Kimiyuki Saito

Allergenic epitopes of ovalbumin (OVA) in patients with hen's egg allergy : inhibition of basophil histamine release by haptenic ovalbumin peptide

We studied allergenic determinants that induce hypersensitivity to OVA, the major allergen in egg allergy, using immunoblot and histamine release assays. Immunoblot analysis demonstrated a part of the OVA epitope was in the C‐terminal region comprising residues 347‐385 (OVA347‐385). Histamine was released from basophils of a patient with egg allergy upon stimulation with the OVA fragment corresponding to OVA347–385. Furthermore, detailed epitope mapping using overlapping peptides (residues 347‐366, OVA‐A; residues 357‐376, OVA‐B; and residues 367‐385, OVA‐C) in the OVA 347‐385 region was carried out using the histamine release assay. In order for histamine release from basophils to occur, the allergen must possess two or more allergenic determinants located on the protein molecule at distances that would be equivalent to the distances between IgE molecules on the membrane surface. These results suggest that there are at least two epitopes that bind IgE antibodies on each OVA peptide. In addition, one epitope that binds IgE antibodies in two patients appears to reside in the haptenic peptide OVA357‐366 (OVA‐B1). The histamine release from basophils stimulated by OVA‐B was completely inhibited by OVA‐B1 in one of these patients. Similarly, OVA‐B1 inhibited the histamine release produced by OVA‐A in the other by more than 40%. These results suggest that haptenic synthetic peptides could regulate the allergic reaction in the effector phase if common epitope(s) recognized by IgE antibodies in the patients with egg allergy can be found. These are the first studies that provide an antigen‐specific approach to inhibiting histamine release from basophils by a haptenic peptide recognized by IgE antibodies in an allergic disorder.

Specificities of IgE, IgG and IgA Antibodies to Ovalbumin

We studied the binding activities of IgE, IgG and IgA antibodies in patients with allergy to hens egg white against two different ovalbumin (OVA) preparations, which were physically or chemically denatured OVA and enzyme-digested OVA fragments. The binding activities of IgE antibodies to these OVA preparations with those of IgG or IgA antibodies were compared. It was found that the binding activities of IgE antibodies to denatured OVA by treatment with dithiothreitol, urea or hydrochloric acid were similar to those of IgG or IgA antibodies. In contrast, the binding activities of IgE antibodies to heat-denatured OVA or by treatment with sodium hydroxide at pH 11.0 were different from those of IgG or IgA antibodies to these denatured OVA. Furthermore, we found that the binding activities of anti-OVA antibodies in sera from patients with allergy to hens egg white against fragmented OVA were different between IgE antibodies and IgG or IgA antibodies. Thus, it can be concluded that IgE antibodies to OVA in sera from patients with allergy to egg white differ from IgG or IgA antibodies in respect to binding activities against different preparations of denatured or fragmented OVA, probably due to differences in fine specificities of these antibodies against OVA.

Anti-thyroid peroxidase antibodies in sera from healthy subjects and from patients with chronic thyroiditis: differences in the ability to inhibit thyroid peroxidase activities

A significant percentage (6.4%) of healthy subjects was found to contain anti‐thyroid peroxidase (TPO) antibodies in their sera. However, in contrast with IgG from sera of patients with chronic thyroiditis, IgG from sera of healthy subjects did not inhibit TPO activities both in guaiacol and iodide assays. In addition, anti‐TPO antibodies from healthy subjects did not block the inhibition of enzyme activities by anti‐TPO antibodies from patients. These findings suggest that anti‐TPO antibodies from healthy subjects do not bind to the epitopes relating to substrate‐combining sites of TPO. Thus, the specificities of anti‐TPO antibodies in healthy subjects may differ from those in cases of chronic thyroiditis.

Anti-thyroglobulin autoantibodies in sera from patients with chronic thyroiditis and from healthy subjects: differences in cross-reactivity with thyroid peroxidase

A significant portion (about 12·7%) of healthy subjects was found to contain anti‐thyroglobulin (anti‐Tg) antibodies in their sera. We compared the binding activities of these antibodies and of anti‐Tg autoantibodies from sera of patients with chronic thyroiditis with human thyroid peroxidase (TPO). The results obtained by ELISA indicated that out of 10 healthy subjects with anti‐Tg antibodies, only four had anti‐Tg antibodies capable of binding to TPO, whereas anti‐Tg autoantibodies from almost all patients with chronic thyroiditis possessed high binding activities to TPO. By use of the immunoprecipitation method, it was also shown that although all anti‐Tg autoantibodies from patients precipitated TPO, a majority of anti‐Tg antibodies from healthy subjects could not precipitate TPO. Such findings cannot be ascribed to the differences in levels of anti‐Tg autoantibodies and anti‐TPO autoantibodies in sera and the differences in avidities of anti‐Tg antibodies in sera between healthy subjects and patients with chronic thyroiditis. Thus, it can be concluded that anti‐Tg antibodies from healthy subjects differ from those of patients with chronic thyroiditis with respect to TPO binding, probably due to difference in fine specificities of these anti‐Tg antibodies.

The clinical features of Sjögren's syndrome in Japanese children

Sjögrens syndrome (SS) is thought to be uncommon in children. An epidemiological study to describe the clinical features distinguishing SS in Japanese children was performed by sending questionnaires to hospitals. A total of 61 cases of SS were reported from 1290 hospitals. The diagnosis of SS was based on histopathological changes and/or sialographic changes in the salivary glands. Forty‐two cases had primary SS and 19 were secondary SS with other autoimmune disorders. Fourteen cases (65%) of secondary SS were associated with systemic lupus erythematosus. In primary SS, the initial symptoms were systemic manifestations (fever, exanthema, arthralgia, etc) except for sicca symptoms. In laboratory studies, antinuclear antibodies, elevated serum IgG, rheumatoid factor, anti‐Ro/SS‐A antibodies and anti‐La/SS‐B antibodies were frequently observed.

