Toshio Katsunuma

Evaluation of the staphylococcal exotoxins and their specific IgE in childhood atopic dermatitis

BACKGROUND Superantigenic exotoxins produced by Staphylococcus aureus and their specific IgE antibodies are thought to be important precipitating factors of atopic dermatitis (AD), but there are few reports evaluating these 2 factors at the same time. OBJECTIVE We examined whether the presence of the exotoxins sampled from the skin of patients with AD and the levels of anti-exotoxin IgE antibodies in their sera correlated with their severity of AD. METHODS Patients with mild-to-severe AD, 1 to 22 years of age, were evaluated by using Leicesters scoring system. Specific IgE antibodies against these exotoxins were determined by using ELISA. S aureus was isolated from 3 different areas of the skin. We examined whether the exotoxin (staphylococcal enterotoxin [SE]A, SEB, SEC, SED, and toxic shock syndrome toxin-1) could be detected. RESULTS The levels of SEB-specific IgE were correlated with the severity of AD. Five of 6 patients having very high SEB-specific IgE antibody titers were under 6 years of age, and SEB was most frequently isolated (41%). There was no difference in severity between patients with or without exotoxin-producing S aureus. The severity of 9 patients who had both exotoxin-producing S aureus on the skin and specific IgE antibody against the same exotoxin in sera was significantly higher than that of the other patients. CONCLUSIONS Anti-SEB IgE titers correlate well with the severity of AD. The presence of exotoxin-producing S aureus may precipitate AD through its specific IgE antibody.

Identification of highly expressed genes in peripheral blood T cells from patients with atopic dermatitis.

Background: Analysis of genes that are differentially expressed in patients with atopic dermatitis (AD) and normal individuals will provide important information on the underlying molecular pathogenetic mechanisms of AD. Methods: Transcript of freshly isolated peripheral blood T cells from 59 individuals were analyzed with a fluorescent differential display (FDD) method. Ninety-two differentially expressed genes were identified in this manner. Additionally, real-time quantitative RT-PCR was employed to investigate the expression of the FDD-selected genes and also genes related to T cell function. Results: A number of genes, including CC chemokine receptor 4, T cell-specific tyrosine kinase (Emt/Itk), integrin β1, integrin α6, IQGAP1 and MAR/SAR DNA-binding protein (SATB1), were shown to be more highly expressed in patients with moderate and/or severe AD than in controls or patients with mild AD. Because the products of these upregulated genes influence chemotaxis, adhesion, migration and Th2 polarization, it is suggested that in more severe AD, circulating T cells may function differently in this regard. Several other genes, the role of which in T cell function is currently unknown, were also found to be differentially expressed in AD. These included the heat shock protein 40 and vasopressin-activated calcium-mobilizing receptor 1. Conclusion: The upregulated genes identified in this work may serve as useful markers for moderate to severe AD as opposed to normal or mild AD and also as markers indicating progression to more severe AD. Further functional characterization will provide a better understanding of the pathophysiology of circulating T cells in AD.

Peripheral Blood Mononuclear Cells from Patients with Bronchial Asthma Show Impaired Innate Immune Responses to Rhinovirus in vitro

Background: Asthmatic patients have a higher susceptibility to rhinovirus (RV) infection, and impaired IFN-β and IFN-λ production has been demonstrated in bronchial epithelial cells from asthmatic adults upon exposure to RV. However, the mechanisms underlying the increased susceptibility of asthmatic patients to RV infection remain poorly understood. The present study aimed to elucidate the characteristics of the immune responses of asthmatic patients’ peripheral blood mononuclear cells (PBMCs) to RV exposure. Methods: PBMCs obtained from 3 different age groups (2–6 years: young-children group; 7–19 years: youth group; ≧20 years: adult group) of asthmatic patients and nonasthmatic control subjects were stimulated with RV-14 for 72 h. Healthy adults with a history of childhood asthma were also enrolled. The concentrations of IFN-α, IL-6, TNF-α, IL-10, and soluble Fas ligand (sFasL) in the culture supernatants were measured by ELISA. Results: When compared with age-matched control subjects, IFN-α production was significantly lower in the asthmatic youth group. IL-6, TNF-α, IL-10, and sFasL productions were significantly lower in both the asthmatic youth group and the adult group. Such impaired responses were not found in healthy adults with a history of childhood asthma. No significantly different responses were found between the asthmatics and controls in the young-children group, whereas young asthmatic children with persistent wheeze during a 2-year follow-up showed significantly lower IL-10 production than those without wheeze. Conclusions: These results imply the involvement of impaired production of both IFN-α and inflammatory cytokines seen in asthmatic patients’ PBMCs upon exposure to RV in the higher susceptibility of those patients to RV infection.

