Publications by authors named "Yonghua Zhuang"

17 Publications

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Plasma Metabolomic Signatures of Chronic Obstructive Pulmonary Disease and the Impact of Genetic Variants on Phenotype-Driven Modules.

Netw Syst Med 2020 Dec 31;3(1):159-181. Epub 2020 Dec 31.

National Jewish Health, Denver, Colorado, USA.

Small studies have recently suggested that there are specific plasma metabolic signatures in chronic obstructive pulmonary disease (COPD), but there have been no large comprehensive study of metabolomic signatures in COPD that also integrate genetic variants. Fresh frozen plasma from 957 non-Hispanic white subjects in COPDGene was used to quantify 995 metabolites with Metabolon's global metabolomics platform. Metabolite associations with five COPD phenotypes (chronic bronchitis, exacerbation frequency, percent emphysema, post-bronchodilator forced expiratory volume at one second [FEV]/forced vital capacity [FVC], and FEV percent predicted) were assessed. A metabolome-wide association study was performed to find genetic associations with metabolite levels. Significantly associated single-nucleotide polymorphisms were tested for replication with independent metabolomic platforms and independent cohorts. COPD phenotype-driven modules were identified in network analysis integrated with genetic associations to assess gene-metabolite-phenotype interactions. Of metabolites tested, 147 (14.8%) were significantly associated with at least 1 COPD phenotype. Associations with airflow obstruction were enriched for diacylglycerols and branched chain amino acids. Genetic associations were observed with 109 (11%) metabolites, 72 (66%) of which replicated in an independent cohort. For 20 metabolites, more than 20% of variance was explained by genetics. A sparse network of COPD phenotype-driven modules was identified, often containing metabolites missed in previous testing. Of the 26 COPD phenotype-driven modules, 6 contained metabolites with significant met-QTLs, although little module variance was explained by genetics. A dysregulation of systemic metabolism was predominantly found in COPD phenotypes characterized by airflow obstruction, where we identified robust heritable effects on individual metabolite abundances. However, network analysis, which increased the statistical power to detect associations missed previously in classic regression analyses, revealed that the genetic influence on COPD phenotype-driven metabolomic modules was modest when compared with clinical and environmental factors.
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http://dx.doi.org/10.1089/nsm.2020.0009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109053PMC
December 2020

Identifying Protein-metabolite Networks Associated with COPD Phenotypes.

Metabolites 2020 Mar 25;10(4). Epub 2020 Mar 25.

Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.

Chronic obstructive pulmonary disease (COPD) is a disease in which airflow obstruction in the lung makes it difficult for patients to breathe. Although COPD occurs predominantly in smokers, there are still deficits in our understanding of the additional risk factors in smokers. To gain a deeper understanding of the COPD molecular signatures, we used Sparse Multiple Canonical Correlation Network (SmCCNet), a recently developed tool that uses sparse multiple canonical correlation analysis, to integrate proteomic and metabolomic data from the blood of 1008 participants of the COPDGene study to identify novel protein-metabolite networks associated with lung function and emphysema. Our aim was to integrate -omic data through SmCCNet to build interpretable networks that could assist in the discovery of novel biomarkers that may have been overlooked in alternative biomarker discovery methods. We found a protein-metabolite network consisting of 13 proteins and 7 metabolites which had a -0.34 correlation (-value = 2.5 × 10) to lung function. We also found a network of 13 proteins and 10 metabolites that had a -0.27 correlation (-value = 2.6 × 10) to percent emphysema. Protein-metabolite networks can provide additional information on the progression of COPD that complements single biomarker or single -omic analyses.
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http://dx.doi.org/10.3390/metabo10040124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241079PMC
March 2020

Bronchoalveolar Lavage Fluid from COPD Patients Reveals More Compounds Associated with Disease than Matched Plasma.

Metabolites 2019 Jul 25;9(8). Epub 2019 Jul 25.

School of Medicine, University of Colorado, Aurora, CO 80045, USA.

