RI (p=0.0029) and more severe GI (p=0.0001) than non-allergic subjects. This preliminary study provides evidence that allergic subjects may have more numerous RI and prolonged GI than non-allergic subjects.

Antibiotic Treatment Alone for Acute Rhinosinusitis Gives a Poor Response in Allergic Children

Acute rhinosinusitis (ARS) is frequent in children. Patients with allergic rhinitis show signs of more impaired paranasal sinus functioning than normal subjects during viral colds. This study evaluated the effectiveness of administering antibiotics alone to treat ARS in both allergic and non-allergic children. We obtained informed consent from the parents of each patient enrolled in the study. 97 children, 55 males and 42 females aged between 4 and 9 years (46 of whom were allergic), suffering from ARS, were treated with amoxicilline-clavulanate (50 mg/Kg bid) for 14 days. Symptoms and endoscopic signs of illness were evaluated at baseline and immediately after treatment. Symptoms improved significantly (p<0.001) after treatment in 84 patients, equally distributed between allergic and non-allergic subjects. On the contrary, endoscopic signs disappeared only in 49 children, 9 of whom were allergic (OR 14.9, 95%CI 4.6-40.1, p<0.001). Slight to fair agreement was observed between clinical symptoms and endoscopic signs (agreement 64% , Kappa=0.28, McNemar test p<0.001). Antibiotic therapy alone in the treatment of ARS may be generally insufficient to resolve symptoms, mainly endoscopic signs. Moreover, in allergic children this issue appears to be more evident.

Eosinophil infiltration and activation in nasal polyposis

Conclusions. Infiltration and activation of eosinophils is a characteristic of nasal polyposis. Allergic reaction is a risk factor for the accumulation of eosinophils in this disease. T-cell-derived interleukin 5 (IL-5) and autosecretion of IL-5 from activated eosinophils may be causative reasons for the extension and persistence of eosinophil inflammation. Objectives. To investigate whether eosinophils were accumulated and activated in nasal polyposis, and the roles of IL-5, eotaxin, and T cells in this process. Materials and methods. A retrospective study was conducted on 17 tissue samples from patients with nasal polyposis with allergy and 26 cases of non-allergic polyposis. Immunohistochemical staining by specific antibodies was carried out using the alkaline phosphatase anti-alkaline phosphatase method and the avidin-biotin complex technique.Results. The number of EG1-positive cells (pan eosinophil marker) was similar to the number of EG2-positive cells (activated eosinophil marker) in all tissue samples, although EG1- and EG2-positive cells were richer in allergic patients than those in non-allergic patients. Both EG1- and EG2-positive cells were correlated with CD3-positive cells (pan T cell marker) and IL-5-producing cells in allergic or non-allergic polyposis. A large proportion of IL-5 producing cells were eosinophils. Eotaxin protein was detected in all tissue samples and dominantly located in epithelial cells. Eotaxin expression between allergic and non-allergic subjects was not significantly different.

Thrombocytopoesis in allergic asthma

Allergic asthma is characterized by bronchial inflammation and simultaneous repair processes that results in increased airway obstruction. Recent studies indicate that platelets could play an important role in this process. The aim of the present study was to evaluate the platelet count, the percentage of reticulated platelets, plasma thrombopoietin and interleukin 6 (IL-6) concentrations in patients with moderate bronchial asthma. The study was performed in 12 allergic chronic asthma patients and 12 nonallergic chronic asthma patients. Patients were treated according to the GINA 2004 recommendations for chronic moderate asthma. Seven healthy individuals served as negative controls. Blood was collected on CTAD from antecubital vein in the morning. The platelet count was statistically higher in allergic asthma patients compared with healthy controls. In nonallergic asthma patients the platelet count was also higher but the difference was not statistically significant. The percentage of the reticulated platelets in allergic asthma patients was statistically higher as compared to nonallergic and healthy subjects, who had similiar values. The concentration of IL-6 was significantly elevated in allergic asthma patients as compared to healthy controls. There was no difference in IL-6 levels between both asthma patient groups. In nonallergic asthma patients plasma thrombopoietin levels were slightly higher, but not statistically significant, in comparison with allergic asthma patients and controls. Our findings suggest that platelets may be involved in allergic inflammation and play a significant role in remodeling of the airways. In patients with allergic asthma we found increased reticulated platelet count suggesting enhanced megakaryocytopoiesis. Further studies on a role of platelets in the pathogenesis of asthma and their potential clinical implications are warranted and may contribute to the development of novel therapies.

