Human Allergic Responses Research Articles

Modification of the human allergic immune response by allergen-DNA–transfected dendritic cells in vitro

Background Atopic-allergic diseases are characterized by T H2-dominated immune responses, resulting in IgE production. DNA-based immunotherapies have been shown to shift the immune response toward a T H1-type response in animal models. Objective The aim of the study was to analyze whether dendritic cells (DCs) transfected with allergen-DNA conjugates are able to stimulate human autologous CD4 + T cells, CD8 + T cells, or both from atopic individuals to produce T H1 cytokines instead of T H2 cytokines. Methods For this purpose, human mature DCs from atopic donors were transfected with an adenovirus encoding the allergen Phl p 1. Autologous CD4 + and CD8 + T cells were stimulated with these transfected DCs, and proliferation and cytokine production were measured. Results By using an adenoviral vector, a transfection rate of 92% could be achieved. The proliferative response of CD4 + T cells stimulated with autologous transfected DCs was concentration dependent and almost as high as that of T cells stimulated with mature allergen-pulsed DCs. The proliferation of CD8 + T cells stimulated with transfected DCs, however, was higher than that of cells stimulated with allergen-pulsed DCs. The cytokine pattern showed a shift toward a T H1 immune response compared with T cells stimulated with allergen-pulsed DCs. Conclusions Human DCs can be transfected with allergen-DNA conjugates very efficiently by using an adenoviral vector yielding DCs with high T-cell stimulatory capacities, directing the atopic-allergic immune response from T H2 dominance toward T H1 dominance.

Immunoassays of soy proteins.

Proteins of soybeans (Glycine max) are widely used in animal and human nutrition. In addition to the bulk of the seed storage proteins, which are classified as albumins and globulins, approximately 6% of soybean proteins are classified as inhibitors of trypsin and chymotrypsin and approximately 0.5% are sugar-binding lectins. The two major classes of inhibitors are the Kunitz trypsin inhibitor, which inhibits trypsin, and the Bowman-Birk inhibitor (BBI), which inhibits both trypsin and chymotrypsin. Unless removed or inactivated, these inhibitors and lectins can impair the nutritional quality and safety of soy-based diets. On the other hand, several studies suggest that BBI can also function as an anticarcinogen, possibly through interaction with a cellular serine protease. Good-quality soybean proteins contribute to the nutritional value of many specialty foods including infant soy formulas and milk replacers for calves, and provide texture to many processed foods. However, they may also induce occasional allergic responses in humans. This paper outlines immunoassays developed to analyze for soy proteins in different soybean lines, in processed foods, and in nonsoy foods fortified with soy proteins. An assessment of the current status of immunoassays, especially of enzyme-linked immunosorbent assays for soybean inhibitors of digestive enzymes, soy globulins, and soy lectins, demonstrates the usefulness of these methods in plant and food sciences and in medicine.

IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells.

In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor alpha subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13's effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.

Inhibition of human allergic T-cell responses by IL-10–treated dendritic cells: Differences from hydrocortisone-treated dendritic cells

Background: Dendritic cells (DCs) are able to induce human allergic TH1 responses as well as TH2 responses. Objective: In this study, we examined the effect of antiinflammatory agents such as IL-10 and hydrocortisone (HC) on the accessory function of DCs and the resulting T-cell response, especially that of TH2 cells. Methods: Naive and memory CD4+ T cells from atopic donors were stimulated with autologous allergen-pulsed DCs generated from CD14+ monocytes by culture with GM-CSF/IL-4 and fully matured with IL-1β, TNF-α, and PGE2 in the presence or absence of IL-10 or HC. Results: IL-10–treated DCs and, to a lesser extent, HC-treated DCs showed a decreased expression of MHC II molecules, the costimulatory molecule CD86, and the DC-specific marker CD83, as well as a strongly reduced IL-12 secretion. Consequently, T-cell proliferation was reduced after stimulation with IL-10– or HC-treated DCs alike. However, pretreatment of DCs with IL-10 inhibited the production of TH1 and TH2 cytokines by T cells, whereas HC-treated DCs inhibited production of IFN-γ but induced an increased release of IL-4 and no change in IL-5. Both effects were long-lasting; cytokine production remained low (which was due not to enhanced apoptosis but to functional hyporesponsiveness) or even increased after restimulation with fully matured DCs. Conclusion: These data indicate that IL-10– or HC-treated DCs differ in their ability to influence human allergic T-cell responses. This has major implications for therapeutic strategies aiming at the downregulation of proallergic TH2 responses. (J Allergy Clin Immunol 2001;108:242-9.)