Smoking causes chronic obstructive pulmonary disease (COPD). Though recent studies identified a COPD metabolomic signature in blood, no large studies examine the metabolome in bronchoalveolar lavage (BAL) fluid, a more direct representation of lung cell metabolism. We performed untargeted liquid chromatography-mass spectrometry (LC-MS) on BAL and matched plasma from 115 subjects from the SPIROMICS cohort. Regression was performed with COPD phenotypes as the outcome and metabolites as the predictor, adjusted for clinical covariates and false discovery rate. Weighted gene co-expression network analysis (WGCNA) grouped metabolites into modules which were then associated with phenotypes. K-means clustering grouped similar subjects. We detected 7939 and 10,561 compounds in BAL and paired plasma samples, respectively. FEV/FVC (Forced Expiratory Volume in One Second/Forced Vital Capacity) ratio, emphysema, FEV % predicted, and COPD exacerbations associated with 1230, 792, eight, and one BAL compounds, respectively. Only two plasma compounds associated with a COPD phenotype (emphysema). Three BAL co-expression modules associated with FEV/FVC and emphysema. K-means BAL metabolomic signature clustering identified two groups, one with more airway obstruction (34% of subjects, median FEV/FVC 0.67), one with less (66% of subjects, median FEV/FVC 0.77; < 2 × 10). Associations between metabolites and COPD phenotypes are more robustly represented in BAL compared to plasma.
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http://dx.doi.org/10.3390/metabo9080157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724137PMC
July 2019

Unsupervised discovery of phenotype-specific multi-omics networks.

Bioinformatics 2019 11;35(21):4336-4343

Department of Biostatistics and Informatics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Motivation: Complex diseases often involve a wide spectrum of phenotypic traits. Better understanding of the biological mechanisms relevant to each trait promotes understanding of the etiology of the disease and the potential for targeted and effective treatment plans. There have been many efforts towards omics data integration and network reconstruction, but limited work has examined the incorporation of relevant (quantitative) phenotypic traits.

Results: We propose a novel technique, sparse multiple canonical correlation network analysis (SmCCNet), for integrating multiple omics data types along with a quantitative phenotype of interest, and for constructing multi-omics networks that are specific to the phenotype. As a case study, we focus on miRNA-mRNA networks. Through simulations, we demonstrate that SmCCNet has better overall prediction performance compared to popular gene expression network construction and integration approaches under realistic settings. Applying SmCCNet to studies on chronic obstructive pulmonary disease (COPD) and breast cancer, we found enrichment of known relevant pathways (e.g. the Cadherin pathway for COPD and the interferon-gamma signaling pathway for breast cancer) as well as less known omics features that may be important to the diseases. Although those applications focus on miRNA-mRNA co-expression networks, SmCCNet is applicable to a variety of omics and other data types. It can also be easily generalized to incorporate multiple quantitative phenotype simultaneously. The versatility of SmCCNet suggests great potential of the approach in many areas.

Availability And Implementation: The SmCCNet algorithm is written in R, and is freely available on the web at https://cran.r-project.org/web/packages/SmCCNet/index.html.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6931269PMC
November 2019

Interferon Beta Contributes to Astrocyte Activation in the Brain following Reovirus Infection.

J Virol 2019 05 1;93(10). Epub 2019 May 1.

Department of Neurology, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado, USA.

Reovirus encephalitis in mice was used as a model system to investigate astrocyte activation (astrogliosis) following viral infection of the brain. Reovirus infection resulted in astrogliosis, as evidenced by increased expression of glial fibrillary acidic protein (GFAP), and the upregulation of genes that have been previously associated with astrocyte activation. Astrocyte activation occurred in regions of the brain that are targeted by reovirus but extended beyond areas of active infection. Astrogliosis also occurred following reovirus infection of brain slice cultures (BSCs), demonstrating that factors intrinsic to the brain are sufficient to activate astrocytes and that this process can occur in the absence of any contribution from the peripheral immune response. In agreement with previous reports, reovirus antigen did not colocalize with GFAP in infected brains, suggesting that reovirus does not infect astrocytes. Reovirus-infected neurons produce interferon beta (IFN-β). IFN-β treatment of primary astrocytes resulted in both the upregulation of GFAP and cytokines that are associated with astrocyte activation. In addition, the ability of media from reovirus-infected BSCs to activate primary astrocytes was blocked by anti-IFN-β antibodies. These results suggest that IFN-β, likely released from reovirus-infected neurons, results in the activation of astrocytes during reovirus encephalitis. In areas where infection and injury were pronounced, an absence of GFAP staining was consistent with activation-induced cell death as a mechanism of inflammation control. In support of this, activated Bak and cleaved caspase 3 were detected in astrocytes within reovirus-infected brains, indicating that activated astrocytes undergo apoptosis. Viral encephalitis is a significant cause of worldwide morbidity and mortality, and specific treatments are extremely limited. Virus infection of the brain triggers neuroinflammation; however, the role of neuroinflammation in the pathogenesis of viral encephalitis is unclear. Initial neuroinflammatory responses likely contribute to viral clearance, but prolonged exposure to proinflammatory cytokines released during neuroinflammation may be deleterious and contribute to neuronal death and tissue injury. Activation of astrocytes is a hallmark of neuroinflammation. Here, we show that reovirus infection of the brain results in the activation of astrocytes via an IFN-β-mediated process and that these astrocytes later die by Bak-mediated apoptosis. A better understanding of neuroinflammatory responses during viral encephalitis may facilitate the development of new treatment strategies for these diseases.
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http://dx.doi.org/10.1128/JVI.02027-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498044PMC
May 2019