Responses of IgE, IgG1, and IgG4 to concanavalin A-binding Blomia tropicalis antigens in allergic patients

Blomia tropicalis (Bt) and Dermatophagoides pteronyssinus (Dp) are the prevalent house dust mites in tropical countries and are associated with allergic diseases. Glycosylated antigens are highly immunogenic and involved in different pathologies. We evaluated the presence of IgE, IgG1, and IgG4 to concanavalin A-binding antigens (Bt-Con-A) isolated from Bt-total extract in sera of allergic and non-allergic subjects. Bt-total and Bt-Con-A extracts were evaluated by SDS-PAGE and ELISA for reacting with IgE, IgG1, and IgG4 in sera of 121 patients with allergic rhinitis and 36 non-allergic individuals. All subjects were skin prick tested with Bt-total extract and inhibition tests were performed for IgE, IgG1, and IgG4 using both extracts (Bt-total and Bt-Con-A). Skin prick test showed that 58% of the patients were sensitized to Bt (Bt+), with 52% reactive to both mites (Bt and Dp) and 6% to Bt only. A broad spectrum of proteins (14-152 kDa) was visualized in Bt-total and components >27 kDa for the Bt-Con-A extract. ELISA showed a similar profile of IgE, IgG1 and IgG4 levels in response to Bt-total and Bt-Con-A extracts in different groups, although Bt+ patients showed a lower IgG4 reactivity to Bt-Con-A extract. Specific IgG1 levels were higher in Bt+ patients than in control subjects, and IgG4 levels showed no significant difference among groups. ELISA inhibition showed a partial IgE and total IgG1 and IgG4 cross-reactivity with Dp extract for Bt-total and Bt-Con-A extracts. We conclude that Con-A-binding components isolated from Bt constitute major allergens and are involved in both allergen sensitization (IgE response) and homeostasis maintenance (IgG1 and IgG4 responses).

Differential accumulation of pulmonary and cardiac mast cell-subsets and eosinophils between fatal anaphylaxis and asthma death: A postmortem comparative study

The distribution profile of infiltrated mast cell-subpopulations and eosinophils in the lung and heart sections of the patients who died of severe allergic hyperresponsiveness, was investigated. Four study groups were designed comprising 9 cases who died in systemic anaphylaxis (Group I), 10 asthmatic individuals whose death were assigned to acute and severe bronchial asthma (Group II), 10 asthmatic cases who died from non-immunological diseases (Group III). Twenty consecutive autopsies of non-allergic subjects who died of unnatural causes (Group IV) served as control group in this study. Utilizing antibodies against human tryptase and chymase and a double immunohistochemical staining method, we distinguished successfully all three subsets of mast cells (MC), MC-TC (containing both tryptase and chymase), MC-T (containing only tryptase) and MC-C (containing only chymase) types, subdivided on the basis of the protease compositions of their secretory granules. In order to immunostaining eosinophils, we used antibody to major basic protein as a marker. We also measured postmortem blood tryptase, specific and total serum IgE. The intriguing finding of this study was the marked differences of cellular composition in the lung between fatal anaphylaxis and asthma death. Significant augmentation of MCs infiltrated in lung and heart sections of anaphylaxis patients and drastic infiltration of bronchial eosinophils in asthmatic death and consequent release of their related inflammatory mediators might explain the differential expression of the associated symptoms in these two groups. The anaphylactic deaths did show neither emphysema nor significant mucous bronchial secretions whereas all asthmatic deaths did. The degree of pulmonary congestion and edema was also more severe in anaphylaxis. This corresponded with the histological findings and the location and number of mast cell-subsets and eosinophils in the different compartments of the lungs. We have demonstrated that the third type of mast cell MC-C is only found in the lungs in anaphylactic deaths. The practical consequence of our study will be that it is now possible to confirm a suspicion of anaphylaxis death not only by measurements of serum mast cell tryptase, but also by immunohistochemical methods.

