Publications by authors named "Aaron Winkler"

27 Publications

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and functional characterization of human HLA-DRB1∗04 restricted T cell receptors.

J Transl Autoimmun 2021 3;4:100087. Epub 2021 Mar 3.

Division of Rheumatology, Department of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden.

Recent advances in single-cell sequencing technologies enable the generation of large-scale data sets of paired TCR sequences from patients with autoimmune disease. Methods to validate and characterize patient-derived TCR data are needed, as well as relevant model systems that can support the development of antigen-specific tolerance inducing drugs. We have generated a pipeline to allow streamlined generation of 'artificial' T cells in a robust and reasonably high throughput manner for and studies of antigen-specific and patient-derived immune responses. Hereby chimeric (mouse-human) TCR alpha and beta constructs are re-expressed in three different formats for further studies: () transiently in HEK cells for peptide-HLA tetramer validation experiments, () stably in the TCR-negative 58 ​T cell line for functional readouts such as IL-2 production and NFAT-signaling, and lastly () in human HLA-transgenic mice for studies of autoimmune disease and therapeutic interventions. As a proof of concept, we have used human HLA-DRB1∗04:01 restricted TCR sequences specific for a type I diabetes-associated GAD peptide, and an influenza-derived HA peptide. We show that the same chimeric TCR constructs can be used in each of the described assays facilitating sequential validation and prioritization steps leading to humanized animal models.
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http://dx.doi.org/10.1016/j.jtauto.2021.100087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980064PMC
March 2021

Cannabis Legalization and College Mental Health.

Curr Psychiatry Rep 2021 Mar 3;23(4):17. Epub 2021 Mar 3.

College Mental Health Program, Department of Psychiatry & Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, USA.

Purpose Of Review: To assess how the changing landscape of marijuana use affects the developing brain and mental health of college students.

Recent Findings: Legalization of cannabis may facilitate use in the college population, with 38% of college students, whose brains are still maturing, regularly using marijuana products. Earlier and increased use, higher potency, pre-existing issues, and genetic predispositions increase negative outcomes by precipitating or worsening mental illness and ultimately impacting academic success. In the USA, the sharpest increase in cannabis users following legalization has been in the college age population (18-25 years of age). This population is especially vulnerable to the negative impacts and risks associated with cannabis use, including risk for the onset of major psychiatric illness. College mental health practitioners should remain informed about health effects of cannabis use, assess patient use on a regular basis, provide education and be familiar with interventions to reduce harm.
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http://dx.doi.org/10.1007/s11920-021-01231-1DOI Listing
March 2021

College Student Perspectives of Telemental Health: a Review of the Recent Literature.

Curr Psychiatry Rep 2021 Jan 6;23(2). Epub 2021 Jan 6.

Department of Psychiatry & Behavioral Sciences, Stanford University School of Medicine, Stanford, CA, USA.

Purpose Of Review: We review the recent literature regarding college student experiences with and attitudes toward telemental health (TMH). We examine their perspectives of the advantages and drawbacks to this form of mental healthcare and their willingness to engage in TMH.

Recent Findings: College students view TMH as convenient, accessible, easy to use, and helpful. TMH helps to overcome the barrier of stigma associated with seeking mental health treatment. Despite positive reviews, many students find a lack of customization or connection to the provider to be drawbacks to some forms of TMH. Willingness to engage in TMH varies based on prior experience with mental health treatment, ethnicity, and severity of symptoms. The recent literature highlights the potential for TMH to play a key role in mental health services for college students. It also highlights some of its shortcomings, which are indicative of the continued need for in-person services. Future studies should continue to track college student perspectives toward and utilization of TMH.
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http://dx.doi.org/10.1007/s11920-020-01215-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785477PMC
January 2021

Optimized protocols for studying the NLRP3 inflammasome and assessment of potential targets of CP-453,773 in undifferentiated THP1 cells.

J Immunol Methods 2019 04 5;467:19-28. Epub 2019 Feb 5.

