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author:("Han, jinyan")
1.  Step-Wise Epigenetic and Phenotypic Alterations Poise CD8+ T Cells to Mediate Airway Hyperresponsiveness and Inflammation 
The functional plasticity of CD8+ T cells in an atopic environment, encompassing a spectrum from IFN-γ- to IL-13-producing cells, is pivotal in the development of allergic airway hyperresponsiveness (AHR) and inflammation and yet remains mechanistically undefined. We demonstrate that CD8+ T cell IL-13 induction proceeded through a series of distinct IL-4/GATA3-regulated stages characterized by gene expression and epigenetic changes. In vivo, CD8+ T cells exposed to an environment rich in IL-4 displayed epigenetic changes at the GATA3 and IL-13 promoter indicative of transcriptional activation and IL-13 production. In vitro, IL-4 triggered the step-wise molecular conversion of CD8+ T cells from IFN-γ to IL-13 production. During the initial stage, IL-4 suppressed T-bet and induced GATA3 expression, characterized by enhanced activating histone modifications and RNA Pol II recruitment to the GATA3 locus. Notably, recruitment of GATA3 and RNA Pol II to the IL-13 promoter was also detected at this initial stage. However, enhanced IL-13 transcription only occurred at a later stage following TCR stimulation, indicating that IL-4 induced GATA3 recruitment poises the IL-13 locus for TCR-mediated transcription. Thus, both in vivo and in vitro an atopic (IL-4) environment poises CD8+ T cells via step-wise epigenetic and phenotypic mechanisms for pathogenic conversion to IL-13 production, which is ultimately triggered via an allergen-mediated TCR stimulus.
PMCID: PMC3622148  PMID: 23509358
CD8 T cells; IL-4; plasticity; IL-13; asthma
2.  Ultra Pseudo-Stokes Shift Near Infrared Dyes Based on Energy Transfer 
Tetrahedron letters  2013;54(6):502-505.
Novel fluorescent dyes with ultra pseudo-Stokes Shift were prepared based on intramolecular energy transfer between a fluorescent donor and a Cyanine-7 acceptor. The prepared dyes could be excited at ~ 320 nm and emit fluorescence at ~ 780 nm. The energy transfer efficiencies of the system are found to be > 94 %.
PMCID: PMC3539804  PMID: 23316093
Ultra Pseudo-Stokes Shift (UPSS); Energy Transfer; Cyanine-7 Dyes; 7-hydroxycoumarine; benzothiazole
3.  Responsiveness to respiratory syncytial virus in neonates is mediated through thymic stromal lymphopoietin and OX40 ligand 
Recent studies revealed a critical role for thymic stromal lymphopoietin (TSLP) released from epithelial cells and OX40 ligand (OX40L) expressed on dendritic cells (DCs) in TH2 priming and polarization.
We sought to determine the importance of the TSLP-OX40L axis in neonatal respiratory syncytial virus (RSV) infection.
Mice were initially infected with RSV as neonates or adults and reinfected 5 weeks later. Anti-OX40L or anti-TSLP were administered during primary or secondary infection. Outcomes included assessment of airway function and inflammation and expression of OX40L, TSLP, and IL-12.
OX40L was expressed mainly on CD11c+MHC class II (MHCII)+CD11b+ DCs but not CD103+ DCs. Treatment of neonates with OX40L antibody during primary RSV infection prevented the subsequent enhancement of airway hyperresponsiveness and the development of airway eosinophilia and mucus hyperproduction on reinfection. Administration of anti-TSLP before neonatal RSV infection reduced the accumulation of lung DCs, decreased OX40L expression on lung DCs, and attenuated the enhancement of airway responses after reinfection.
In mice initially infected as neonates, TSLP expression induced by RSV infection is an important upstream event that controls OX40L expression, lung DC migration, and TH2 polarization, accounting for the enhanced response on reinfection.
PMCID: PMC3593657  PMID: 23036746
Respiratory syncytial virus; OX40 ligand; thymic stromal lymphopoietin
4.  Microbial Heat Shock Protein 65 Attenuates Airway Hyperresponsiveness and Inflammation by Modulating the Function of Dendritic Cells 
Heat shock proteins (HSPs), produced in response to stress are suppressive in disease models. We previously showed that Mycobacterium leprae HSP65 prevented development of airway hyperresponsiveness and inflammation in mice. Our goal here was to define the mechanism responsible for the suppressive effects of HSP. In one in vivo approach, BALB/c mice were sensitized to ovalbumin (OVA) followed by primary OVA challenges. Several weeks later, HSP65 was administered prior to a single, provocative secondary challenge. In a second in vivo approach, the secondary challenge was replaced by intratracheal instillation of allergen-pulsed bone marrow-derived dendritic cells (BMDCs). The in vitro effects of HSP65 on BMDCs were examined in co-culture experiments with CD4+ T cells. In vivo, HSP65 prevented development of airway hyperresponsiveness and inflammation. As well, Th1 cytokine levels in bronchoalveolar lavage (BAL) fluid were increased. In vitro, HSP65 induced notch receptor ligand Delta1 expression on BMDCs and HSP65-treated BMDCs skewed CD4+ T cells to Th1 cytokine production. Thus, HSP65-induced effects on allergen-induced airway hyperresponsiveness and inflammation were associated with increased Delta 1 expression on DCs, modulation of DC function, and CD4+ Th1 cytokine production.
