Chiyako Oshikata

Cytokine Production Profile of CD4+ T Cells from Patients with Active Churg-Strauss Syndrome Tends Toward Th17

Background: Churg-Strauss syndrome (CSS) is a rare systemic necrotizing vasculitis that develops in some asthma patients. What types of asthma make patients prone to develop this condition remains unknown. We found that inhibition of regulatory T cells (Treg) differentiation, especially by Tr1 cells, due to a decreased ability of responder T cells to generate IL-2, is associated with the onset and pathogenesis of CSS. In contrast, recent evidence suggests that IL-17-producing Th17 cells play a crucial role in autoimmune inflammation. However, few studies have addressed the role of Th17 in the pathogenesis of CSS. Methods: Mononuclear leukocytes were obtained from healthy subjects, patients with bronchial asthma (BA), asthma-accompanying chronic eosinophilic pneumonia (BA+CEP) and CSS. The cells were stimulated for 4–5 h with PMA and ionomycin in the presence of brefeldin A to generate and accumulate cytokines. Intracellular cytokines were detected after fixation and permeabilization of these cells. Tr1 cells were evaluated as CD4+CD25+ T cells dominantly producing IL-10 and TGF-β, nTreg as CD4+CD25+ T cells expressing Foxp3, a master transcriptional factor, and Th17 were also evaluated as CD4+ T cells mainly producing IL-17. Patients with CSS were classified into an active group and an inactive group in accordance with the disease state after treatment. Results: The frequency of Th17 in peripheral blood was significantly higher in active CSS patients than in healthy subjects, BA, BA+CEP, and inactive CSS patients. In contrast, the Tr1 cell detection frequency was remarkably decreased in active CSS in comparison with BA, BA+CEP, and inactive CSS patients. Also, there was a significant relation between the condition of a given CSS patient and the detection frequencies of both CD4+ helper T cells when CSS cases were divided into inactive and active stages. The Th17 detection frequency was increased in the active stage of CSS, while the Tr1 frequency decreased sharply but then reversed in the inactive stage of CSS. Conclusion: Active CSS patients have elevated intracellular IL-17 and decreased IL-10 levels, which correlate with measures of disease activity, suggesting that Th17 and Tr1 may play important roles in the pathogenesis of CSS.

Differences in regulatory T cells between Churg-Strauss syndrome and chronic eosinophilic pneumonia with asthma

BACKGROUND Chronic eosinophilic pneumonia (CEP) with asthma precedes the onset of Churg-Strauss syndrome (CSS) in half of all patients with CSS. It is not known what determines whether patients with CEP after asthma will have CSS. OBJECTIVE We examined whether activation of regulatory T cells in patients with CEP inhibits CSS development and is otherwise involved in the mechanism of CSS disease. METHODS In patients with CSS (n = 38), CEP with asthma (n = 20), and general adult asthma (n = 108), we examined the number of CD4(+)CD25(+) T cells in peripheral blood, as well as levels of expression of the cytokines IL-2, IL-5, IL-10, and TGF-beta by CD4(+)CD25(+) T cells, CD4(+)CD25(-) T cells, or both. RESULTS At disease onset, patients with CSS, unlike patients with CEP, had significantly fewer CD4(+)CD25(+) T cells than patients with any step of asthma. CD4(+)CD25(+) T cells producing IL-10 were rarely detected in patients with CSS at disease onset or relapse, whereas the numbers of IL-10-producing T cells in patients with CEP were high at disease onset. There were fewer CD4(+)CD25(-) T cells producing IL-2 in patients with CSS before treatment than in patients with CEP at disease onset. The proportions of CD4(+)CD25(+) T cells producing IL-10 and CD4(+)CD25(-) T cells producing IL-2 in patients with CSS increased at remission. CONCLUSIONS Maintenance of the numbers of regulatory T cells in patients with CEP with asthma might inhibit CSS development through the action of cytokines, such as IL-10 and IL-2, produced by CD4(+)CD25(+) or CD4(+)CD25(-) T cells. This might be part of a mechanism that influences progression and prognosis in these diseases.