Inflammation & Immunology Research Unit, Pfizer, Cambridge, MA 02139, United States. Electronic address:

The NLRP3 inflammasome is a complex multimeric signaling apparatus that regulates production of the pro-inflammatory cytokine IL-1β. To overcome both the variability among primary immune cells and the limitations of genetic manipulation of differentiated human or murine macrophages, we developed a simplified, reliable and relevant cell-based model for studying the NLRP3 inflammasome using the undifferentiated human myelomonocytic cell line THP1. Undifferentiated THP1 cells constitutively express NLRP3, and NLRP3 inflammasome activation occurred in response to canonical NLRP3 activation stimuli including nigericin, ATP, and urea crystals, culminating in pro-IL-1β cleavage, extracellular release of mature IL-1β, and pyroptosis. We used this THP1 cell system to investigate potential targets of the potent, NLRP3 inflammasome selective inhibitor CP-456,773. We optimized a viral shRNA transduction method for gene expression knockdown (KD), and the KD of NLRP3 itself eliminated inflammasome activation and IL-1β production. NLRP3 inflammasome activation and CP-453,773 pharmacology were not altered in ABCb7- or ABCb10-deficient THP1 cells, eliminating these gene products as candidate pharmacological targets of CP-453,773. For ABCb10, we confirmed our results using CRISPR/CAS9-mediated ABCb10 knockout (KO) THP1 sub-lines. In summary, undifferentiated THP1 cells are fully competent for activation of the NLRP3 inflammasome and production of IL-1β, without differentiation into macrophages, and we describe optimized KD and KO methodologies to manipulate gene expression in these cells. As an example of the utility of undifferentiated THP1 cells for investigations into the biology of the NLRP3 inflammasome, we have used this cell system to rule out ABCb7 and ABCb10 as potential targets of the NLRP3 inflammasome inhibitor CP-453,773.
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http://dx.doi.org/10.1016/j.jim.2019.02.002DOI Listing
April 2019

B Cell-Intrinsic Role for IRF5 in TLR9/BCR-Induced Human B Cell Activation, Proliferation, and Plasmablast Differentiation.

Front Immunol 2017 10;8:1938. Epub 2018 Jan 10.

Center for Autoimmune Musculoskeletal and Hematopoietic Diseases, The Feinstein Institute for Medical Research, Manhasset, NY, United States.

Upon recognition of antigen, B cells undergo rapid proliferation followed by differentiation to specialized antibody secreting cells (ASCs). During this transition, B cells are reliant upon a multilayer transcription factor network to achieve a dramatic remodeling of the B cell transcriptional landscape. Increased levels of ASCs are often seen in autoimmune diseases and it is believed that altered expression of regulatory transcription factors play a role in this imbalance. The transcription factor interferon regulatory factor 5 (IRF5) is one such candidate as polymorphisms in associate with risk of numerous autoimmune diseases and correlate with elevated expression. genetic risk has been widely replicated in systemic lupus erythematosus (SLE), and loss of ameliorates disease in murine lupus models, in part, through the lack of pathogenic autoantibody secretion. It remains unclear, however, whether IRF5 is contributing to autoantibody production through a B cell-intrinsic function. To date, IRF5 function in healthy human B cells has not been characterized. Using human primary naive B cells, we define a critical intrinsic role for IRF5 in B cell activation, proliferation, and plasmablast differentiation. Targeted IRF5 knockdown resulted in significant immunoglobulin (Ig) D retention, reduced proliferation, plasmablast differentiation, and IgG secretion. The observed decreases were due to impaired B cell activation and clonal expansion. Distinct from murine studies, we identify and confirm new IRF5 target genes, , and , and pathways that mediate IRF5 B cell-intrinsic function. Together, these results identify IRF5 as an early regulator of human B cell activation and provide the first dataset in human primary B cells to map IRF5 dysfunction in SLE.
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http://dx.doi.org/10.3389/fimmu.2017.01938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768180PMC
January 2018

IRAK4 kinase activity controls Toll-like receptor-induced inflammation through the transcription factor IRF5 in primary human monocytes.

J Biol Chem 2017 11 18;292(45):18689-18698. Epub 2017 Sep 18.