Prevalence of arthropod antibodies in Korean patients with allergic rhinitis.

Arthropod antigens are main causative agents which induce allergic responses in humans. However, little information is known about the prevalence of specific arthropod allergens in Koreans with allergic diseases. The current study was designed to determine the positive rates of arthropod antibodies by the Korean inhalant panel of MAST-CLA. One hundred sixty patients, who were diagnosed with allergic rhinitis from an out-patient center at the Soonchunhyang University Chunan Hospital, were studied between August 1998 to July 2000. The overall positive rate, at least more than one specific antibody of arthropods such as Dermatophagoides farinae (Df), Dermatophagoides pteronyssinus (Dp), and cockroach mix (Cm), was 46.9%. Each positive rate of Df, Dp, and Cm was 45.0%, 43.1%, and 8.8%, respectively. A significant agreement among arthropod allergens was observed (Df and Dp: 95.6%, Kappa = 0.911, P < 0.001). Our data supported the fact that arthropods were the most common allergens in Korean patients with allergic rhinitis; however, the MAST-CLA should be modified to increase specificity of arthropod allergens.

Allergen-specific production of interferon-gamma by peripheral blood mononuclear cells and CD8 T cells in allergic disease and following immunotherapy.

CD8 T cells are important immunoregulatory cells in animal models of allergic disease, but their role in human allergic immune responses has not been defined. With the development of novel immunotherapeutic reagents, it is clearly important to ascertain whether CD8 T-cell responses are altered following conventional allergen-specific immunotherapy (SIT) and hence targets for future developments/strategies. To study the allergen-specific cytokine release of freshly isolated CD8 T cells from the blood of separate groups of house dust mite- (HDM) allergic patients, patients post-SIT and control nonatopic donors. CD8 T cells were isolated by positive selection with immunomagnetic beads and cultured with the affinity purified major mite allergen Der p 1 or with different mitogens, using irradiated autologous peripheral blood mononuclear cells as antigen-presenting cells (APCs). Supernatants were collected at a number of time points and assayed by ELISA for the cytokines interleukin (IL) -4, IL-5 and interferon-gamma (IFNgamma). CD8 T cells stimulated with Der p 1 produced significant quantities of IFNgamma with cells from HDM-allergic subjects releasing considerably more IFNgamma than cells from nonatopic subjects, an average of 804 +/- 283 pg/mL of supernatant compared with 30.2 +/- 18.8 pg/mL (P = 0.006). The cytokine was detected in cultures of 16/17 of the allergic subjects and 4/7 of the nonatopic. CD8 T cells from 6/10 patients who had received HDM-SIT released IFNgamma at an average of 363 +/- 202 pg/mL, which was less than the allergic group but still higher than the nonatopic (P = 0.05). Equivalent levels of IFNgamma were detected when the cells were stimulated with the mitogen PHA and this was the same in all groups. Reliable allergen-specific release of significant quantities of IL-4 or IL-5 was not detected from CD8 T cells. Allergen-specific IFNgamma is produced at far greater levels from CD8 T cells of HDM-sensitive allergic patients than from nonatopic control individuals and this level is reduced following SIT.

A Human-SCID Mouse Model for Allergic Immune Responses: Bacterial Superantigen Enhances Skin Inflammation and Suppresses IgE Production

Chronic skin colonization with Staphylococcus aureus is a well-known feature in atopic dermatitis. The aim of this study was to develop a human-SCID mouse model to analyze the possible role of bacterial superantigens in human allergic immune responses under in vivo conditions. SCID mice were reconstituted with peripheral blood mononuclear cells (between 2 and 9 x 10(7) cells per mouse) from atopic dermatitis patients sensitized to house dust mite allergen (Der p). Total and Der p specific antibody production required the following conditions: (i) injection of Der p; (ii) presence of CD14+ antigen-presenting cells; and (iii) IL-4 as shown by the inhibitory effect of human soluble IL-4 receptor on immunoglobulin E production. This model was used to study the immunomodulatory effects of the superantigen staphylococcal enterotoxin B in comparison with Der p. In intraperitoneally reconstituted human-SCID mice, topical treatment was ineffective in inducing skin inflammation. Therefore, additionally to intraperitoneal transfer, peripheral blood mononuclear cells from atopic donors were also injected intradermally. Such reconstituted SCID mice were then exposed via the skin to either Der p, staphylococcal enterotoxin B, or a combination of both. Maximal effects on epidermal inflammation and dermal T cell infiltration were obtained with staphylococcal enterotoxin B and Der p. Staphylococcal enterotoxin B alone was less effective and Der p only stimulated dermal T cell infiltration. These findings support the hypothesis that bacterial superantigens can act as trigger factors in allergic skin inflammation.