High-dose Intravenous Immunoglobulin Treatment Increases Regulatory T Cells in Patients with Eosinophilic Granulomatosis with Polyangiitis

Objective. We studied the effects of intravenous immunoglobulin (IVIG) treatment on clinical symptoms and regulatory T (Treg) cell frequency in patients with eosinophilic granulomatosis with polyangiitis (EGPA). Methods. Twenty-two EGPA patients with severe mononeuritis multiplex or cardiac dysfunction received IVIG therapy combined with conventional therapy (corticosteroid, immunosuppressants, or both). As a control, 24 EGPA patients without severe vasculitic symptoms were treated with conventional therapy. Before, during, and after treatment, we determined percentages of Treg cells and other relevant cells in patients’ peripheral blood. Results. The frequency of CD25+ among CD4+ T cells was lower at onset in the study group than in controls but increased significantly after IVIG treatment, relative to controls. The frequency of CD25+ among CD4+ T cells correlated with the frequency of FOXP3+ among CD4+ T cells and interleukin 10 produced by CD25+CD4+ T cells. Conclusion. The increase in Treg cells seen with the combination of IVIG and conventional therapy may promote remission in EGPA.

The Proportion of Regulatory T Cells in the Peripheral Blood Reflects the Relapse or Remission Status of Patients with Churg-Strauss Syndrome

Background: Churg-Strauss syndrome (CSS) is a rare, systemic, necrotizing vasculitis that develops in some asthma patients. We previously reported that maintenance of the proportion of type 1 regulatory T (Tr1) cells in patients with chronic eosinophilic pneumonia and asthma might inhibit the development of CSS through the action of cytokines, such as interleukin (IL)-10 and IL-2, produced by Tr1 and responder T cells. We also reported that IL-17-producing CD4+ helper T cells (Th17 cells) are involved in the pathogenesis of CSS because a higher proportion of Th17 cells was observed in CSS patients during relapses than during remissions. However, few studies have addressed the role of both Tr1 cells and Th17 cells in the status of CSS. Methods: We recruited 40 patients (25 in remission and 15 in relapse) for participation in this study. CSS was diagnosed on the basis of American College of Rheumatology criteria. Remission was defined as the absence of any clinical symptoms of active vasculitis. Tr1 cells were defined as CD4+CD25+ T cells that predominantly produce IL-10 when costimulated with phorbol myristate acetate (PMA) and ionomycin. Naturally occurring Treg (nTreg) cells were defined as CD4+CD25+ T cells that expressed Forkhead box P3 (FOXP3) and cytotoxic T-lymphocyte antigen 4 (CTLA-4). Th17 cells were identified as CD4+ T cells that mainly produced IL-17 and IL-22. Peripheral blood mononuclear cells (PBMCs) obtained from the subjects were costimulated with PMA and ionomycin, and intracellular cytokines were detected after fixing and permeabilizing the cells. Indoleamine 2,3-dioxygenase (IDO) expression was measured in PBMCs that had been treated with IFN-γ and then stimulated overnight with lipopolysaccharide (LPS) or lipopeptide Pam3CSK. Results: Lower expression of CTLA-4 was observed on the surface of CD4+CD25+ T cells obtained from patients with relapsed CSS versus patients in remission. Both FOXP3-expressing nTreg cells and IL-10-producing Tr1 cells were detected in a lower proportion in patients with a relapse compared to patients in remission, but the proportion of CD4+ T cells producing IL-17 was higher during relapse than during remission. In addition, the proportion of CD4+ T cells that produced IL-25, which promotes Th2 inflammation, was also higher in the relapsed patients. We observed a lower percentage of CD14+ monocytes expressing both TLR2 and TLR4 obtained from patients with a relapse of CSS versus patients in remission. Stimulation of CD14+ monocytes with LPS or Pam3CSK reduced IDO expression by the cells from patients with relapsed CSS. The level of IDO expression was positively correlated with the proportion of Tr1 cells in the peripheral blood and inversely correlated with the percentage of Th17 cells. Conclusion: CSS relapse may be linked to increased numbers of CD4+ T cells producing IL-25, which promotes Th2 inflammation, and to a decline in the Tr1 cell subpopulation resulting from lower IDO expression in monocytes. Thus, the proportions of Tr1 cells and Th17 cells reflect the status of CSS.

Decreases in the Numbers of Peripheral Blood Regulatory T Cells, and Increases in the Levels of Memory and Activated B Cells, in Patients with Active Eosinophilic Granulomatosis and Polyangiitis