From the Departments of Inflammation and Immunology and

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate immune signaling by Toll-like receptors (TLRs), and loss of IRAK4 activity in mice and humans increases susceptibility to bacterial infections and causes defects in TLR and IL1 ligand sensing. However, the mechanism by which IRAK4 activity regulates the production of downstream inflammatory cytokines is unclear. Using transcriptomic and biochemical analyses of human monocytes treated with a highly potent and selective inhibitor of IRAK4, we show that IRAK4 kinase activity controls the activation of interferon regulatory factor 5 (IRF5), a transcription factor implicated in the pathogenesis of multiple autoimmune diseases. Following TLR7/8 stimulation by its agonist R848, chemical inhibition of IRAK4 abolished IRF5 translocation to the nucleus and thus prevented IRF5 binding to and activation of the promoters of inflammatory cytokines in human monocytes. We also found that IKKβ, an upstream IRF5 activator, is phosphorylated in response to the agonist-induced TLR signaling. Of note, IRAK4 inhibition blocked IKKβ phosphorylation but did not block the nuclear translocation of NFκB, which was surprising, given the canonical role of IKKβ in phosphorylating IκB to allow NFκB activation. Moreover, pharmacological inhibition of either IKKβ or the serine/threonine protein kinase TAK1 in monocytes blocked TLR-induced cytokine production and IRF5 translocation to the nucleus, but not nuclear translocation of NFκB. Taken together, our data suggest a mechanism by which IRAK4 activity regulates TAK1 and IKKβ activation, leading to the nuclear translocation of IRF5 and induction of inflammatory cytokines in human monocytes.
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http://dx.doi.org/10.1074/jbc.M117.796912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682975PMC
November 2017

Smoke exposure of human macrophages reduces HDAC3 activity, resulting in enhanced inflammatory cytokine production.

Pulm Pharmacol Ther 2012 Aug 18;25(4):286-92. Epub 2012 May 18.

Department of Inflammation and Remodeling, BioTherapeutics Research and Development, Pfizer, Inc., 200 Cambridge Park Dr., Cambridge, MA 02474, USA.

Chronic obstructive pulmonary disease (COPD) is a debilitating condition resulting from exposure to pollutants such as cigarette smoke. Pulmonary macrophages secrete a plethora of inflammatory mediators that are increased in the lungs of COPD patients, but whether this phenotype results directly from smoke exposure remains unknown. Using an in vitro model for alveolar macrophages (AM) derived from human peripheral blood monocytes with granulocyte-macrophage stimulating factor (GM-MØ), we analyzed the mechanistic connection between cigarette smoke exposure and histone deacetylase (HDAC) regulation, hypothesized to be a contributing factor in COPD pathophysiology. Here we show that acute smoke exposure inhibits HDAC enzymatic activity in GM-MØ. Analysis of mRNA and total cellular proteins for expression of class I (1, 2, 3 and 8), class II (4, 5, 6, 7, 9, 10), and class IV (11) HDAC revealed no effect of smoke exposure, whereas nuclear HDAC3 protein content was reduced. To better understand the physiological significance of reduced HDAC3 activity, we utilized siRNA to knockdown HDAC1, 2 and 3 individually. Interestingly, siRNA-mediated reduction of HDAC3 resulted in increased production of IL8 and IL1β in response to LPS stimulation, while HDAC2 knockdown had no effect on either cytokine. Lower nuclear content of HDAC3 in the context of equivalent total HDAC protein levels following smoke exposure may reflect increased nuclear export of HDAC3, allowing increased nuclear factor kappa b (NF-κB ) driven cytokine expression that can contribute to inflammation.
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http://dx.doi.org/10.1016/j.pupt.2012.05.003DOI Listing
August 2012

Acidic mammalian chitinase is not a critical target for allergic airway disease.

Am J Respir Cell Mol Biol 2012 Jan;46(1):71-9

Inflammation and Immunology Research Unit, Pfizer, Cambridge, Massachusetts 02140, USA.

The expression of acidic mammalian chitinase (AMCase) is associated with Th2-driven respiratory disorders. To investigate the potentially pathological role of AMCase in allergic airway disease (AAD), we sensitized and challenged mice with ovalbumin or a combination of house dust mite (HDM) plus cockroach allergen. These mice were treated or not treated with small molecule inhibitors of AMCase, which significantly reduced allergen-induced chitinolytic activity in the airways, but exerted no apparent effect on pulmonary inflammation per se. Transgenic and AMCase-deficient mice were also submitted to protocols of allergen sensitization and challenge, yet we found little or no difference in the pattern of AAD between mutant mice and wild-type (WT) control mice. In a separate model, where mice were challenged only with intratracheal instillations of HDM without adjuvant, total bronchoalveolar lavage (BAL) cellularity, inflammatory infiltrates in lung tissues, and lung mechanics remained comparable between AMCase-deficient mice and WT control mice. However BAL neutrophil and lymphocyte counts were significantly increased in AMCase-deficient mice, whereas concentrations in BAL of IL-13 were significantly decreased compared with WT control mice. These results indicate that, although exposure to allergen stimulates the expression of AMCase and increased chitinolytic activity in murine airways, the overexpression or inhibition of AMCase exerts only a subtle impact on AAD. Conversely, the increased numbers of neutrophils and lymphocytes in BAL and the decreased concentrations of IL-13 in AMCase-deficient mice challenged intratracheally with HDM indicate that AMCase contributes to the Th1/Th2 balance in the lungs. This finding may be of particular relevance to patients with asthma and increased airway neutrophilia.
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http://dx.doi.org/10.1165/rcmb.2011-0095OCDOI Listing
January 2012