Hookworm-induced Hypersensitivity: A Response

Since the recent discovery that the ubiquitous nematode parasite of dogs, Ancylostoma caninum, causes eosinophilic enteritis (EE) in people, research into zoonotic hookworm infections is enjoying a `rebirth'. The association between A. caninum (a parasite that is endemic in dogs throughout most of the tropic and sub-tropic regions of the world) and enteric disease in people, seemed to have escaped notice up until the late 1980s[1]. In this issue of Parasitology Today, John Croese has reviewed the subject in some detail, but appears to have omitted a key issue in our understanding (or lack thereof) of hookworm-mediated allergy. When considering the intense nature of the human allergic response to canine hookworms, Croese refers to A. caninum as a parasite that is poorly `adapted' to growth and survival in humans. His explanation centres on canine hookworms inducing allergy in people (characterized by IgE and eosinophilia) by inoculating into the mucosa, antigens that are either subtly different from, or not found at all, in anthropophilic hookworms leading to a lack of tolerance of this parasite. While at a glance this may appear to be a valid argument, Croese fails to mention crucial findings described in one of his references[2], where Maxwell and colleagues described the clinical and immunologic response of five volunteers to low dose infections with 50 infective larvae of the anthropophilic hookworm, Necator americanus. In this study, four of the patients complained of abdominal pain; the fifth, however, had such severe intestinal pain that the infection had to be treated and the experiment terminated. An interesting feature of this one patient's infection was an absence of eggs in the faeces, a notable feature of human enteric infection with A. caninum. Maxwell claimed that this finding was surprising based on the description by others of an association between gastrointestinal pain and heavy worm burdens[3, 4]. Another piece of evidence that does not support Croese's hypotheses actually comes from his own research; of nine patients where a solitary adult A. caninum was recovered, one had no gastrointestinal symptoms and underwent colonoscopy for anxiety about bowel cancer[5]; in addition, this patient mounted a strong IgG and IgE response to secretory antigens of the parasite. This finding suggests that a far greater proportion of the population may be asymptomatically infected with canine hookworms than first realized, lending support to the notion that A. caninum is no more likely to induce gut hypersensitivity in people than anthropophilic hookworms.

Immunologic Investigations of T-Cell Regulation of Human IgE Antibody Secretion and Allergic Responses

The pathophysiology of allergic disease is multifactorial, involving an intricate network of interactions among cells, mediators, and cytokines. Substantial progress has been made in defining the role of antigen-specific T cells and cytokines in the regulation of immunoglobulin E (IgE) synthesis and the atopic diseases. The development of antigen-specific T-cell lines and clones has facilitated efforts to characterize human T-cell subsets and their cytokine repertoires. Molecular methods currently available include techniques for the quantitative analysis of cytokine gene expression and secretion from activated T cellsex vivoas well as in tissues. The availability of these newly developed techniques has become essential to the investigation of the pharmacologic regulation of T cells and cytokines bothin vitroandin vivo.Future investigations will contribute to our understanding of the differential regulation of T-cell subsets and their relationships to allergic diseases, ultimately leading to a better understanding of the molecular pathogenesis of allergic diseases and the design of more effective therapeutic interventions.

INTERLEUKIN (IL)-13 EXPRESSION AT THE SITES OF ALLERGEN CHALLENGE IN PATIENTS WITH ASTHMA

IL-13 is in the same gene cluster as IL-4, IL-5, and GM-CSF and is increased after allergen exposure. Other studies have shown IL-13 shares function with IL-4 inducing immunoglobulin E synthesis. A number of cytokines are relevant to the human allergic response.

The skin as a model to study the pathogenesis of IgE-mediated acute and late-phase responses

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Mechanisms of the Human Allergic Response: Clinical Implications

During the past decade, there have been enormous strides made in our understanding of the mechanisms underlying the human allergic response. In particular, interleukin-4 and interleukin-5 play a critical role in this process. This article details the understanding of the mechanisms underlying the regulation of allergic response, which is critical to the development of new treatment of allergic diseases.

Antigenic analyses of Sugi basic protein by monoclonal antibodies: I. Distribution and characterization of B-cell-tropic epitopes of Cry j I molecules.