Mitochondrial p53 Contributes to Reovirus-Induced Neuronal Apoptosis and Central Nervous System Injury in a Mouse Model of Viral Encephalitis.

J Virol 2016 09 12;90(17):7684-91. Epub 2016 Aug 12.

Department of Neurology, University of Colorado Denver, Aurora, Colorado, USA Department of Immunology and Microbiology, University of Colorado Denver, Aurora, Colorado, USA Department of Medicine, University of Colorado Denver, Aurora, Colorado, USA Denver VA Medical Center, Denver, Colorado, USA.

Unlabelled: The tumor suppressor p53 plays a critical part in determining cell fate both as a regulator of the transcription of several proapoptotic genes and through its binding interactions with Bcl-2 family proteins at mitochondria. We now demonstrate that p53 protein levels are increased in infected brains during reovirus encephalitis. This increase occurs in the cytoplasm of reovirus-infected neurons and is associated with the activation of caspase 3. Increased levels of p53 in reovirus-infected brains are not associated with increased expression levels of p53 mRNA, suggesting that p53 regulation occurs at the protein level. Increased levels of p53 are also not associated with the increased expression levels of p53-regulated, proapoptotic genes. In contrast, upregulated p53 accumulates in mitochondria. Previous reports demonstrated that the binding of p53 to Bak at mitochondria causes Bak activation and results in apoptosis. We now show that Bak is activated and that activated Bak is bound to p53 during reovirus encephalitis. In addition, survival is enhanced in reovirus-infected Bak(-/-) mice compared to controls, demonstrating a role for Bak in reovirus pathogenesis. Inhibition of the mitochondrial translocation of p53 with pifithrin μ prevents the formation of p53/Bak complexes following reovirus infection of ex vivo brain slice cultures and results in decreased apoptosis and tissue injury. These results suggest that the mitochondrial localization of p53 regulates reovirus-induced pathogenesis in the central nervous system (CNS) through its interactions with Bak.

Importance: There are virtually no specific treatments of proven efficacy for virus-induced neuroinvasive diseases. A better understanding of the pathogenesis of virus-induced CNS injury is crucial for the rational development of novel therapies. Our studies demonstrate that p53 is activated in the brain following reovirus infection and may provide a therapeutic target for virus-induced CNS disease.
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http://dx.doi.org/10.1128/JVI.00583-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988164PMC
September 2016

IL-4 confers resistance to IL-27-mediated suppression on CD4+ T cells by impairing signal transducer and activator of transcription 1 signaling.

J Allergy Clin Immunol 2013 Oct 16;132(4):912-21.e1-5. Epub 2013 Aug 16.

Division of Allergy and Immunology, Department of Medicine, National Jewish Health, Denver, Colo; Zhangshan Hospital, Fudan University, Shanghai, China.

Background: TH2 cells play a critical role in the pathogenesis of allergic asthma. Established TH2 cells have been shown to resist reprogramming into TH1 cells. The inherent stability of TH2 cells poses a significant barrier to treating allergic diseases.

Objective: We sought to understand the mechanisms by which CD4(+) T cells from asthmatic patients resist the IL-27-mediated inhibition.