Complement C3a, CpG oligos, and DNA/C3a complex stimulate IFN-α production in a receptor for advanced glycation end product-dependent manner.

J Immunol 2010 Oct 3;185(7):4213-22. Epub 2010 Sep 3.

Inflammation and Immunology, Pfizer Biotherapeutics Research and Development, Cambridge, MA 02140, USA.

The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor implicated in a number of diseases including autoimmune diseases. To further understand the pathogenic mechanism of RAGE in these diseases, we searched for additional ligands. We discovered that C3a bound to RAGE with an EC(50) of 1.9 nM in an ELISA, and the binding was increased both in magnitude (by >2-fold) and in affinity (EC(50) 70 pM) in the presence of human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs). Surface plasmon resonance and fluorescence anisotropy analyses demonstrated that hCpGAs could bind directly to RAGE and C3a and form a ternary complex. In human PBMCs, C3a increased IFN-α production in response to low levels of hCpGAs, and this synergy was blocked by soluble RAGE or by an Ab directed against RAGE. IFN-α production was reduced in response to mouse CpGAs and C3a in RAGE(-/-) mouse bone marrow cells compared wild-type mice. Taken together, these data demonstrate that RAGE is a receptor for C3a and CpGA. Through direct interaction, C3a and CpGA synergize to increase IFN-α production in a RAGE-dependent manner and stimulate an innate immune response. These findings indicate a potential role of RAGE in autoimmune diseases that show accumulation of immunostimulatory DNA and C3a.
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http://dx.doi.org/10.4049/jimmunol.1000863DOI Listing
October 2010

Evolutionary and biochemical differences between human and monkey acidic mammalian chitinases.

Gene 2010 Mar 28;452(2):63-71. Epub 2009 Dec 28.

Pfizer Biotherapeutics Research and Development, Department of Inflammation and Immunology, 200 CambridgePark Drive, Cambridge, MA 02140, USA.

Acidic mammalian chitinase (AMCase), an enzyme implicated in the pathology of asthma, is capable of chitin cleavage at a low pH optimum. The corresponding gene (CHIA) can be found in genome databases of a variety of mammals, but the enzyme properties of only the human and mouse proteins were extensively studied. We wanted to compare enzymes of closely related species, such as humans and macaques. In our attempt to study macaque AMCase, we searched for CHIA-like genes in human and macaque genomes. We found that both genomes contain several additional CHIA-like sequences. In humans, CHIA-L1 (hCHIA-L1) is an apparent pseudogene and has the highest homology to CHIA. To determine which of the two genes is functional in monkeys, we assessed their tissue expression levels. In our experiments, CHIA-L1 expression was not detected in human stomach tissue, while CHIA was expressed at high levels. However, in the cynomolgus macaque stomach tissue, the expression pattern of these two genes was reversed: CHIA-L1 was expressed at high levels and CHIA was undetectable. We hypothesized that in macaques CHIA-L1 (mCHIA-L1), and not CHIA, is a gene encoding an acidic chitinase, and cloned it, using the sequence of human CHIA-L1 as a guide for the primer design. We named the new enzyme MACase (Macaca Acidic Chitinase) to emphasize its differences from AMCase. MACase shares a similar tissue expression pattern and pH optimum with human AMCase, but is 50 times more active in our enzymatic activity assay. DNA sequence of the mCHIA-L1 has higher percentage identity to the human pseudogene hCHIA-L1 (91.7%) than to hCHIA (84%). Our results suggest alternate evolutionary paths for human and monkey acidic chitinases.
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http://dx.doi.org/10.1016/j.gene.2009.12.005DOI Listing
March 2010

In vitro modeling of human alveolar macrophage smoke exposure: enhanced inflammation and impaired function.

Exp Lung Res 2008 Nov;34(9):599-629

Wyeth Research, Cambridge, Massachusetts 02140, USA.

