Cho-Rong Lee

The kinase PDK1 is essential for B-cell receptor mediated survival signaling.

Phosphoinositide-dependent kinase 1 (PDK1) plays an important role in integrating the T cell antigen receptor (TCR) and CD28 signals to achieve efficient NF-κB activation. PDK1 is also an important regulator of T cell development, mediating pre-TCR induced proliferation signals. However, the role of PDK1 in B cell antigen receptor (BCR) signaling and B cell development remains largely unknown. In this study we provide genetic evidence supporting the role of PDK1 in B cell survival. We found PDK1 is required for BCR mediated survival in resting B cells, likely through regulation of Foxo activation. PDK1-dependent signaling to NF-κB is not crucial to resting B cell viability. However, PDK1 is necessary for triggering NF-κB during B cell activation and is required for activated B cell survival. Together these studies demonstrate that PDK1 is essential for BCR-induced signal transduction to Foxo and NF-κB and is indispensable for both resting and activated B cell survival.

Epigenetic regulation of Kcna3-encoding Kv1.3 potassium channel by cereblon contributes to regulation of CD4+ T-cell activation.

Significance In resting T cells, CRBN normally represses expression of the Kv1.3 potassium channel by regulating histone modifications to prevent hyperactivation of T cells. It does this by controlling recruitment of EZH1 to the potassium channel region of the Kcna3 gene (or locus). However, lack of CRBN causes up-regulation of Kv1.3 expression, which in turn increases potassium flux, thereby triggering increased calcium flux during T-cell activation. The role of cereblon (CRBN) in T cells is not well understood. We generated mice with a deletion in Crbn and found cereblon to be an important antagonist of T-cell activation. In mice lacking CRBN, CD4+ T cells show increased activation and IL-2 production on T-cell receptor stimulation, ultimately resulting in increased potassium flux and calcium-mediated signaling. CRBN restricts T-cell activation via epigenetic modification of Kcna3, which encodes the Kv1.3 potassium channel required for robust calcium influx in T cells. CRBN binds directly to conserved DNA elements adjacent to Kcna3 via a previously uncharacterized DNA-binding motif. Consequently, in the absence of CRBN, the expression of Kv1.3 is derepressed, resulting in increased Kv1.3 expression, potassium flux, and CD4+ T-cell hyperactivation. In addition, experimental autoimmune encephalomyelitis in T-cell–specific Crbn-deficient mice was exacerbated by increased T-cell activation via Kv1.3. Thus, CRBN limits CD4+ T-cell activation via epigenetic regulation of Kv1.3 expression.

Peroxisome-localized hepatitis Bx protein increases the invasion property of hepatocellular carcinoma cells

HBx acts as a multifunctional regulator that modulates various cellular responses, which can lead to development and progression of hepatocellular carcinoma (HCC). Here, we show that the HBx protein is also localized to peroxisomes, and this increases cellular reactive oxygen species (ROS) to levels that are higher than when HBx is localized to other organelles. The elevated ROS strongly activated nuclear factor (NF)-κB. In addition, the peroxisome-localized HBx increased the expressions of matrix metalloproteinases and decreased the expression of E-cadherin, which increased the invasive ability of HCC cells. Thus, a specific distribution of HBx to peroxisomes may contribute to HCC progression by increasing the invasive ability of HCC cells through elevation of the cellular ROS level.