Methods: We isolated and cultured CD4(+) T cells from both healthy subjects and allergic asthmatic patients to test whether IL-27 can inhibit IL-4 production by the cultured CD4(+) T cells using ELISA. Culturing conditions that resulted in resistance to IL-27 were determined by using both murine and human CD4(+) T-cell culture systems. Signal transducer and activator of transcription (STAT) 1 phosphorylation was analyzed by means of Western blotting and flow cytometry. Suppressor of cytokine signaling (Socs) mRNA expression was measured by using quantitative PCR. The small interfering RNA method was used to knockdown the expression of Socs3 mRNA.

Results: We demonstrated that CD4(+) T cells from asthmatic patients resisted the suppression of IL-4 production mediated by IL-27. We observed that repeated exposure to TH2-inducing conditions rendered healthy human CD4(+) T cells resistant to IL-27-mediated inhibition. Using an in vitro murine culture system, we further demonstrated that repeated or higher doses of IL-4 stimulation, but not IL-2 stimulation, upregulated Socs3 mRNA expression and impaired IL-27-induced STAT1 phosphorylation. The knockdown of Socs3 mRNA expression restored IL-27-induced STAT1 phosphorylation and IL-27-mediated inhibition of IL-4 production.

Conclusions: Our findings demonstrate that differentiated TH2 cells can resist IL-27-induced reprogramming toward TH1 cells by downregulating STAT1 phosphorylation and likely explain why the CD4(+) T cells of asthmatic patients are resistant to IL-27-mediated inhibition.
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http://dx.doi.org/10.1016/j.jaci.2013.06.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788709PMC
October 2013

Antagonistic regulation by the transcription factors C/EBPα and MITF specifies basophil and mast cell fates.

Immunity 2013 Jul 18;39(1):97-110. Epub 2013 Jul 18.

Department of Medicine, Division of Allergy and Immunology, National Jewish Health, 1400 Jackson Street, Denver, CO 80206, USA.

It remains unclear whether basophils and mast cells are derived from a common progenitor. Furthermore, how basophil versus mast cell fate is specified has not been investigated. Here, we have identified a population of granulocyte-macrophage progenitors (GMPs) that were highly enriched in the capacity to differentiate into basophils and mast cells while retaining a limited capacity to differentiate into myeloid cells. We have designated these progenitor cells "pre-basophil and mast cell progenitors" (pre-BMPs). STAT5 signaling was required for the differentiation of pre-BMPs into both basophils and mast cells and was critical for inducing two downstream molecules: C/EBPα and MITF. We have identified C/EBPα as the critical basophil transcription factor for specifying basophil cell fate and MITF as the crucial transcription factor for specifying mast cell fate. C/EBPα and MITF silenced each other's transcription in a directly antagonistic fashion. Our study reveals how basophil and mast cell fate is specified.
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http://dx.doi.org/10.1016/j.immuni.2013.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3755602PMC
July 2013

IFN-γ suppresses permissive chromatin remodeling in the regulatory region of the Il4 gene.

Cytokine 2013 Apr 13;62(1):91-5. Epub 2013 Mar 13.

Division of Allergy and Immunology, Department of Medicine, National Jewish Health, Denver, CO 80206, USA.

In order to develop the most effective T helper type-1 (Th1) immunity, naïve CD4(+) T cells must acquire the capacity to express IFN-γ while silencing T helper type-2 (Th2) cytokine-producing potential. An Il4 gene silencer has been described. However, it is not completely understood how the silencer works. In this study, we examine whether IFN-γ can suppress permissive chromatin remodeling of regulatory region of the Il4 gene. We demonstrate that IFN-γ suppresses H3K4 dimethylation at the intronic enhancer region of the Il4 gene. The IFN-γ-mediated suppression of permissive chromatin remodeling is IFN-γ receptor-, STAT1-, and T-bet-dependent. Our study reveals a novel mechanism of how Th1 cells silence the Il4 gene.
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http://dx.doi.org/10.1016/j.cyto.2013.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3615094PMC
April 2013

Daxx upregulation within the cytoplasm of reovirus-infected cells is mediated by interferon and contributes to apoptosis.

J Virol 2013 Mar 9;87(6):3447-60. Epub 2013 Jan 9.

University of Colorado, Denver, Anschutz Medical Campus, Aurora, CO, USA.