Diminished interferon‐gamma (IFN‐γ) production by bacterial antigen‐specific T cells in atopic patients

In this study, we established and studied cytokine production of T cell lines (TCL) specific to either a purified protein derivative of Mycobacterium tuberculosis (PPD) or Dermatophagoides farinae (Df) from atopic patients and non‐atopic healthy subjects. IFN‐γ was detected in the culture supernatants of all of 36 PPD‐specific TCL established from healthy controls, whereas only 24 of 38 PPD‐specific TCL from patients produced IFN‐γ. Furthermore, the amounts of IFN‐γ produced by PPD‐specific TCL from patients were significantly lower than those from healthy controls. No IL‐4 was detected in any PPD‐specific TCL from either healthy controls or atopic patients. The amounts of IL‐4 production from Df‐specific TCL from atopic patients were much higher than from healthy controls, while few TCL produced IFN‐γ. These results suggest that the skewing to the Th2‐type T cell response in atopic patients is a response not only to allergens, but also to bacterial antigens, compared with non‐atopic subjects. Activation of PPD‐specific TCL from patients with calcium ionophore A23187 plus phorbol myristate acetate resulted in much higher IFN‐γ production than in TCL established from healthy controls, indicating that the low production of IFN‐γ by PPD‐specific T cells from atopic patients is not due to an intrinsic T cell defect but to some regulatory mechanisms.

Detection of House Dust Mite (HDM)-Specific IgE Antibodies on Nasal Mast Cells from Asthmatic Patients Whose Skin Prick Test and RAST Are Negative for HDM

Mast cells are found in nasal smears of pediatric patients with perennial bronchial asthma whose skin prick test and radioallergosorbent test (RAST) are negative for inhalant allergens. IgE antibodies were demonstrated on these mast cells by monoclonal anti-human IgE antibodies, whereas IgG antibodies were not detected by monoclonal anti-IgG antibodies. In order to pursue the causative allergen for asthma in these patients, binding potential between IgE antibodies on nasal mast cells and house dust mite (HDM), the most prevalent aeroallergen, was examined by an immunochemical technique. Out of 9 patients whose skin prick test and RAST were negative for HDM, 7 were found to have HDM-specific IgE antibodies on their nasal smear mast cells. None of these 7 patients had IgE antibodies to cedar pollen, a negative control aero-allergen, on their mast cells. Specific binding of HDM on the mast cells was further confirmed by the fact that nasal mast cells from patients with egg allergy bound egg white but not HDM on their surface. Preincubation of mast cells with anti-IgE antibodies inhibited binding of HDM on the mast cells, indicating that HDM was bound to surface IgE antibodies on the mast cells. These experiments enabled us to expeditiously identify sensitization to an inhalant allergen, such as HDM, in young asthmatic patients whose allergen cannot be found by conventional laboratory diagnostic procedures.

Thyroglobulin-specific t cell line from a healthy individual does not produce proinflammatory cytokines on antigenic stimulation: an implication for possible fail-safe mechanism to avoid autoimmunity

In order to investigate the regulation of autoimmune response to thyroglobulin (Tg), one of the thyroid autoantigens, we established a Tg-specific T cell line by stimulation of peripheral blood mononuclear cells from a healthy volunteer with Tg and characterized its cytokine production pattern. The Tg-specific T cell line, designated DH5D1, obtained from a limiting dilution culture bore alpha beta T cell receptor and was CD4 and CD45RO positive. Upon stimulation with Tg, DH5D1 secreted little or no titers of IL-2, TNF-alpha, and IFN-gamma, whereas activation with combination of phorbol myristate acetate and calcium ionophore produced measurable levels of these cytokines. These results indicate that the Tg-specific T cell line is not defective in its capacity to produce proinflammatory cytokines and suggest that the inability of cytokine production by autoreactive T cells of healthy individuals is one fail-safe mechanism for preventing aggression of harmful autoimmune response.

Pranlukast reduces asthma exacerbations during autumn especially in 1- to 5-year-old boys

Background Leukotriene receptor antagonists have been used to prevent virus-induced asthma exacerbations in autumn. Its efficacy, however, might differ with age and sex. Objective This study aimed to investigate whether pranlukast added to usual asthma therapy in Japanese children during autumn, season associated with the peak of asthma, reduces asthma exacerbations. It was also evaluated the effect of age and sex on pranlukasts efficacy. Methods A total of 121 asthmatic children aged 1 to 14 years were randomly assigned to receive regular pranlukast or not according to sex, and were divided in 2 age groups, 1–5 years and 6–14 years. The primary outcome was total asthma score calculated during 8 weeks by using a sticker calendar related to the days in which a child experienced a worsening of asthma symptoms. This open study lasted 60 days from September 15 to November 14, 2007. Results Significant differences in pranlukast efficacy were observed between sex and age groups. Boys aged 1 to 5 years had the lower total asthma score at 8 weeks (p = 0.002), and experienced fewer cold episodes (p = 0.007). There were no significant differences between pranlukast and control group in total asthma score at 8 weeks (p = 0.35), and in the days in which a child experienced a worsening of asthma symptoms (p = 0.67). Conclusion There was a substantial benefit of adding pranlukast to usual therapy in asthmatic children, especially in boys aged 1 to 5 years, during autumn season.