Allergic Tears Promote Upregulation of Eosinophil Adhesion to Conjunctival Epithelial Cells in an Ex Vivo Model: Inhibition with Olopatadine Treatment

The mechanism by which eosinophils adhere to the ocular surface during allergic inflammation is unknown. This study examined whether the incubation of human conjunctival epithelial cells (HCEs) with tears from allergic subjects promotes eosinophil adhesion, and it examined the effect of treatment with olopatadine on this process. Allergic subjects (n = 6) and nonallergic subjects (n = 4) were treated in season for 1 week with olopatadine in one eye while the other eye remained untreated. Tears were collected from both eyes with the use of a microcapillary tube. HCEs were acquired by enzymatic digestion of cadaveric conjunctival tissues. Confluent cultures of HCEs were treated with diluted tears for 24 hours before incubation with peripheral blood eosinophils (purified with negative magnetic bead selection). Eosinophil adhesion was measured with an eosinophil peroxidase assay. Incubation of HCEs with tears from allergic subjects significantly upregulated eosinophil adhesion compared with eosinophil adhesion to untreated HCEs or with HCEs treated with nonallergic tears and untreated HCEs (P < 0.05). Eosinophil adhesion to HCEs treated with tears from olopatadine-treated allergic subjects was inhibited (P < 0.01) compared with tear-stimulated adhesion observed from untreated eyes. Percentage of inhibition was 43.3% +/- 13.9% (mean +/- SD). Blocking antibodies demonstrated that eosinophil adhesion to HCEs in vitro involved beta2 integrins on eosinophils but not intercellular adhesion molecule-1 on human HCEs. Tears collected from allergic subjects contain bioactivity capable of upregulating eosinophil adhesion to HCEs in vitro. Inhibition of this process by treatment of subjects with olopatadine suggests that some of the cellular targets of this drug may play a role in promoting eosinophil adhesion.

Evidence of in vivo basophil activation in chronic idiopathic urticaria

Approximately 40% of chronic idiopathic urticaria (CIU) subjects have autoantibodies to either FcepsilonRIalpha or IgE. The effect of such autoantibodies on circulating basophil activation status is unknown. The expression of cell surface activation markers on basophils from CIU, non-allergic, and allergic subjects were compared. Further, the relationship between marker expression and serum factors reported in CIU, such as histamine-releasing activity (HRA) and immunoreactivity to FcepsilonRIalpha were examined. Peripheral blood was obtained from CIU, allergic, and non-allergic donors and fractionated by density gradients. Enriched basophils (1-12%) were analysed by flow cytometry for expression of activation markers including CD63, CD69, and CD203c. Dilutions of serum (5-50%) were analysed for HRA on basophils from a normal donor. Serum was tested for immunoreactivity by western blotting to a standard cell lysate prepared from an RBL-SX38 cell line transfected with human FcepsilonRIalpha. CIU subjects (n=9) and allergic subjects (n=8) exhibited enhanced expression of CD63 and CD69, as compared with non-allergic subjects (n=7); however, no difference was seen among groups for CD203c expression. Five CIU and two non-allergic subjects had evidence of significant serum HRA (>20%), whereas two CIU, two allergic, and three non-allergic subjects had evidence of serum immunoreactivity to FcepsilonRIalpha. Serum HRA and serum immunoreactivity to FcepsilonRIalpha were not associated with enhanced surface marker expression. Basophil activation marker expression is increased in CIU subjects and is not associated with serum factors. In addition, serum HRA and FcepsilonRIalpha immunoreactivity are not unique to CIU, or related to enhanced circulating basophil marker expression.