PMCID: PMC3448847  PMID: 22933632
HSP65; asthma; dendritic cells; T cells
5.  Inhibition of Pim1 kinase prevents peanut allergy by enhancing Runx3 expression and suppressing TH2 and TH17 T-cell differentiation 
The provirus integration site for Moloney murine leukemia virus (Pim) 1 kinase is an oncogenic serine/threonine kinase implicated in cytokine-induced cell signaling, whereas Runt-related transcription factor (Runx) has been implicated in the regulation of T-cell differentiation. The interaction of Pim1 kinase and Runx3 in the pathogenesis of peanut allergy has not been defined.
We sought to determine the effects of Pim1 kinase modulation on Runx3 expression and TH2 and TH17 cell function in an experimental model of peanut allergy. Methods: A Pim1 kinase inhibitor was administered to peanut-sensitized and challenged wild-type and Runx3+/− mice. Symptoms, intestinal inflammation, and Pim1 kinase and Runx3 mRNA expression and protein levels were assessed. The effects of Pim1 kinase inhibition on TH1, TH2, and TH17 differentiation in vivo and in vitro were also determined.
Peanut sensitization and challenge resulted in accumulation of inflammatory cells and goblet cell metaplasia and increased levels of Pim1 kinase and TH2 and TH17 cytokine production but decreased levels of Runx3 mRNA and protein in the small intestines of wild-type mice. All of these findings were normalized with Pim1 kinase inhibition. In sensitized and challenged Runx3+/− mice, inhibition of Pim1 kinase had less effect on the development of the full spectrum of intestinal allergic responses. In vitro inhibition of Pim1 kinase attenuated TH2 and TH17 cell differentiation and expansion while maintaining Runx3 expression in T-cell cultures from wild-type mice; these effects were reduced in T-cell cultures from Runx3+/− mice.
These data support a novel regulatory axis involving Pim1 kinase and Runx3 in the control of food-induced allergic reactions through the regulation of TH2 and TH17 differentiation.
PMCID: PMC3652680  PMID: 22944483
Pim1 kinase; Runx3; peanut; intestinal allergy; TH2; TH17
6.  Fluorescent Proton Sensors Based On Energy Transfer 
The Journal of organic chemistry  2011;76(13):5219-5228.
Photophysical data and orbital energy levels (from electrochemistry) were compared for molecules with the same BODIPY acceptor part (red) and perpendicularly oriented xanthene or BODIPY donor fragments (green). Transfer of energy, hence the photophysical properties of the cassettes, including the pH dependant fluorescence in the xanthene containing molecules, correlates with the relative energies of the frontier orbitals in these systems.
Intracellular sensing of protons is often achieved via sensors that switch off completely at certain pH values, but probes of this type are not easy to locate inside cells in their “off-state”. A communication from these laboratories (J. Am. Chem. Soc., 2009, 131, 1642 – 3) described how the energy transfer cassette 1 could be used for intracellular imaging of pH. This probe is fluorescent whatever the pH, but its exact photophysical properties are governed by the protonation-states of the xanthene donors. This work was undertaken to further investigate correlations between structure, photophysical properties, and pH for energy transfer cassettes. To achieve this, three other cassettes 2 – 4 were prepared another one containing pH-sensitive xanthene donors (2), and two “control cassettes” that each have two BODIPY-based donors (3 and 4). Both the cassettes 1 and 2 with xanthene-based donors fluoresce red under slightly acidic conditions (pH < ca 6), and green when the medium is more basic (> ca 7), whereas the corresponding cassettes with BODIPY donors give almost complete energy transfer regardless of pH. The cassettes that have BODIPY donors by contrast, show no significant fluorescence from the donor parts, but the overall quantum yields of the cassettes when excited at the donor (observation of acceptor fluorescence) are high (ca 0.6 and 0.9). Electrochemical measurements were performed to elucidate orbital energy level differences between the pH-fluorescence profiles of cassettes with xanthene donors, relative to the two with BODIPY donors. These studies confirm energy transfer in the cassettes is dramatically altered by analytes that perturb relative orbital levels. Energy transfer cassettes with distinct fluorescent donor and acceptor units provide a new, and potentially useful, approach to sensors for biomedical applications.
PMCID: PMC3130547  PMID: 21618970
pH probe; fluorescent sensors; BODIPY; fluorescein; energy transfer
8.  Montelukast during Primary Infection Prevents Airway Hyperresponsiveness and Inflammation after Reinfection with Respiratory Syncytial Virus 
Rationale: Respiratory syncytial virus (RSV) bronchiolitis in infants may be followed by the development of asthma-like symptoms. Age at first infection dictates consequences upon reinfection. Reinfection of mice initially exposed as neonates to RSV enhanced development of airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus hyperproduction. RSV lower respiratory tract disease is associated with activation of the leukotriene pathway.