High-Density Oligonucleotide Array Analysis of mRNA Transcripts in Peripheral Blood Cells of Severe Atopic Dermatitis Patients

Background: There are few laboratory tests for evaluating atopic dermatitis (AD) with the exception of IgE levels or the eosinophil count. We attempted to identify new diagnostic markers by screening the genome-wide expression of transcripts in peripheral blood mononuclear cells (PBMC). Methods: For this study, we enrolled 7 nonatopic healthy volunteers, 5 AD patients who responded well to treatment and 6 who responded poorly. We compared genome-wide transcript levels in PBMC derived from patients with severe AD and healthy volunteers using high-density oligonucleotide arrays (GeneChip, Affymetrix). After the first screening with GeneChip, we employed real-time quantitative PCR to confirm differential expression levels. Results: Screening with GeneChip showed that the levels of a total of 92 transcripts increased at least 3-fold in one population compared to another. After further evaluation of these genes with real-time quantitative PCR, the levels of 4 transcripts were confirmed to be significantly different in PBMC from AD patients compared to controls, namely IFN-γ, TRAIL (TNF-related apoptosis-inducing ligand), ISGF-3 (STAT1) and defensin-1. With the exception of IFN-γ, none of these genes has previously been implicated in AD pathology. Conclusion: These 4 transcripts in PBMC are expected to be useful markers for evaluating AD.

Japanese pediatric guidelines for the treatment and management of bronchial asthma 2008

The fourth version of the Japanese Pediatric Guidelines for the Treatment and Management of Bronchial Asthma 2008 (JPGL 2008) was published by the Japanese Society of Pediatric Allergy and Clinical Immunology in December 2008. In JPGL 2008, the recommendations were revised on the basis of the JPGL 2005. The JPGL 2008 is different to the Global Initiative for Asthma guideline in that it contains the following items: a classification system of asthma severity; recommendations for long‐term management organized by age; a special mention of infantile asthma; and an emphasis on prevention and early intervention. Here we show a summary of the JPGL 2008 revising our previous report concerning JPGL 2005.

Analysis of Highly Expressed Genes in Monocytes from Atopic Dermatitis Patients

Background: Monocytes, macrophages, and antigen-presenting cells (APCs) are key effectors of both innate and acquired immune responses. Such cells have been implicated in the pathogenesis of some inflammatory diseases. Differential gene expression in CD14-positive cells from patients with atopic dermatitis (AD) was studied using real-time quantitative RT-PCR to measure transcription levels of selected genes. Methods: PBMCs were prepared by Ficoll gradient separation from 30 AD patients (the anti-mite-specific IgE RAST score: 0.75 to >100 UA/ml) and 10 healthy adult individuals (the RAST score: <0.34–0.37 UA/ml) and CD14-positive cells were selected. A total of 64 genes was selected for study from groups of genes with different molecular function. Results: Genes involved in MHC class I antigen presentation, such as β2-microglobulin, subunits of an immunoproteasome and ATP-binding cassette transporter TAP2 were expressed at higher levels in the AD patients than in the healthy controls. The genes for Toll-like receptors, CD36 and IFNγ receptor were also upregulated in the AD patients. These genes are involved in MHC class I antigen presentation, recognition of bacterial pathogens and apoptotic cells. Conclusions: The upregulation of genes suggests that circulating monocytes in AD patients may be primed to differentiate into effector cells by conditions associated with AD. The upregulation of genes may prove to be a useful marker for AD.

Association between the Results of the Childhood Asthma Control Test and Objective Parameters in Asthmatic Children

Objective. The Childhood Asthma Control Test (C-ACT), a seven-item, self-administered questionnaire, has been used as a tool to assess the control level in children with asthma. The aim of this study was to determine whether the C-ACT reflects airflow limitation and airway inflammation in addition to clinical manifestations. Methods. Asthmatic children aged 5–11 years who were able to perform the lung function test and fractional exhaled nitric oxide (FeNO) evaluation correctly were recruited during their regular visits. Children and their parents were asked to answer the officially developed Japanese version of the C-ACT. Results. Among 258 children (176 boys, median age 9 years), there was a significant positive correlation between the C-ACT score and the percent predicted forced expiratory volume in 1 s (%FEV1) (r = 0.317, p < .001). The accuracy of the C-ACT for identifying asthmatic subjects with normal lung function (%FEV1 >80%) described as the area under the receiver operating characteristic curve was 71.5% (95% CI = 62.8–80.2%, p < .001), and based on the Youden index the optimal cutoff score was 23 (sensitivity of 78% and specificity of 54%). In contrast, there was no relationship between the C-ACT score and the FeNO value. Conclusions. These results suggest that a cutoff score of 23 for the C-ACT could be useful for identifying children with well-controlled asthma and normal lung function. Further studies are warranted to develop an easy-to-use questionnaire to assess the extent of airway inflammation in children.

Adrenocortical function in patients with severe atopic dermatitis.

