Identification of biomarker sets for predicting the efficacy of sublingual immunotherapy against pollen-induced allergic rhinitis.

Sublingual immunotherapy (SLIT) is effective against allergic rhinitis, although a substantial proportion of individuals is refractory. Herein, we describe a predictive modality to reliably identify SLIT non-responders (NRs). We conducted a 2-year clinical study in 193 adult patients with Japanese cedar pollinosis, with biweekly administration of 2000 Japanese allergy units of cedar pollen extract as the maintenance dose. After identifying high-responder (HR) patients with improved severity scores and NR patients with unchanged or exacerbated symptoms, differences in 33 HR and 34 NR patients were evaluated in terms of peripheral blood cellular profiles by flow cytometry and serum factors by ELISA and cytokine bead array, both pre- and post-SLIT. Improved clinical responses were seen in 72% of the treated patients. Pre-therapy IL-12p70 and post-therapy IgG1 serum levels were significantly different between HR and NR patients, although these parameters alone failed to distinguish NR from HR patients. However, the analysis of serum parameters in the pre-therapy samples with the Adaptive Boosting (AdaBoost) algorithm distinguished NR patients with high probability within the training data set. Cluster analysis revealed a positive correlation between serum Th1/Th2 cytokines and other cytokines/chemokines in HR patients after SLIT. Thus, processing of pre-therapy serum parameters with AdaBoost and cluster analysis can be reliably used to develop a prediction method for HR/NR patients.

Protein phosphatase 1 is involved in IL-2-induced IL-5 and IL-13 expression in human T cells.

IL-2 plays an important role in immunological and other biological functions. This cytokine directly induces the production of several cytokines, such as IL-5 and IL-13. The mechanisms of IL-2-mediated cytokine synthesis are mostly unclear; however, the involvement of IL-2 receptor (IL-2R)ß has been suggested. In this study, the signaling molecule downstream of IL-2Rß was investigated, employing a proteomic approach. Full-length IL-2Rß and its mutant in which the intracellular component was truncated were introduced in an IL-2Rα- and IL-2Rγ-stably transfected T cell hybridoma, S1. The differential phosphorylation profiles of protein tyrosine residues in these cells upon IL-2 stimulation were examined by two-dimensional gel electrophoresis. The candidate phosphoproteins of interest were re-covered, in-gel digested and mass spectrometry fingerprinted. Among proteins specifically phosphorylated in full-length IL-2Rß-expressing cells in response to IL-2 stimulation, protein phosphatase (PP)1ß and FK506-binding protein 4 were identified. Particularly, PP1ß augmented IL-5 and IL-13 expression stimulated by IL-2 but not by anti-CD3 antibody in human peripheral CD4+ T cells upon ectopic expression. IL-2-induced cytokine expression was suppressed by overexpression of PP1 regulatory subunit 2. A PP1 inhibitor, tautomycin, but not a PP2A inhibitor, okadaic acid, also inhibited the IL-2R-mediated responses. It was conclusively shown that PP1 is crucially involved in IL-2-mediated IL-5 and IL-13 synthesis in human T cells.

Proteomic Approach to FcepsilonRI aggregation-initiated signal transduction cascade in human mast cells.

BACKGROUND: Mast cells (MCs) play a central role in allergic reactions through high-affinity IgE receptor (FcepsilonRI)-mediated responses. Many attempts have been performed to investigate MC functions, though molecular bases of the intracellular signaling cascade through FcepsilonRI, especially in human MCs, remain scant and unexplored. METHODS: Human MCs were differentiated from CD34+ cells by culture with stem cell factor, IL-6 and IL-3. The differential phosphorylation profiles of protein tyrosine residues in the resulting MCs with or without FcepsilonRI aggregation were examined by two-dimensional gel electrophoresis. The candidate phosphoproteins of interest were picked, in-gel digested and mass spectrometry fingerprinted. RESULTS: Approximately 40 proteins in MCs were phosphorylated on their tyrosine residues in response to activation and some of them were identified. Particularly IL-31 receptor alpha, solute carrier family 39, syntaxin 5 and heterogeneous nuclear ribonucleoprotein are newly identified as phosphoproteins that are potentially involved in the MC signaling cascade through FcepsilonRI. CONCLUSION: Our present phosphoproteome data may provide the clue to understand the molecular mechanisms for the activation of human MCs.

T-box 21 transcription factor is responsible for distorted T(H)2 differentiation in human peripheral CD4+ T cells.