Pulmonary macrophages (MØs) are essential for clearance of inhaled particles, innate immunity, and lung tissue maintenance. However, the products of activated MØs have also been implicated in inflammation and tissue destruction, including in chronic obstructive pulmonary disease (COPD). Primary human alveolar macrophages (AMs) are available in limited numbers via bronchoalveolar lavage (BAL) or sputum induction, and BAL macrophages are not commonly available to all researchers. A readily available, plentiful, but representative surrogate for AMs would advance understanding of the contribution of macrophages to lung pathophysiology. Herein the authors describe a method for the in vitro derivation of AM-like cells using primary human peripheral blood monocytes differentiated in suspension with granulocyte-macrophage colony-stimulating factor (GM-CSF). The method produces a cell population with a consistent and stable phenotype. Flow cytometry reveals that GM-CSF-derived macrophages (GM-MØs) express lineage markers, immunoglobulin gamma (Fc gamma) receptors, adhesion molecules, antigen presentation coreceptors, and scavenger receptors akin to AMs. Functionally, cigarette smoke activates extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, enhances interleukin 8 (IL8) production from GM-MØs and inhibits phagocytosis, phenotypes previously described for smokers' AMs. Global transcriptional profiling revealed significant overlap in regulated genes between smokers' AMs and GM-MØs treated with cigarette smoke preparations in vitro.
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http://dx.doi.org/10.1080/01902140802366261DOI Listing
November 2008

Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: analysis by scanning cytometry.

Respir Res 2008 Aug 7;9:59. Epub 2008 Aug 7.

Harvard School of Public Health, Molecular and Integrative Physiological Sciences Program, 655 Huntington Ave, Building II, 2nd Floor, Boston, MA 02115, USA.

Background: Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Mice with genetic deletions of scavenger receptors, such as SR-A and MARCO, are susceptible to infection or inflammation from inhaled pathogens or dusts. However, the signaling pathways required for scavenger receptor-mediated phagocytosis of unopsonized particles have not been characterized.

Methods: We developed a scanning cytometry-based high-throughput assay of macrophage phagocytosis that quantitates bound and internalized unopsonized latex beads. This assay allowed the testing of a panel of signaling inhibitors which have previously been shown to target opsonin-dependent phagocytosis for their effect on unopsonized bead uptake by human in vitro-derived alveolar macrophage-like cells. The non-selective scavenger receptor inhibitor poly(I) and the actin destabilizer cytochalasin D were used to validate the assay and caused near complete abrogation of bead binding and internalization, respectively.

Results: Microtubule destabilization using nocodazole dramatically inhibited bead internalization. Internalization was also significantly reduced by inhibitors of tyrosine kinases (genistein and herbimycin A), protein kinase C (staurosporine, chelerythrine chloride and Gö 6976), phosphoinositide-3 kinase (LY294002 and wortmannin), and the JNK and ERK pathways. In contrast, inhibition of phospholipase C by U-73122 had no effect.

Conclusion: These data indicate the utility of scanning cytometry for the analysis of phagocytosis and that phagocytosis of unopsonized particles has both shared and distinct features when compared to opsonin-mediated phagocytosis.
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http://dx.doi.org/10.1186/1465-9921-9-59DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2527561PMC
August 2008

Expression of the cysteine protease legumain in vascular lesions and functional implications in atherogenesis.

Atherosclerosis 2008 Nov 21;201(1):53-66. Epub 2008 Feb 21.

Department of Cardiovascular and Metabolic Diseases, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA.

Objective: The present study was conducted to characterize the expression of the cysteine protease legumain in murine and human atherosclerotic tissues, and to explore the molecular mechanisms by which legumain may contribute to the pathophysiology of atherosclerosis.

Methods And Results: Using microarray analysis, legumain mRNA expression was found to increase with development of atherosclerosis in the aorta of aging Apolipoprotein E deficient mice while expression remained at low level and unchanged in arteries of age-matched C57BL/6 control mice. In situ hybridization and immunohistochemical analysis determined that legumain was predominantly expressed by macrophages in the atherosclerotic aorta, in lesions at the aortic sinus and in injured carotid arteries of Apolipoprotein E deficient mice as well as in inflamed areas in advanced human coronary atherosclerotic plaques. In vitro, M-CSF differentiated human primary macrophages were shown to express legumain and the protein could also be detected in the culture media. When tested in migration assays, legumain induced chemotaxis of primary human monocytes and human umbilical vein endothelial cells.