Tofacitinib relieves symptoms of stimulator of interferon genes (STING)–associated vasculopathy with onset in infancy caused by 2 de novo variants in TMEM173

4. Kukkonen AK, Pelkonen AS, M€akinen-Kiljunen S, Voutilainen H, M€akel€a MJ. Ara h 2 and Ara 6 are the best predictors of severe peanut allergy: a double-blind placebo-controlled study. Allergy 2015;11:1239-45. 5. Nozawa A, Okamoto Y, Mov erare R, Borres MP, Kurihara K. Monitoring Ara h 1, 2 and 3-sIgE and sIgG4 antibodies in peanut allergic children receiving oral rush immunotherapy. Pediatr Allergy Immunol 2014;25:323-8. 6. Vickery BP, Scurlock AM, Kulis M, Steele PH, Kamilaris J, Berglund JP, et al. Sustained unresponsiveness to peanut in subjects who have completed peanut oral immunotherapy. J Allergy Clin Immunol 2014;133:468-75. 7. Krause S, Reese G, Randow S, Zennaro D, Quaratino D, Palazzo P, et al. Lipid transfer protein (Ara h 9) as a new peanut allergen relevant for a Mediterranean allergic population. J Allergy Clin Immunol 2009;124:771-8.e5. 8. Glaumann S, Nilsson C, Asarnoj A, Mov erare R, Johansson SG, Borres MP, et al. IgG4 antibodies and peanut challenge outcome in children IgE-sensitized to peanut. Pediatr Allergy Immunol 2015;26:386-9. 9. Santos AF, James LK, Bahnson HT, Shamji MH, Couto-Francisco NC, Islam S, et al. IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens. J Allergy Clin Immunol 2015;135:1249-56.

Secretion of IL‐1β from imatinib‐resistant chronic myeloid leukemia cells contributes to BCR–ABL mutation‐independent imatinib resistance

Some cases of chronic myelogenous leukemia are resistant to tyrosine kinase inhibitors (TKIs) independently of mutation in BCR–ABL, but the detailed mechanism underlying this resistance has not yet been elucidated. In this study, we generated a TKI‐resistant CML cell line, K562R, that lacks a mutation in BCR–ABL. Interleukin‐1β (IL‐1β) was more highly expressed in K562R than in the parental cell line K562S, and higher levels of IL‐1β contributed to the imatinib resistance of K562R. In addition, IL‐1β secreted from K562R cells affected stromal cell production of CXCL11, which in turn promoted migration of K562R cells into the stroma. Thus, elevated IL‐1β production from TKI‐resistant K562R cells may contribute to TKI resistance by increasing cell viability and promoting cell migration.

Effect of fluorescent whitening agent on the transcription of cell damage-related genes in zebrafish embryos

7‐Diethylamino‐4‐methylcoumarin (DEMC) is a fluorescent whitening agent (FWAs). There have been some studies on DEMCs protective effects against biological activity but there are few papers about the in vivo toxicity of DEMC. In this study, we used wild‐type zebrafish embryos 3 days post fertilization (dpf). Test solutions with DEMC concentrations were negative control (without vehicle), 0 (with vehicle, 0.01% v/v ethanol), 0.25, 0.5, 0.75, 1.0, 1.25, 1.5 and 2 ppm. Embryos and larvae were counted for survival rate and hatching rate. Heart rates were also counted at 2.5 and 3.0 dpf. At 3.0 dpf, quantitative RT‐PCR was performed with some samples (0, 0.25, 0.75 and 1.25 ppm) to determine the toxic effect to DEMC by detecting the expression levels of toxic‐responsive genes. We used 11 genes, which included oxidative stress‐related genes [sod(Mn), sod(Cu,Zn) and hsp70], mitochondrial metabolism‐related genes (coxI, pyc, cyt and cyclinG1) and apoptosis‐related genes (c‐jun, bcl2, bax and p53). High‐concentration DEMC‐treated groups showed significant different survival rate, hatching rate and heart rate compared with low‐concentration DEMC‐treated groups. The LC50 of this chemical, 0.959 ppm, was calculated. We also confirmed that some genes in the DEMC exposure groups showed significantly up‐regulations in expression levels compared with control groups. We concluded that the fluorescence agent, DEMC, has possible developmental toxicities and alteration effect of gene expression, which are related to oxidative stress, mitochondrial metabolism and apoptosis in zebrafish embryos. Copyright

Characterization of protoberberine analogs employed as novel human P2X7 receptor antagonists