Reovirus infection is a well-characterized experimental system for the study of viral pathogenesis and antiviral immunity within the central nervous system (CNS). We have previously shown that c-Jun N-terminal kinase (JNK) and the Fas death receptor each play a role in neuronal apoptosis occurring in reovirus-infected brains. Death-associated protein 6 (Daxx) is a cellular protein that mechanistically links Fas signaling to JNK signaling in several models of apoptosis. In the present study, we demonstrate that Daxx is upregulated in reovirus-infected brain tissue through a type I interferon-mediated mechanism. Daxx upregulation is limited to brain regions that undergo reovirus-induced apoptosis and occurs in the cytoplasm and nucleus of neurons. Cytoplasmic Daxx is present in Fas-expressing cells during reovirus encephalitis, suggesting a role for Daxx in Fas-mediated apoptosis following reovirus infection. Further, in vitro expression of a dominant negative form of Daxx (DN-Daxx), which binds to Fas but which does not transmit downstream signaling, inhibits apoptosis of reovirus-infected cells. In contrast, in vitro depletion of Daxx results in increased expression of caspase 3 and apoptosis, suggesting that Daxx plays an antiapoptotic role in the nucleus. Overall, these data imply a regulatory role for Daxx in reovirus-induced apoptosis, depending on its location in the nucleus or cytoplasm.
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http://dx.doi.org/10.1128/JVI.02324-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592169PMC
March 2013

Ara h 2 and Ara h 6 have similar allergenic activity and are substantially redundant.

Int Arch Allergy Immunol 2013 16;160(3):251-8. Epub 2012 Oct 16.

Division of Allergy and Clinical Immunology, Department of Medicine, University of Colorado Denver, Aurora, CO 80045, USA.

Background: The moderately homologous (approx. 60%) proteins Ara h 2 and Ara h 6 are the most potent peanut allergens. This study was designed to define the relative individual contributions of Ara h 2 and Ara h 6 to the overall allergenic activity of a crude peanut extract (CPE).

Methods: Ara h 2 and Ara h 6 were removed from CPE by gel filtration chromatography. Ara h 2.01, Ara h 2.02 and Ara h 6 were further purified (>99%). The potency of each allergen and the ability of these allergens to reconstitute the allergenic activity of CPE depleted of Ara h 2 and Ara h 6 was measured with RBL SX-38 cells sensitized with IgE from sensitized peanut allergic patients.

Results: The potency of the native proteins were significantly different (p < 0.0001) although not dramatically so, with a rank order of Ara h 2.01 > Ara h 2.02 > Ara h 6. The addition of either purified Ara h 2 or Ara h 6 independently at their original concentration to CPE depleted of both Ara h 2 and Ara h 6 restored 80-100% of the original CPE allergenic activity. Addition of both Ara h 2 and Ara h 6 consistently completely restored the allergenic activity of CPE.

Conclusions: These studies indicate that either Ara h 2 or Ara h 6 independently can account for most of the allergenic activity in a CPE and demonstrate important redundancy in the allergenic activity of these related molecules.
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http://dx.doi.org/10.1159/000341642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3652334PMC
April 2013

Redefining the major peanut allergens.

Immunol Res 2013 Mar;55(1-3):125-34

Division of Allergy and Clinical Immunology, Department of Medicine, University of Colorado School of Medicine, Aurora, CO 80045, USA.

Food allergy has become a major public health concern in westernized countries, and allergic reactions to peanuts are particularly common and severe. Allergens are defined as antigens that elicit an IgE response, and most allergenic materials (e.g., pollens, danders, and foods) contain multiple allergenic proteins. This has led to the concept that there are "major" allergens and allergens of less importance. "Major allergens" have been defined as allergens that bind a large amount of IgE from the majority of patients and have biologic activity. However, the ability of an allergen to cross-link complexes of IgE and its high-affinity receptor FcεRI (IgE/FcεRI), which we have termed its allergic effector activity, does not correlate well with assays of IgE binding. To identify the proteins that are the most active allergens in peanuts, we and others have employed in vitro model assays of allergen-mediated cross-linking of IgE/FcεRI complexes and have demonstrated that the most potent allergens are not necessarily those that bind the most IgE. The importance of a specific allergen can be determined by measuring the allergic effector activity of that allergen following purification under non-denaturing conditions and by specifically removing the allergen from a complex allergenic extract either by chromatography or by specific immunodepletion. In our studies of peanut allergens, our laboratory has found that two related allergens, Ara h 2 and Ara h 6, together account for the majority of the effector activity in a crude peanut extract. Furthermore, murine studies demonstrated that Ara h 2 and Ara h 6 are not only the major elicitors of anaphylaxis in this system, but also can effectively desensitize peanut-allergic mice. As a result of these observations, we propose that the definition of a major allergen should be based on the potency of that allergen in assays of allergic effector activity and demonstration that removal of that allergen from an extract results in loss of potency. Using these criteria, Ara h 2 and Ara h 6 are the major peanut allergens.
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http://dx.doi.org/10.1007/s12026-012-8355-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4451826PMC
March 2013