Conclusions: Legumain is expressed in both murine and human atherosclerotic lesions. The macrophage-specific expression of legumain in vivo and ability of legumain to induce chemotaxis of monocytes and endothelial cells in vitro suggest that legumain may play a functional role in atherogenesis.
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http://dx.doi.org/10.1016/j.atherosclerosis.2008.01.016DOI Listing
November 2008

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export.

Appl Environ Microbiol 2008 May 14;74(9):2766-77. Epub 2008 Mar 14.

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.
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http://dx.doi.org/10.1128/AEM.02591-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2394876PMC
May 2008

Pharmacologic inhibition of tpl2 blocks inflammatory responses in primary human monocytes, synoviocytes, and blood.

J Biol Chem 2007 Nov 11;282(46):33295-33304. Epub 2007 Sep 11.

Department of Inflammation, Wyeth Research, Cambridge, Massachusetts, 02140. Electronic address:

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.
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http://dx.doi.org/10.1074/jbc.M703694200DOI Listing
November 2007

Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component.

Adv Biochem Eng Biotechnol 2007 ;108:179-204

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands.

Metabolic engineering of Saccharomyces cerevisiae for ethanol production from D-xylose, an abundant sugar in plant biomass hydrolysates, has been pursued vigorously for the past 15 years. Whereas wild-type S. cerevisiae cannot ferment D-xylose, the keto-isomer D-xylulose can be metabolised slowly. Conversion of D-xylose into D-xylulose is therefore crucial in metabolic engineering of xylose fermentation by S. cerevisiae. Expression of heterologous xylose reductase and xylitol dehydrogenase does enable D-xylose utilisation, but intrinsic redox constraints of this pathway result in undesirable byproduct formation in the absence of oxygen. In contrast, expression of xylose isomerase (XI, EC 5.3.1.5), which directly interconverts D-xylose and D-xylulose, does not have these constraints. However, several problems with the functional expression of various bacterial and Archaeal XI genes have precluded successful use of XI in yeast metabolic engineering. This changed with the discovery of a fungal XI gene in Piromyces sp. E2, expression of which led to high XI activities in S. cerevisiae. When combined with over-expression of the genes of the non-oxidative pentose phosphate pathway of S. cerevisiae, the resulting strain grew anaerobically on D-xylose with a doubling time of ca. 8 h, with the same ethanol yield as on glucose. Additional evolutionary engineering was used to improve the fermentation kinetics of mixed-substrate utilisation, resulting in efficient D-xylose utilisation in synthetic media. Although industrial pilot experiments have already demonstrated high ethanol yields from the D-xylose present in plant biomass hydrolysates, strain robustness, especially with respect to tolerance to inhibitors present in hydrolysates, can still be further improved.
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http://dx.doi.org/10.1007/10_2007_057DOI Listing
December 2007

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose.

Appl Environ Microbiol 2007 Aug 1;73(15):4881-91. Epub 2007 Jun 1.

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.
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http://dx.doi.org/10.1128/AEM.00177-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951023PMC
August 2007

An inverse relationship between peroxisome proliferator-activated receptor gamma and allergic airway inflammation in an allergen challenge model.

Ann Allergy Asthma Immunol 2005 Nov;95(5):468-73

Respiratory Disease, Wyeth Research, Cambridge, Massachusetts, USA.

Background: Peroxisome proliferator-activated receptor gamma (PPAR-gamma) expression has not been evaluated in bronchoalveolar lavage (BAL) cells from allergic asthmatic patients.

Objective: To determine whether inappropriate down-regulation of PPAR-gamma in alveolar macrophages may contribute to persistent airway inflammation in allergic asthma.

Methods: We used segmental allergen challenge as a model of in vivo experimental allergic asthmatic exacerbation and airway inflammation. PPAR-y gene expression was evaluated at baseline and 24 hours later in asthmatic patients and controls using real-time polymerase chain reaction. Immunofluorescence was used to determine cellular location of the PPAR-gamma protein.

Results: We demonstrate for the first time to our knowledge that PPAR-gamma messenger RNA and protein, which are highly expressed in alveolar macrophages of healthy individuals, are significantly reduced in asthmatic patients after segmental allergen challenge. In allergic asthmatic patients (n=9), PPAR-gamma gene expression decreased significantly from baseline to postchallenge BAL (median decrease, 45%; P = .008). Furthermore, immunofluorescence staining demonstrated that PPAR-gamma protein was associated with alveolar macrophages and not with inflammatory eosinophils and neutrophils.