Using 23 monoclonal antibodies raised against Sugi basic protein (SBP, major allergen of Japanese cedar pollen), composed of Cry j I and Cry j II, analyses of B-cell-tropic epitopes of Cry j I were performed. The following results were obtained. (1) As far as the mAbs were used, no major cross-reactive determinants were detected between Cry j I and Cry j II molecules. (2) 21 of the 23 mAbs were specific for Cry j I, and the anti-Cry j II mAbs were classified into four groups by their fine specificities, suggesting that Cry j I bears at least four antigenic determinant regions. (3) Cry j I molecules were found to take a monomeric form in solution and to display no repeating antigenic epitopes on their surfaces. (4) Some of the determinants seemed to be located in the interior of a Cry j I molecule, and when the Ag is coated on a plastic plate, the determinants become exposed on its surface. (5) Binding of human IgE antibodies to Cry j I and Cry j II was blocked by some of the obtained mAbs, suggesting that these epitopes recognized by the mAbs might have an important role in human allergic response against the cedar pollen.

EDX microanalysis of cellular components in the pollen of an angiosperm, Plumbago zeylanica

Pollen grains in flowering plants recognize compatible stigmas and transmit the sperm cells that they contain to the egg. The pollen grains bear specific proteins and lipids on their outer walls, which are the principal sources of both pollen-stigma recognition and human allergic response; they also contain the organelles required to form the pollen tube and the two sperm cells that participate in double fertilization. Although limited information is available about x-ray microanalysis of pollen grains, no information is available about the cellular distribution of elements.The contents of pollen of Plumbago zeylanica L. were burst in double-distilled water onto formvar-coated Cu slot grids ( 1 × 2 mm) and air-dried at room temperature. Grids were then examined at 40 kV in a JEOL 2000 STEM (LaB6 e' source) equipped with a high-angle 10 mm2 Kevex Quantum detector and Delta analyzer. Spectra were collected for 100-500 livetime sec; digital x-ray maps were collected at resolutions of up to 128 × 256 (dwell time: 30 msec [10-15 collections], 15-30% deadtime at ca. 1,000 cps, adjusted using the free lens control).

IgE Response and Its Regulation in Allergic Diseases

The distinguishing feature of the allergic person is his or her elevation of serum IgE. This propensity to develop a sustained IgE response is determined genetically. The biologic effects of IgE are mediated via Fc receptors (Fc epsilon R) present on mast cells and basophils (Fc epsilon R type 1) and subpopulations of monocytes, macrophages, eosinophils, and platelets (Fc epsilon R type 2). Interaction of allergen with IgE on these cells results in receptor "bridging" and the release of histamine and other inflammatory mediators. Fc epsilon R type 2 on lymphocytes and monocytes are upregulated in atopic disease and may play a role in the allergic inflammatory reaction. The activation of B cells to synthesize IgE requires several stages (see Fig. 2). T cells play an important role in the regulation of IgE synthesis. In vitro activation of resting B cells to synthesize IgE requires direct cellular interaction with T cells or the presence of IL4 for activation. The latter effect is inhibited by alpha-interferon. Preactivated B cells are influenced in an isotype-specific manner by T-cell-derived IgE binding factors (IgE-BF), which may act as IgE-potentiating or IgE-suppressive factors, depending on their degree of glycosylation. The regulation of IgE synthesis is an important area of investigation. It provides us with an understanding of the basis of the human allergic response and ultimately may provide the basis for novel strategies in the treatment of allergic diseases.

Mediators of Immediate Hypersensitivity Reactions

PATIENTS with immediate hypersensitivity reactions are commonly encountered by physicians in both primary care and various specialty practices. Such patients present a broad spectrum of illnesses, ranging in severity from mild urticaria or seasonal rhinitis to fatal anaphylactic shock. For many years the pathophysiology of these illnesses has been known to involve the release of inflammatory mediators by mast cells and basophils. Over the past 10 years, considerable progress has been made in elucidating the structure and function of these mediators. Thus, many of the signs and symptoms of allergic responses in humans can now be attributed to specific mast-cell . . .

(5Z,13E)-(15S)-9 alpha,11 beta,15-trihydroxyprosta-5,13-dien-1-oic acid (9 alpha,11 beta-prostaglandin F2): formation and metabolism by human lung and contractile effects on human bronchial smooth muscle.