Objectives: To determine the effects of montelukast (MK), a cysteinyl leukotriene (cysLT) receptor antagonist, in primary and secondary RSV-infected newborn and adult mice.
Methods: BALB/c mice were infected with RSV at 1 week (neonate) or 6 to 8 weeks (adult) of age and reinfected 5 weeks later. MK was administered 1 day before the initial infection and through Day 6 after infection. Seven days after primary or secondary infection, airway function was assessed by lung resistance to increasing doses of inhaled methacholine; lung inflammation, goblet cell metaplasia, and cytokine levels in bronchoalveolar lavage fluid were monitored.
Measurements and Main Results: RSV infection induced cysLT release in bronchoalveolar lavage fluid. MK decreased RSV-induced AHR, airway inflammation, and increased IFN-γ production in primary infected adult and neonatal mice. MK, administered during initial infection of neonates but not during secondary infection, prevented subsequent enhancement of AHR, airway eosinophilia, and mucus hyperproduction upon reinfection.
Conclusions: MK attenuated the initial responses to primary RSV infection in both age groups and altered the consequences of RSV reinfection in mice initially infected as neonates. These data support an important role for cysLT in RSV-induced AHR and inflammation.
PMCID: PMC2937239  PMID: 20442434
airway; inflammation; RSV; cysteinyl leukotrienes
9.  The Role of RSV Infection in Asthma Initiation and Progression: Findings in a Mouse Model 
Pulmonary Medicine  2011;2011:748038.
Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract diseases (bronchiolitis and pneumonia) during infancy and early childhood. There is increasing evidence which indicates that severe pulmonary disease caused by RSV infection in infancy is associated with recurrent wheezing and development of asthma later in childhood. However, the underlying mechanisms linking RSV infection to persistent airway hyperresponsiveness and dysfunction are not fully defined. To study these processes in ways which are not available in humans, animal models have been established and have provided valuable insight into the pathophysiology of RSV-induced disease. In this paper, we discuss experimental models of RSV infection in mice and highlight a new investigative approach in which mice are initially infected as neonates and then reinfected later in life. The findings shed light on the mechanisms underlying the association between early severe RSV infection and development of asthma later in childhood.
PMCID: PMC3135221  PMID: 21766019
10.  A Ratiometric pH Reporter For Imaging Protein-dye Conjugates In Living Cells 
A molecule that transfers energy through bonds from a donor to an acceptor was prepared with a pH sensitive donor function (fluorescein). At pH values above 6.5, minimal energy transfer occurred, and the probe emits green fluorescence (ca 520 nm) when excited at the donor (488 nm). Below pH 6 however, energy transfer is efficient hence excitation at the donor causes emission at the acceptor part (600 nm). This probe was used to image a conjugate of the probe with bovine serum albumin that was imported into endosomes or in the cytosol using the non-covalently bound carrier, Pep-1, at 37 and 4 °C, respectively. The more acidic environment of the endosomes was conspicuous from the red fluorescence of the probe.
PMCID: PMC2720788  PMID: 19146412
11.  Extracellular High-Mobility Group Box 1 Acts as an Innate Immune Mediator to Enhance Autoimmune Progression and Diabetes Onset in NOD Mice 
Diabetes  2008;57(8):2118-2127.
OBJECTIVE—The implication of innate immunity in type 1 diabetes development has long been proposed. High-mobility group box 1 (HMGB1), an evolutionarily conserved chromosomal protein, was recently recognized to be a potent innate inflammatory mediator when released extracellularly. We sought to test the hypothesis that HMGB1 acts as an innate immune mediator implicated in type 1 diabetes pathogenesis.
RESEARCH DESIGN AND METHODS—Eight- and 12-week-old NOD mice were treated with an HMGB1 neutralizing antibody once a week until 25 weeks of age and monitored for insulitis progression and diabetes onset. The underlying mechanisms of HMGB1 regulation of autoimmune response were further explored.
RESULTS—During autoimmunity, HMGB1 can be passively released from damaged pancreatic β-cells and actively secreted by islet infiltrated immune cells. Extracellular HMGB1 is potent in inducing NOD dendritic cell maturation and stimulating macrophage activation. Blockade of HMGB1 significantly inhibited insulitis progression and diabetes development in both 8- and 12-week-old NOD mice. HMGB1 antibody treatment decreased the number and maturation of pancreatic lymph node (PLN) CD11c++CD11b+ dendritic cells, a subset of dendritic cells probably associated with autoantigen presentation to naïve T-cells, but increased the number for PLN CD4+Foxp3+ regulatory T-cells. Blockade of HMGB1 also decreased splenic dendritic cell allo-stimulatory capability associated with increased tolergenic CD11c+CD8a+ dendritic cells. Interestingly, the number of CD8+interferon-γ+ (Tc1) T-cells was increased in the PLNs and spleen after blockade of HMGB1, which could be associated with retarded migration of activated autoreactive T-cells into the pancreatic islets.
CONCLUSIONS—Extracellular HMGB1 functions as a potent innate immune mediator contributing to insulitis progression and diabetes onset.
PMCID: PMC2494682  PMID: 18477810

Results 1-11 (11)