Allergen-Specific MHC Class II Tetramer+ Cells Are Detectable in Allergic, but Not in Nonallergic, Individuals

Allergen-specific cells are present in very low frequency in peripheral blood of humans, and differ in function in allergic and nonallergic individuals. We report in this study that soluble class II MHC tetramers can be used to directly identify and study such allergen epitope-specific CD4+ T cells in humans. We identified the major antigenic epitope of rye grass allergen Lol p 1 in HLA-DRB1*0401 individuals using HLA-DR*0401 transgenic mice and peripheral blood cells from HLA-DR*0401 individuals. Using DRB1*0401 tetramers loaded with this major epitope of Lol p 1, we detected allergen-specific CD4+ T cells in the peripheral blood of DRB1*0401 rye grass allergic individuals after ex vivo expansion with allergen. These tetramer-positive cells produced IL-4, but little IFN-gamma. In contrast, we were unable to detect rye grass tetramer-positive cells in cultures from HLA-DR*0401 nonallergic individuals, even after expansion with IL-2. Thus, our results suggest that rye grass allergen-specific T cells in DR*0401 nonallergic subjects are present at very low levels (e.g., because of deletion or suppression), differ in a fundamental way in their requirement for ex vivo expansion (e.g., they may be anergic), or use TCRs distinct from those of allergic individuals. Thus, analysis using DRB1*0401 tetramers loaded with a major epitope of Lol p 1 indicates that allergen-specific CD4+ T cells in nonallergic individuals are distinct from those in allergic subjects.

Increased bone fracture prevalence in postmenopausal women suffering from pollen-allergy

Our aim was to investigate whether pollen-allergy can affect bone mass and fractures in postmenopausal women. A total of 125 postmenopausal pollen-allergic women (mean age: 61.26 yr) were split into four groups: (1) treated with neither H1 histamine receptor (H1R) antagonist nor inhaled corticosteroid (n=43); (2) treated only with H1R antagonist (n=53); (3) treated with both H1R antagonist and inhaled corticosteroid (n=17); (4) treated with only inhaled corticosteroid (n=12). Treatment, in the appropriate groups, had occurred for at least 5 years, seasonally. One-hundred non-allergic postmenopausal subjects matched for age, body mass index (BMI), and age at menopause served as controls. Overweight and obesity (25 kg/m(2) < or =BMI) were common among the allergic women (76%). Allergic patients without treatment had a slightly lower bone density than their non-allergic counterparts. The rate (34.9%) of prevalent low-energy fractures (distal forearm, hip, and clinical vertebral fractures) in untreated allergic patients was almost triple that observed in non-allergic women (13%, chi(2) p=0.003). Bone fracture occurred more often in H1R-only treated patients (30.19%) than in controls (chi(2) p=0.01); however, clinical vertebral or hip fractures developed neither in those treated only with H1R antagonist nor in those who received both H1R antagonist and inhaled corticosteroid. Bone fractures were more frequent among patients with inhaled steroid treatment than among patients with a combined treatment of inhaled steroid and antihistamine (50 versus 29.4%). BMI predicted prevalent fractures at 1.278 (95% CI: 1.047-1.559, p=0.016) for a 1 kg/m(2) increase among untreated allergic patients. In conclusion, we found a high prevalence of low-energy fractures among pollen-allergic postmenopausal women which was associated with obesity. It is possible that the H1R antagonists compensate for both the negative effect of pollen-allergy and the adverse effect of inhaled corticosteroid treatment on bone fracture risk.