The P2X(7) receptor (P2X(7)R), a member of the ATP-gated ion channel family, is regarded as a promising target for therapy of immune-related diseases including rheumatoid arthritis and chronic pain. A group of novel protoberberine analogs (compounds 3-5), discovered by screening of chemical libraries, was here investigated with respect to their function as P2X(7)R antagonists. Compounds 3-5 non-competitively inhibited BzATP-induced ethidium ion influx into hP2X(7)-expressing HEK293 cells, with IC(50) values of 100-300nM. This antagonistic action on the channel further confirmed that both BzATP-induced inward currents and Ca(2+) influx were strongly inhibited by compounds 3-5 in patch-clamp and Ca(2+) influx assays. The antagonists also effectively suppressed downstream signaling of P2X(7) receptors including IL-1β release and phosphorylation of ERK1/2 and p38 proteins in hP2X(7)-expressing HEK293 cells or in differentiated human monocytes (THP-1 cells). Moreover, IL-2 secretion from CD3/CD28-stimulated Jurkat T cell was also dramatically inhibited by the antagonist. These results imply that novel protoberberine analogs may modulate P2X(7) receptor-mediated immune responses by allosteric inhibition of the receptor.

Characterization of Multiple Cytokine Combinations and TGF-β on Differentiation and Functions of Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSCs) regulate T cell immunity, and this population is a new therapeutic target for immune regulation. A previous study showed that transforming growth factor-β (TGF-β) is involved in controlling MDSC differentiation and immunoregulatory function in vivo. However, the direct effect of TGF-β on MDSCs with various cytokines has not previously been tested. Thus, we examined the effect of various cytokine combinations with TGF-β on MDSCs derived from bone marrow cells. The data show that different cytokine combinations affect the differentiation and immunosuppressive functions of MDSCs in different ways. In the presence of TGF-β, interleukin-6 (IL-6) was the most potent enhancer of MDSC function, whereas granulocyte colony-stimulating factors (G-CSF) was the most potent in the absence of TGF-β. In addition, IL-4 maintained MDSCs in an immature state with an increased expression of arginase 1 (Arg1). However, regardless of the cytokine combinations, TGF-β increased expansion of the monocytic MDSC (Mo-MDSC) population, expression of immunosuppressive molecules by MDSCs, and the ability of MDSCs to suppress CD4+ T cell proliferation. Thus, although different cytokine combinations affected the MDSCs in different ways, TGF-β directly affects monocytic-MDSCs (Mo-MDSCs) expansion and MDSCs functions.

Design and Evaluation of Four-Stage Low-Pressure Cascade Impactor Using Electrical Measurement System

The low-pressure cascade impactor has been used to collect ultrafine particles that cannot be measured by conventional cascade impactors. Low-pressure cascade impactors resemble ordinary impactors, but are operated at reduced pressures of 0.05 ∼ 0.4 atm. Many kinds of low-pressure impactors have been developed by different researchers. However, it is still difficult to accurately design and evaluate the low-pressure cascade impactor. In this study, a four-stage low-pressure cascade impactor for measuring the size distribution of submicron aerosol particles was designed and evaluated. To evaluate particle collection efficiency of each stage, an electrical measurement system was used. The cut-point diameters of Stages 1 through 4 were 0.238, 0.173, 0.111, and 0.063 μm in aerodynamic diameter. Stage 2 showed poor steepness of the collection efficiency curve and larger cut-point Stokes number than theory, which may be attributed to high nozzle velocity. The fluorometric method for particle collection efficiency measurement was shown to be unreliable for ultrafine particles. The solid particle collection efficiency of the designed impactor was examined with different substrate conditioning methods. Porous metal substrate and silicon-coated substrate were tested with NaCl particles. It was shown that silicon coating did not effectively reduce the particle bounce because of high nozzle velocity, whereas the porous metal substrate considerably enhanced the particle collection efficiency.