PurposeEosinophilic granulomatosis with polyangiitis (EGPA), a rare disease characterized by the presence of allergic granulomatosis and necrotizing vasculitis, is often effectively treated with corticosteroids. However, relapse rates are high and, for unknown reasons, some EGPA patients suffer frequent relapses after entry into initial remission. Regulatory T (Treg) cells and B cells are implicated in the development and progression of EGPA. Here, we explored the influence of Treg cells and a co-stimulatory factor present on B cells on the development and course of EGPA.MethodsWe studied 45 EGPA patients (19 of whom experienced frequent relapses and 26 of whom seldom relapsed) and 67 (control) patients with general asthma. We determined the counts or percentages of whole-blood cells exhibiting the following characteristics: FOXP3+ cells among CD4+ Treg cells; CTLA-4+ cells among CD4+/CD25+ Treg cells; and CD27+, CD80+, CD86+, or CD95+ cells among CD19+ B cells. We also measured serum IgG concentrations.ResultsCompared with patients with asthma or seldom-relapsing EGPA, frequently relapsing EGPA patients with active disease exhibited decreased counts of Treg cells and increased percentages of B cells that scored as CD80+, CD27+, or CD95+. Patients with frequently relapsing EGPA had increased percentages of CD27+ and CD95+ B cells, and fewer CD19+ B cells, than did patients in the other two groups. Lower CD19+ B cell counts were associated with reduced Treg cell counts and a lower serum IgG concentration.ConclusionIn patients with frequently relapsing EGPA, decreases in Treg cell numbers and increased percentages of activated B cells may induce apoptosis of B cells.

Bronchial hyperresponsiveness to histamine correlates with airway remodelling in adults with asthma

BACKGROUND Chronic eosinophilic inflammation may promote bronchial hyperresponsiveness (BHR), which involves reversible reduction of airflow and airway remodelling such as a thickening of the reticular basement membrane (RBM) and hypertrophy and hyperplasia of airway smooth muscle (ASM). BHR to histamine (Hist) and acetylcholine (ACh) cannot differentiate airway inflammation and remodelling. OBJECTIVE To examine the correlations between eosinophilic inflammation or airway remodelling and BHR to Hist or ACh in adults with severe asthma. METHODS We examined eosinophils in the sputum of 50 adult patients with severe asthma before inhaled corticosteroid (ICS) treatment. Airway responses to ACh and Hist were measured on separate days after the first hospital visit and before bronchofiberscopy. Bronchial specimens were obtained by bronchofibrescopy for evaluation of RBM and ASM thickening after systemic corticosteroid treatment. RESULTS Eosinophil scores in the sputum before ICS treatment were correlated with BHR to ACh but not to Hist. Asthma duration was inversely correlated with % forced expiratory volume in 1 s, %V(50), %V(25), BHR to Hist, and ASM thickness, but not BHR to ACh or RBM. A multivariate logistic regression model showed that Long duration of asthma affected ASM thickness more than it affected %V(50). ASM thickness was inversely correlated with BHR to Hist but not to ACh. CONCLUSION In adult patients with severe asthma, BHR to ACh is related to the degree of eosinophilic airway inflammation, whereas BHR to Hist indicates airway remodeling, particularly ASM hypertrophy.

Obesity and aspirin intolerance are risk factors for difficult‐to‐treat asthma in Japanese non‐atopic women

Asthma is a clinical syndrome characterized by variabilities in disease expression and severity. The pathophysiological mechanism underlying anti‐asthma treatment resistance is also assumed to be different between disease phenotypes.

Bronchial Reactivity to Histamine Is Correlated With Airway Remodeling in Adults With Moderate to Severe Asthma

Background. Chronic eosinophilic inflammation may promote airway remodeling, including thickening of the reticular basement membrane (RBM), hypertrophy and hyperplasia of the airway smooth muscles (ASM), and an increase in the production of tenascin. The authors examined the correlation between airway remodeling and bronchial reactivity to histamine (Hist) and acetylcholine (ACh) in patients with moderate to severe asthma. Methods. In 30 adult patients with asthma, the authors assessed bronchial hyperresponsiveness (BHR) to various concentrations of ACh and Hist by measuring decreases in forced expiratory volume in one second (FEV1) of >20% from the preprovocation state, and % recoveries of FEV1 after inhalation of β-stimulant. After corticosteroid therapy, the authors evaluated the thickening of RBM and ASM and the production of tenascin in bronchial specimens. Results. The % decrease in FEV1 was correlated with the % recovery in FEV1 after provocation by ACh or Hist. Hypertrophy of ASM was correlated with the % decrease in FEV1 after provocation by Hist, but not by ACh. Thickening of ASM, up-regulation of tenascin in RBM, and duration of asthma were inversely correlated with the % recovery of FEV1 after provocation by Hist, but not by ACh. Conclusions. In adult patients with moderate to severe asthma, a strong bronchial contraction provoked by Hist and a subsequent small recovery indicate airway remodeling.