BACKGROUND Adrenocortical suppression is a potential complication of the use of topical corticosteroids in patients with atopic dermatitis (AD). OBJECTIVES To determine whether or not the adrenocortical suppression observed in patients with severe AD is a sole result of the application of topical steroids. METHODS A total of 45 patients with severe AD that required hospitalization for treatment were enrolled. These patients were divided into two groups according to the treatment received before hospitalization: group 1 had not used topical corticosteroids for at least three months (n = 17), while group 2 had used topical corticosteroids daily (n = 28). Otherwise, these two groups were matched to clinical characteristics. A rapid ACTH test was performed upon hospital admission. Topical corticosteroids were then applied to both groups. The second ACTH test was performed just before discharge, an average of 23 days after the first test. RESULTS The basal serum cortisol levels as well as the response to ACTH stimulation in the first examination were significantly lower in the AD patients than in the controls (P < .001), although there were no significant differences in the results between groups 1 and 2. The followup study of adrenocortical function at hospital discharge showed that morning basal serum cortisol levels were significantly increased in group 1 (P < .01), despite their topical corticosteroid treatment, while no significant increase or decrease was seen in group 2. CONCLUSION Our findings suggest that the adrenocortical suppression seen in patients with AD may be caused by the percutaneous absorption of topical corticosteroids as well as by other factors related to the disease.

Hypoproteinemia in severe childhood atopic dermatitis: A serious complication

As a complication of atopic dermatitis (AD), the incidence of hypoproteinemia is increasing among infants with severe AD in Japan. It can be a life‐threatening condition owing to hypovolemic shock as a result of hypoproteinemia and vascular infarction as a result of thrombocythemia. However, the pathophysiology of this condition remains unclear. The objectives of the present study were two‐fold. The first objective was to determine the main route of protein loss, i.e. through the damaged skin or the gastrointestinal tract, or as a result of insufficient food intake. The second objective was to identify whether allergy or infection was the cause of severe skin inflammation. Fifteen patients with AD were enrolled who had serum protein levels of 3.2–5.8 g/dl. Specific immunoglobulin E (IgE) and skin test to allergens, stool eosinophils, α1‐antitrypsin clearance, skin Staphylococcus aureus colonization and superantigens (SAgs) produced by the organism, serum SAg‐specific IgE antibodies, serum interleukin (IL)‐5, IL‐6, IL‐12, and interferon‐γ (IFN‐γ) were evaluated. Prominent serous skin discharge was seen in all of the patients and was found to have almost the same protein concentration as serum. Marked thrombocytosis, with a maximum of 1,060 × 103/ml, was seen. Skin culture revealed S. aureus colonization in all patients. SAg‐producing S. aureus were found in 84.6% of the patients. The concentration of serum IL‐5 was significantly increased and correlated well with the blood eosinophil count. Hence, the main route of protein loss was believed to be through damaged skin. The cause of severe inflammation was thought to be a combination of allergic inflammation and skin colonization by SAg‐producing S. aureus. Serum cytokines showed a T helper 2 (Th2) T‐cell‐mediated pattern. To prevent hypovolemic shock, vascular occlusion, and growth retardation, it is of vital importance to diagnose hypoproteinemia at an early stage and start appropriate therapy.

Analysis of gene expression in peripheral blood eosinophils from patients with atopic dermatitis and in vitro cytokine-stimulated blood eosinophils

Investigation of differentially expressed genes in eosinophils of patients with allergic diseases such as atopic dermatitis (AD) will provide important information for elucidating possible mechanisms of pathology. To identify novel genes that are expressed in AD, we compared gene expression in samples of peripheral blood eosinophils from AD patients and healthy volunteers. RNA was extracted from peripheral blood eosinophils. The expression of various genes, such as those for cytokine receptors, eosinophil activation marker, platelet activating factor (PAF) receptor, eosinophil‐specific granular proteins and apoptosis‐related genes, was confirmed using real‐time reverse transcription–polymerase chain reaction (RT‐PCR). Peripheral blood eosinophils of healthy volunteers were also isolated and stimulated for introduction of various cytokines. RNA was extracted and gene expression was monitored. Several genes, such as those for cytokine receptors (granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) receptor α and β chain and interleukin (IL)‐3 receptor α chain), CD44 and PAF receptor were expressed at significantly higher levels in AD patients than in healthy volunteers. In addition, the anti‐apoptotic genes, bcl‐2 and bcl‐xL, were expressed at increased levels in AD patients. No single gene expression correlated with clinical markers, such as eosinophil count or IgE levels. Expression of GM‐CSF receptor β chain and IL‐3 receptor α chain in isolated blood eosinophils of healthy volunteers was stimulated by IL‐5, IL‐4, interferon (IFN)‐γ and GM‐CSF. Expression of bcl‐2 and bcl‐xL was also increased after stimulation with IL‐5, IL‐4 or IFN‐γ. The in vitro enhancement of cytokine‐stimulated gene expression correlated well with the enhancement observed in clinical samples of eosinophils, suggesting that cytokines may affect gene expression in vivo in eosinophils of patients with AD.