Expression of recombinant Ara h 6 in Pichia pastoris but not in Escherichia coli preserves allergic effector function and allows assessment of specific mutations.

Mol Nutr Food Res 2012 Jun;56(6):986-95

Department of Medicine, Division of Allergy and Clinical Immunology, University of Colorado Denver, Aurora, CO 80045, USA.

Scope: Ara h 6 has recently been recognized as an important peanut allergen. Recombinant allergens have been used for analysis of IgE binding, but have not been used to analyze the allergic effector activity that is more relevant to allergic reactions.

Methods And Results: Ara h 6 was expressed as a recombinant protein in both Escherichia coli and Pichia pastoris (rAra h 6-E. coli and rAra h 6-Pichia, respectively). Effector activity was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from patients with severe peanut allergy. Compared to native Ara h 6 (nAra h 6), rAra h 6-Pichia had intact effector function whereas rAra h 6-E. coli had significantly reduced function. The lower effector activity in rAra h 6-E. coli compared to nAra h 6 and rAra h 6-Pichia did not appear to be due to differences in posttranslational modifications (analyzed by mass spectrometry and staining for carbohydrates) and may be due to subtle alteration(s) of folding seen on CD analysis and on nonreduced gels. Finally, we introduced point mutations in four important IgE-binding linear epitopes of Ara h 6 and found dramatically reduced allergic effector activity.

Conclusion: Our studies demonstrate the utility of fully functional rAra h 6-Pichia as a starting point for analysis of specific mutations that adversely affect allergic effector function.
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http://dx.doi.org/10.1002/mnfr.201100827DOI Listing
June 2012

ERK1 is important for Th2 differentiation and development of experimental asthma.

FASEB J 2012 May 18;26(5):1934-45. Epub 2012 Jan 18.

Division of Allergy and Immunology, National Jewish Health, 1400 Jackson Street, Denver, CO 80206, USA.

The ERK1/2 signaling pathway regulates a variety of T-cell functions. We observed dynamic changes in the expression of ERK1/2 during T-helper cell differentiation. Specifically, the expression of ERK1/2 was decreased and increased by IL-12 and IL-4, respectively. To address this subject further, we examined the specific role of ERK1 in Th2 differentiation and development of experimental asthma using ERK1(-/-) mice. ERK1(-/-) mice were unable to mount airway inflammation and hyperreactivity in two different models of asthma, acute and chronic. ERK1(-/-) mice had reduced expression of Th2 cytokines IL-4 and IL-5 but not IL-17A or IFN-γ. They had reduced levels of allergen-specific IgE and blood eosinophils. T cells from immunized ERK1(-/-) mice manifested reduced proliferation in response to the sensitizing allergen. ERK1(-/-) T cells had reduced and short-lived expression of JunB following TCR stimulation, which likely contributed to their impaired Th2 differentiation. Immunized ERK1(-/-) mice showed reduced numbers of CD44(high) CD4 T cells in the spleen. In vitro studies demonstrated that Th2 but not Th1 cells from ERK1(-/-) mice had reduced numbers of CD44(high) cells. Finally, CD4 T cells form ERK1(-/-) mice expressed higher levels of BIM under growth factor-deprived conditions and reduced Mcl-1 on stimulation. As a result, the survival of CD4 T cells, especially CD44(high) Th2 cells, was much reduced in ERK1(-/-) mice. We conclude that ERK1 plays a nonredundant role in Th2 differentiation and development of experimental asthma. ERK1 controls Th2 differentiation and survival through its effect on JunB and BIM, respectively.
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http://dx.doi.org/10.1096/fj.11-196477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3336776PMC
May 2012

Analysis of the effector activity of Ara h 2 and Ara h 6 by selective depletion from a crude peanut extract.