Fatal pneumonia caused by Penicillium digitatum: a case report

BackgroundPenicillium species are among the most common fungi present in the environment and are usually considered non-pathogenic to humans. However, in immunocompromised hosts they can be virulent pathogens and can cause death. Penicillium digitatum is a plant pathogen that commonly causes a postharvest fungal disease of citrus called green mould; it very rarely causes systemic mycosis in humans. Here, we report a case of fatal pneumonia due to P. digitatum infection, as confirmed by repeated examination of cultured sputum.Case presentationA cavity was found in the left upper lung on routine chest X-ray in a 78-year-old undernourished male who had been diagnosed at age 66 with bronchial asthma and pulmonary emphysema. No increased sputum production was present. The presence of antigen-specific precipitating antibodies to Aspergillus flavus and P. digitatum was confirmed in the patient’s serum and also later pleural fluid by using Ouchterlony double immunodiffusion testing with A. flavus and P. digitatum antigens. The patient was treated over a period of months with itraconazole, micafungin, voriconazole, amphotericin B, and antibacterials. However, the cavity enlarged, the pleural effusion increased, and the patient began producing purulent sputum. He died from progressive renal failure. From sputum culture only one fungus was isolated repeatedly on potato-dextrose agar in large quantities. This fungus was confirmed to be P. digitatum by molecular identification. Partial sequences of the beta-tubulin gene were determined by using the primers Bt2a and Bt2b for PCR amplification and sequencing and underwent a BLAST search at the National Centre for Biotechnology Information, these results confirmed that the isolated fungus was P. digitatum.ConclusionTo our knowledge, this is the first report of pulmonary infection with P. digitatum. Our patient had pulmonary emphysema and was elderly, and undernourished. These factors might have facilitated the infection. In his case, antimycotics were ineffective in treating the lung involvement. Although human infection with P. digitatum is considered rare, it appears that this organism can be very virulent and resistant to antimycotics.

Oral Mite Anaphylaxis Caused by Mite-Contaminated Okonomiyaki/Pancake-Mix in Japan: 8 Case Reports and a Review of 28 Reported Cases.

BACKGROUND Anaphylaxis after the ingestion of foods contaminated with mites has recently been recognized. Case series and case reports thus far have shown that mite-contaminated wheat flour is the major cause of oral mite anaphylaxis. However, we have found 8 cases of oral mite anaphylaxis which were caused by mitecontaminated okonomiyaki-mix, a savory Japanese style pancake mix, in our hospital. METHODS In addition to our 8 cases, the databases of MEDLINE and ICHUSHI were systematically searched for patients with oral mite anaphylaxis in Japan. RESULTS Thirty-six patients including our 8 cases with oral mite anaphylaxis were identified. Thirty-four out of 36 cases (94%) ingested okonomiyaki or takoyaki, prepared at home using okonomiyaki-mix or takoyaki-mix which was previously opened and stored for months at ambient temperature. Microscopic examination of culprit mixes of 16 cases including our 1 case revealed contamination of mites such as Dermatophagoides farina (Der f) (5 cases), Tyrophagus putrescentiae (Tyr p) (4 cases), and Dermatophagoides pteronyssinus (Der p) (3 cases). The specific IgE to each mite is generally upregulated in these patients. Especially, the titers of specific IgE to Der p and Der f were more than class 2 in all cases. CONCLUSIONS Mite-contaminated flavored flour is the major cause of oral mite anaphylaxis in Japan.BACKGROUND Anaphylaxis after the ingestion of foods contaminated with mites has recently been recognized. Case series and case reports thus far have shown that mite-contaminated wheat flour is the major cause of oral mite anaphylaxis. However, we have found 8 cases of oral mite anaphylaxis which were caused by mite-contaminated okonomiyaki-mix, a savory Japanese style pancake mix, in our hospital. METHODS In addition to our 8 cases, the databases of MEDLINE and ICHUSHI were systematically searched for patients with oral mite anaphylaxis in Japan. RESULTS Thirty-six patients including our 8 cases with oral mite anaphylaxis were identified. Thirty-four out of 36 cases (94%) ingested okonomiyaki or takoyaki, prepared at home using okonomiyaki-mix or takoyaki-mix which was previously opened and stored for months at ambient temperature. Microscopic examination of culprit mixes of 16 cases including our 1 case revealed contamination of mites such as Dermatophagoides farina (Der f) (5 cases), Tyrophagus putrescentiae (Tyr p) (4 cases), and Dermatophagoides pteronyssinus (Der p) (3 cases). The specific IgE to each mite is generally upregulated in these patients. Especially, the titers of specific IgE to Der p and Der f were more than class 2 in all cases. CONCLUSIONS Mite-contaminated flavored flour is the major cause of oral mite anaphylaxis in Japan.