BACKGROUND: Regardless of T(H)1/T(H)2 theory, CD4(+) T cells of patients with allergic asthma, a typical T(H)2 disease, and those of healthy subjects expressed equivalent levels of IFN-gamma, even though T(H)2 cytokines were significantly upregulated in asthmatic patients. OBJECTIVE: The mechanisms underlying distorted T(H)2 cell polarization in human T cells were elucidated. METHODS: Cytokine-producing activity and the expression of T(H)1/T(H)2-specific transcription factors in naïve, T(H)1/T(H)2, or both CD4(+) T cells derived from human peripheral and cord blood were comparatively analyzed. The mechanisms of the differential expression of T-box 21 transcription factor (T-bet) in the cells were assessed by determining the chromatin accessibility at the TBX21 gene. The functional roles of T-bet and other transcription factors in human T(H)1/T(H)2 differentiation were further investigated. RESULTS: T(H)2 cells derived from naive CD4(+) T cells in peripheral blood but not in cord blood produced IFN-gamma. T-bet was expressed in peripheral, but not cord blood, resting naive T cells. Consistently, the accessibility at the proximal TBX21 gene promoter in peripheral naive T cells was higher than that in cord blood naive T cells. IFN-gamma-producing activity was induced in T(H)2-differentiated cord blood T cells by means of ectopic expression of T-bet. In addition, a reduction of T-bet in peripheral T cells suppressed IFN-gamma production. T-bet not only upregulated IFN-gamma but also downregulated IL-4 and IL-13 gene transcription, independently of the modification of T(H)1/T(H)2 balance. CONCLUSION: The expression of T-bet at a naive stage is crucial for the development of IFN-gamma-producing T cells in human peripheral blood, even in T(H)2-related diseases.

Non-SCN5A related Brugada syndromes: verification of normal splicing and trafficking of SCN5A without exonic mutations.

Recently, it has been reported that under 20% of Brugada syndrome cases are linked to SCN5A mutations. The purpose of this study was to clarify whether abnormalities other than exonic mutations, such as splicing disorders, decreased mRNA expression levels, or membrane transport abnormalities of SCN5A, play a role in the pathogenesis of Brugada syndrome. We analyzed all SCN5A exons and splice sites using genomic DNA from 23 Brugada syndrome patients. We also analyzed the mRNA obtained from RV cardiomyocytes using real time PCR and sequencing, to study the expression levels and splicing patterns of SCN5A. The localization of SCN5A was examined by immunofluorescence analysis. A de novo heterozygous G to A transversion in a 5' splice junction of the intron between exons 21 and 22 was detected in 1 patient. In the mRNA analysis of Brugada syndrome patients without a mutation of SCN5A no splicing abnormalities were detected, and the SCN5A mRNA levels were similar to those of normal controls. Immunofluorescence analyses revealed that SCN5A is located on the surface membrane not only in the RV cardiomyocytes of normal controls but also in those with Brugada syndrome. We can confirm that some Brugada syndrome patients without exonic mutations in SCN5A had no other SCN5A abnormalities, including any involving the location of the SCN5A protein. These results suggest the involvement of other proteins in the pathogenesis in Brugada syndrome.

Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis.

To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.

Microgravity potentiates stem cell proliferation while sustaining the capability of differentiation.

A three-dimensional (3D) clinostat is a device for generating multidirectional G force, resulting in an environment with an average of 10(3) G. Here we report that human mesenchymal stem cells (hMSCs) cultured in a 3D-clinostat (group CL) showed marked proliferation (13-fold in a week) compared with cells cultured under normal conditions of 1 G (group C) (4-fold in a week). Flow cytometry revealed a 6-fold increase in the number of hMSCs double-positive for CD44/CD29 or CD90/CD29 in group CL after 7 days in culture, compared with group C. Telomere length remained the same in cells from both groups during culturing. Group C cells showed increasing expression levels of type II collagen and aggrecan over the culture period, whereas group CL cells showed a decrease to undetectable levels. Pellets of hMSCs from each group were explanted into cartilagedefective mice. The transplants from group CL formed hyaline cartilage after 7 days, whereas the transplants from group C formed only noncartilage tissue containing a small number of cells. These results show that hMSCs cultured in a 3D-clinostat possess the strong proliferative characteristic of stem cells and retain their ability to differentiate into hyaline cartilage after transplantation. On the contrary, cells cultured in a 1-G environment do not maintain these features. Simulated microgravity may thus provide an environment to successfully expand stem cell populations in vitro without culture supplements that can adversely affect stem cell-derived transplantations. This method has significant potential for regenerative medicine and developmental biology.