Beyond Our Pages

Beyond Our Pages

A novel single-nucleotide substitution, Glu 4 Lys, in the leukotriene C4 synthase gene associated with allergic diseases

Cysteinyl leukotrienes (cysLTs) play important roles in bronchial asthma, and can mediate bronchial smooth muscle constriction and increase mucous secretion, vascular permeability and cellular infiltration. We identified a novel heterozygous single-nucleotide substitution 10G>A (Glu 4 Lys) in the first exon of the leukotriene C4 synthase gene (LTC4S). This substitution was detected in 5 of 141 allergic patients, but not in 110 nonallergic subjects. There was a difference in the Glu 4 Lys frequency between the allergic patients and nonallergic subjects (Fisher's exact test, p=0.0460). The five patients with Glu 4 Lys had allergic diseases such as bronchial asthma and/or allergic dermatitis. Furthermore, a familial analysis of Glu 4 Lys revealed a link with allergic diseases. Thus, our results suggest that Glu 4 Lys in the LTC4S might be associated with allergic diseases.

Association of Transforming Growth Factor-β1 Single Nucleotide Polymorphism C-509T with Allergy and Immunological Activities

Background: A single nucleotide polymorphism (SNP) C-509T within the tumor growth factor β1 (TGFβ1) gene has been associated with atopic asthma and asthma severity. To further understand the mechanisms involved, the association of C-509T with allergy, T-lymphocyte proliferation and plasma TGFβ1 concentration has been explored in a case-control study with allergic and non-allergic subjects. Methods: The recruited subjects including allergic (n = 38) and nonallergic (n = 25) participants have been genotyped for C-509T using allele discrimination assay. Association of C-509T with allergy status was examined using logistic regression analysis in both dominant and recessive models. Association of C-509T with T-cell proliferation in control and antigen-stimulated peripheral blood mononuclear cells (PBMCs), plasma TGFβ1 and total IgE level were tested by multiple regression analysis. Results: Individuals with homozygous mutant TT genotype showed a higher risk of allergy (TT: odds ratio = 5.099, 95% confidence limit: 1.355–19.190, p = 0.016) after covariates were adjusted. A trend to increased plasma TGFβ1 in subjects with T allele has been discovered. In the meantime, the T allele is associated with lower T cell proliferation in controls and maximum response to above antigens. A low T-cell proliferation is correlated with higher plasma TGFβ1 concentration (p < 0.01). The in vitro studies confirmed the suppressing effect of TGFβ1 on T-cell proliferation at physiological range. A significant inhibitory effect on IL-4 production was also observed. Conclusions: A C to T base change in TGFβ1 SNP C-509T has been associated with a higher risk of allergy. The mechanisms are not clear. Elevated TGFβ1 levels associated with the C-509T polymorphism might suppress immune activation as well as Th2 cytokine production.

Human CD1-restricted T cell recognition of lipids from pollens

Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex–related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4+ T cell receptor for antigen (TCR)αβ+, some CD4−CD8− TCRγδ+, but rarely Vα24i + natural killer–T cells, and required CD1a+ and CD1d+ antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-γ, in some cases IL-10 and transforming growth factor-β, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis–exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids.

Cytokine/Chemokine HLDA8 Workshop panel report: Analysis of receptors on lymphocytes from cord blood, normal and asthmatic subjects, and HIV positive patients

Chemokines and cytokines are involved in many processes, both physiological and pathological, particularly the recruitment, differentiation, activation, and proliferation of immune cells taking part in ontogenesis, inflammation, and cancer. It was assumed that chemokines and cytokines receptors are expressed in a regulated manner by human lymphocytes during ontogeny and later on, under the environmental stimulation of antigens they contribute to organogenesis, angiogenesis, and tissue remodeling, as well as modulating leukocyte effector functions. Using monoclonal antibodies classified by the Cytokine/Chemokine section of the 8th International Workshop on Human Leukocyte Differentiation Antigens, we analyzed human lymphocytes in blood samples drawn from the umbilical cord, normal adults, allergic and non-allergic asthma patients, HIV infected, and AIDS positive subjects. The main differences noted between adult and cord blood lymphocytes were related to CCR7 and CXCR4 receptors, which were more strongly expressed on cord blood lymphocytes, confirming the important role of these chemokines during development of the immune system. As with the HIV, CXCR4, and CCR5 co-receptors, we found no differences in CXCR4 expression between HIV and AIDS patients. However CCR5 was more strongly expressed in AIDS patients, which is likely to be associated with the evolution of disease. Further studies are needed to gain a better understanding of the functions of these molecules in the underlying pathogenesis of many diseases and to probe the use of the chemokine receptors as targets for therapeutic intervention.