Conclusion: Results implicate down-regulation of PPAR-gamma in BAL cells as a potential factor in dysregulation of lung homeostasis in asthmatic patients. The present findings suggest that PPAR-gamma agonists could have a future role in asthma therapy and warrant further study.
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http://dx.doi.org/10.1016/S1081-1206(10)61173-8DOI Listing
November 2005

Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain.

FEMS Yeast Res 2005 Jul;5(10):925-34

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

We have recently reported about a Saccharomyces cerevisiae strain that, in addition to the Piromyces XylA xylose isomerase gene, overexpresses the native genes for the conversion of xylulose to glycolytic intermediates. This engineered strain (RWB 217) exhibited unprecedentedly high specific growth rates and ethanol production rates under anaerobic conditions with xylose as the sole carbon source. However, when RWB 217 was grown on glucose-xylose mixtures, a diauxic growth pattern was observed with a relatively slow consumption of xylose in the second growth phase. After prolonged cultivation in an anaerobic, xylose-limited chemostat, a culture with improved xylose uptake kinetics was obtained. This culture also exhibited improved xylose consumption in glucose-xylose mixtures. A further improvement in mixed-sugar utilization was obtained by prolonged anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose mixtures. A final single-strain isolate (RWB 218) rapidly consumed glucose-xylose mixtures anaerobically, in synthetic medium, with a specific rate of xylose consumption exceeding 0.9 gg(-1)h(-1). When the kinetics of zero trans-influx of glucose and xylose of RWB 218 were compared to that of the initial strain, a twofold higher capacity (V(max)) as well as an improved K(m) for xylose was apparent in the selected strain. It is concluded that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeast.
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http://dx.doi.org/10.1016/j.femsyr.2005.04.004DOI Listing
July 2005

Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.

FEMS Yeast Res 2005 Feb;5(4-5):399-409

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.
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http://dx.doi.org/10.1016/j.femsyr.2004.09.010DOI Listing
February 2005

Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: possible consequence of energy-dependent lactate export.

Appl Environ Microbiol 2004 May;70(5):2898-905

Department of Biotechnology, Delft University of Technology, NL-2628 BC Delft, The Netherlands.

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC404449PMC
http://dx.doi.org/10.1128/aem.70.5.2898-2905.2004DOI Listing
May 2004

Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle.

FEMS Yeast Res 2004 Mar;4(6):655-64

Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h(-1). After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h(-1), respectively. The anaerobic ethanol yield was 0.42 g ethanol x g xylose(-1) and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.
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http://dx.doi.org/10.1016/j.femsyr.2004.01.003DOI Listing
March 2004

Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast.

Appl Environ Microbiol 2004 Jan;70(1):159-66

Department of Biotechnology, Delft University of Technology, NL-2628 BC Delft. BIRD Engineering B.V., NL-3044 CK Rotterdam, The Netherlands.

The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc(-)) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc(-) S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C(2) compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc(-) strain without these growth defects. An acetate-independent Pdc(-) mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C(2) independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc(-) S. cerevisiae mutant (TAM) that could grow in batch cultures ( micro (max) = 0.20 h(-1)) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter(-1) was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass(-1) h(-1) during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose(-1).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC321313PMC
http://dx.doi.org/10.1128/aem.70.1.159-166.2004DOI Listing
January 2004

High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?

FEMS Yeast Res 2003 Oct;4(1):69-78

Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.
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http://dx.doi.org/10.1016/S1567-1356(03)00141-7DOI Listing
October 2003

Overproduction of threonine aldolase circumvents the biosynthetic role of pyruvate decarboxylase in glucose-limited chemostat cultures of Saccharomyces cerevisiae.

Appl Environ Microbiol 2003 Apr;69(4):2094-9

Kluyver Laboratory of Biotechnology, Delft University of Technology, The Netherlands.

Pyruvate decarboxylase-negative (Pdc(-)) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C(2) requirement of Pdc(-) S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of L-carnitine to growth media did not restore growth of a Pdc(-) strain on glucose, indicating that the C(2) requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc(-) strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc(-) GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc(-) strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC154831PMC
http://dx.doi.org/10.1128/aem.69.4.2094-2099.2003DOI Listing
April 2003