Prostaglandin D2 (PGD2) was recently found to be stereospecifically converted to the compound (5Z,13E)-(15S)-9 alpha,11 beta,15-trihydroxyprosta-5,13-dien-1-oic acid (9 alpha,11 beta-PGF2) by a human liver cytosolic NADPH-dependent 11-ketoreductase enzyme. Because PGD2 is a potent bronchoconstrictor and is released into bronchoalveolar lavage fluid after allergen stimulation in patients with allergic asthma, the ability of human lung to metabolize PGD2 to 9 alpha,11 beta-PGF2 and the contractile effects of 9 alpha,11 beta-PGF2 on human bronchial smooth muscle were investigated. The 100,000 X g supernatant of human lung converted PGD2 in the presence of an NADPH-generating system stereospecifically to 9 alpha,11 beta-PGF2 at a rate of 3.46 +/- 0.94 pmol per min per mg of protein. 9 alpha,11 beta-PGF2 was found to contract human bronchial rings in a dose-dependent fashion with a potency virtually identical with that of both PGD2 and PGF2 alpha, known potent bronchial constrictors. PGD2 was found to be a very poor substrate for human lung 15-hydroxyprostaglandin dehydrogenases and to be preferentially metabolized by lung to 9 alpha,11 beta-PGF2. 9 alpha,11 beta-PGF2 was also found to be a very poor substrate for the lung 15-hydroxyprostaglandin dehydrogenases. Thus, once formed, 9 alpha,11 beta-PGF2 would not be expected to be rapidly inactivated in situ by these metabolic enzymes. These results suggest that 9 alpha,11 beta-PGF2 may participate along with other putative mediators in the pulmonary allergic response in humans.

Regulation of the human allergic response.

Over the last several years our laboratory has been engaged in the in vitro study of the regulation of IgE synthesis. We have found that polyclonal B cell activators fail to induce IgE synthesis in human B cells. Alloreactive T cell helper clones induced synthesis in B cells only under conditions of interaction and in B cells from allergic donors under cognate conditions of bystander stimulation as well. Isotype-specific regulation of the IgE response was mediated in the secretion of IgE-binding factors by T cells suppressing Fc receptors for IgE. Finally, IgE immune complexes in sera from patients with hyper-IgE states were shown to downregulate T cell proliferation to antigen and to stimulate monocytes to resorb 45Ca-labelled bone and to release prostaglandins. The implications of these in vitro findings for disease states in which IgE is elevated are discussed.

Intraepithelial migration of mucosal mast cells in hay fever: ultrastructural observations.

Evidence has been presented suggesting that a migration of nasal mast cells from the mucosal connective tissue stroma into the epithelium is part of the mucosal response in birch pollen allergy. In a previous study, the identification of these intraepithelial cells as tissue mast cells rather than blood basophils was based on light microscopical morphology and histochemistry. We have now studied the ultrastructure of these cells in mucosal biopsies taken before and during the birch pollen season. Intraepithelial cells with basophil or metachromatic granules were only observed in biopsies taken during the season. Some of these cells had the ultrastructural appearance of tissue mast cells, including cytoplasmic lipid droplets and a granular substructure composed of multilamellar arrays and scrolls, serving to distinguish human mast cells from blood basophils. The ultrastructural traits of the remaining cells were heterogeneous, some reminiscent of human blood basophils, others of globule leucocytes of other species, but entirely typical blood basophils could not be identified. The results thus support our previous suggestion that a migration of mucosal mast cells from the connective tissue stroma into the epithelium is part of the human allergic mucosal response. It cannot be determined whether the ultrastructural heterogeneity of these cells is the result of an adaptation to the intraepithelial environment of one single mast cell type or to the existence of an ultrastructurally distinct mucosal mast cell.

Correlations of in vivo mediator release with late cutaneous allergic responses in humans: I. Kinetics of histamine release

To evaluate the contribution of mast cell-derived mediators in the late cutaneous allergic response, the duration and quantity of antigen-induced histamine release was compared to the intensity of the antigen-induced skin reactions in atopic volunteers. Chambers containing either pollen extract or buffer were appended to denuded blister bases for 1 hr and were replaced hourly with buffer for 3 additional hr. These were compared to the extinction dilution skin test titer and to the mean diameters of the 20-minute wheal and induration at 6 and 8 hr after intradermal injection of antigen. Chamber-fluid histamine levels were significantly higher at antigen than at buffer sites throughout the 4 hr. The hourly histamine levels correlated with the size of the induration at 6 and 8 hr but not with the wheal size or skin test titer. We conclude that (1) histamine is released for at least 4 hr at skin sites of antigen challenge as a consequence of prolonged release either from individual or sequentially activated mast cells, and (2) the quantity of histamine released correlates with the intensity of the late-phase skin response. We hypothesize that histamine might be a marker for prolonged release from the mast cell of other mediators that are responsible for the late-phase response.