Component‐resolved in vitro diagnosis in carrot allergy: Does the use of recombinant carrot allergens improve the reliability of the diagnostic procedure?

In Europe, pollen-related food allergy is the most frequent form of food allergy in adults. Reliability of current diagnostic procedures, however, is poor and therapeutic options are not available. In the present study, we created a panel of recombinant allergens from carrot and evaluated its potential in component-resolved in vitro diagnosis of carrot allergy. Recombinant (r) Dau c 1.0104, Dau c 1.0201 and Dau c 4 were cloned by a polymerase chain reaction strategy, expressed in Escherichia coli and purified. Carrot lipid transfer protein (LTP) was expressed in the yeast Pichia pastoris. Sera from 40 carrot-allergic patients were investigated. Twenty-one birch pollen-allergic subjects with negative open provocation to carrot and 20 non-allergic subjects were included as controls. IgE binding to recombinant allergens as well as to cross-reactive carbohydrate determinants (CCD) was measured by ELISA. Cross-reactivity between Dau c 1 isoforms and Bet v 1 was assayed by ELISA inhibition. Biological activity of the recombinant carrot allergens was assessed by histamine release assay and peripheral blood mononuclear cells stimulation. Ninety-eight percent of the carrot-allergic patients were positive to at least one recombinant allergen; 98% reacted to rDau c 1.0104, 65% to rDau c 1.0201, 38% to rDau c 4 and 20% had IgE against CCD. Specificity using the recombinant allergens was high when compared with non-allergic controls, but low compared with birch-sensitized subjects without carrot allergy. Sensitization to Dau c 1.0201, however, proved to be highly specific for clinically relevant sensitization. Inhibition assays indicated the absence of LTP in carrot root extract, and epitope diversity between Dau c 1.0104, Dau c 1.0201 and Bet v 1. Our panel of recombinant allergens from carrot can provide a standardized tool for in vitro diagnosis of carrot allergy, and for epitope studies.

Biased use of V H5 IgE-positive B cells in the nasal mucosa in allergic rhinitis

IgE antibody-producing B cells are enriched in the nasal mucosa in patients with allergic rhinitis because of local class switching to IgE. The expressed IgE VH genes also undergo somatic hypermutation in situ to generate clonal families. The antigenic driving force behind these events is unknown. To examine the possible involvement of a superantigen in allergic rhinitis, we compared the variable (VH) gene use and patterns of somatic mutation in the expressed IgE heavy-chain genes in nasal biopsy specimens and blood from allergic patients and the IgA VH use in the same biopsy specimens and also those from nonallergic controls. We extracted mRNA from the nasal biopsy specimens of 13 patients and 4 nonallergic control subjects and PBMCs from 7 allergic patients. IgE and IgA VH regions were RT-PCR amplified, and the DNA sequences were compared with those of control subjects. We constructed a molecular model of VH5 to locate amino acids of interest. We observed a significantly increased frequency of IgE and IgA VH5 transcripts in the nasal mucosa of the allergic patients compared with the normal PBMC repertoire. Within IgE and IgA VH5 sequences in the nasal mucosa, the distribution of replacement amino acids was skewed toward the immunoglobulin framework regions. Three of 4 nonintrinsic hotspots of mutation identified in the VH5 sequences were in framework region 1. The hotspots and a conserved VH5-specific framework residue form a tight cluster on the surface of VH5. Our results provide evidence for the activity of a superantigen in the nasal mucosa in patients with allergic rhinitis.