J Immunol Methods 2011 Sep 18;372(1-2):65-70. Epub 2011 Jul 18.

Division of Allergy and Clinical Immunology, Department of Medicine, University of Colorado Denver, Aurora, CO 80045, USA.

It is important to know the contribution of specific allergens to a complex allergenic extract and to have a dependable method to assess the effector activity of an extract specifically depleted of that allergen. We have previously shown that removal of the major peanut allergen, Ara h 2, from a crude peanut extract (CPE) minimally altered the effector activity of the extract. Here we describe in detail the methodology used to generate specific rabbit anti-peptide antibodies to remove a related peanut allergen, Ara h 6, from CPE and describe an improvement in the RBL SX-38 cell assay used to assess the effector activity of treated extracts. Our results show that although Ara h 2 and Ara h 6 can be selectively removed from a CPE, removal of each alone from a CPE had no significant effect on the effector activity. However, removal of Ara h 2 and Ara h 6 together significantly reduced the effector activity of CPE.
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http://dx.doi.org/10.1016/j.jim.2011.06.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170491PMC
September 2011

A continuous T-bet expression is required to silence the interleukin-4-producing potential in T helper type 1 cells.

Immunology 2009 Sep;128(1):34-42

Division of Allergy and Immunology, Department of Medicine, National Jewish Health, Denver, CO, USA.

To develop into committed T helper type 1 (Th1) cells, naive CD4(+) T cells not only need to acquire the capacity to produce interferon-gamma (IFN-gamma), but they also need to gain the ability to silence their interleukin-4 (IL-4) -producing potential. How Th1 cells silence their Th2 cytokine-producing potential is an important yet unresolved issue in Th1 immunity. We found that a lack of IL-4 stimulation was not sufficient to silence the IL-4-producing potential in activated CD4(+) T cells and that Th1-promoting factor was required. Although it has been shown that T-bet is a crucial factor in suppressing Il4 gene expression, it is unclear whether a continuous presence of T-bet is required to silence the Il4 gene in Th1 cells. To address this problem, we used an inducible form of T-bet - a T-bet-oestrogen receptor fusion molecule (T-bet-ER). We found that the activation of T-bet during primary or secondary culture was sufficient to silence IL-4-producing potential. On the other hand, the inactivation of T-bet after naïve CD4(+) T cells had differentiated into Th1 cells resulted in derepression of Il4 gene transcription. Additionally, we found that T-bet is required to maintain Ifng expression. Our data demonstrate that the continuous expression of T-bet is required for Th1 cells to silence their IL-4-producing potential.
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http://dx.doi.org/10.1111/j.1365-2567.2009.03049.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2747137PMC
September 2009

Signaling pathways that lead to the silencing of the interleukin-4-producing potential in Th1 cells.

J Interferon Cytokine Res 2009 Jul;29(7):399-406

Division of Allergy and Clinical Immunology, Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.

In order to develop the most effective Th1 immunity, naïve CD4(+) T cells must acquire the capacity to induce the expression of IFN-gamma and to silence Th2 cytokine-producing potential. Although the IFN-gamma-STAT1 and the IL-12-STAT4 pathways have been demonstrated to be important in inducing the IFN-gamma-producing capacity in Th1 cells, their respective roles in silencing the IL-4-producing potential in Th1 cells remain unclear. In this study, we investigated the role of the IFN-gamma and the IL-12 pathways in silencing the IL-4-producing potential in Th1 cells. We found that IFN-gamma was essential to silence the IL-4-producing potential in Th1 cells, while IL-12 only partially suppressed the IL-4-producing potential. IFN-gamma depended on STAT1 and IL-12 depended on STAT4 to suppress the IL-4-producing potential. We showed that the IL-12-STAT4 pathway and the IFN-gamma-STAT1 pathway converge at the point of T-bet. Our study demonstrates that the IFN-gamma-STAT1-T-bet signaling pathway is the major pathway that leads to silencing the IL-4-producing potential of Th1 cells.
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http://dx.doi.org/10.1089/jir.2008.0093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956644PMC
July 2009
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