Publications by authors named "Barbara Giovannone"

29 Publications

  • Page 1 of 1

CXCL4 Links Inflammation and Fibrosis by Reprogramming Monocyte-Derived Dendritic Cells .

Front Immunol 2020 17;11:2149. Epub 2020 Sep 17.

Center for Translational Immunology, Department of Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.

Fibrosis is a condition shared by numerous inflammatory diseases. Our incomplete understanding of the molecular mechanisms underlying fibrosis has severely hampered effective drug development. CXCL4 is associated with the onset and extent of fibrosis development in multiple inflammatory and fibrotic diseases. Here, we used monocyte-derived cells as a model system to study the effects of CXCL4 exposure on dendritic cell development by integrating 65 longitudinal and paired whole genome transcriptional and methylation profiles. Using data-driven gene regulatory network analyses, we demonstrate that CXCL4 dramatically alters the trajectory of monocyte differentiation, inducing a novel pro-inflammatory and pro-fibrotic phenotype mediated via key transcriptional regulators including CIITA. Importantly, these pro-inflammatory cells directly trigger a fibrotic cascade by producing extracellular matrix molecules and inducing myofibroblast differentiation. Inhibition of CIITA mimicked CXCL4 in inducing a pro-inflammatory and pro-fibrotic phenotype, validating the relevance of the gene regulatory network. Our study unveils that CXCL4 acts as a key secreted factor driving innate immune training and forming the long-sought link between inflammation and fibrosis.
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http://dx.doi.org/10.3389/fimmu.2020.02149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527415PMC
September 2020

Leukocyte Associated Immunoglobulin Like Receptor 1 Regulation and Function on Monocytes and Dendritic Cells During Inflammation.

Front Immunol 2020 19;11:1793. Epub 2020 Aug 19.

Center for Translational Immunology, University Medical Center Utrecht, University of Utrecht, Utrecht, Netherlands.

Inhibitory receptors are crucial immune regulators and are essential to prevent exacerbated responses, thus contributing to immune homeostasis. Leukocyte associated immunoglobulin like receptor 1 (LAIR-1) is an immune inhibitory receptor which has collagen and collagen domain containing proteins as ligands. LAIR-1 is broadly expressed on immune cells and has a large availability of ligands in both circulation and tissues, implicating a need for tight regulation of this interaction. In the current study, we sought to examine the regulation and function of LAIR-1 on monocyte, dendritic cell (DC) and macrophage subtypes, using different models. We found that LAIR-1 is highly expressed on intermediate monocytes as well as on plasmacytoid DCs. LAIR-1 is also expressed on skin immune cells, mainly on tissue CD14 cells, macrophages and CD1c DCs. , monocyte and type-2 conventional DC stimulation leads to LAIR-1 upregulation, which may reflect the importance of LAIR-1 as negative regulator under inflammatory conditions. Indeed, we demonstrate that LAIR-1 ligation on monocytes inhibits toll like receptor (TLR)4 and Interferon (IFN)-α- induced signals. Furthermore, LAIR-1 is downregulated on GM-CSF and IFN-γ monocyte-derived macrophages and monocyte-derived DCs. In addition, LAIR-1 triggering during monocyte derived-DC differentiation results in significant phenotypic changes, as well as a different response to TLR4 and IFN-α stimulation. This indicates a role for LAIR-1 in skewing DC function, which impacts the cytokine expression profile of these cells. In conclusion, we demonstrate that LAIR-1 is consistently upregulated on monocytes and DC during the inflammatory phase of the immune response and tends to restore its expression during the resolution phase. Under inflammatory conditions, LAIR-1 has an inhibitory function, pointing toward to a potential intervention opportunity targeting LAIR-1 in inflammatory conditions.
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http://dx.doi.org/10.3389/fimmu.2020.01793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466540PMC
August 2020

Signal Inhibitory Receptor on Leukocytes-1 is highly expressed on lung monocytes, but absent on mononuclear phagocytes in skin and colon.

Cell Immunol 2020 11 28;357:104199. Epub 2020 Aug 28.

Center of Translational Immunology, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, the Netherlands; Oncode Institute, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, the Netherlands. Electronic address:

Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) is expressed on human blood monocytes and granulocytes and inhibits myeloid effector functions. On monocytes, but not granulocytes, SIRL-1 expression is low or absent in individuals with the single nucleotide polymorphism (SNP) rs612529C. The expression of SIRL-1 in tissue and the influence of rs612529 hereon is currently unknown. Here, we used flow cytometry to determine SIRL-1 expression on immune cells in human blood and three barrier tissues; skin, colon and lung. SIRL-1 was expressed by virtually all neutrophils and eosinophils in these tissues. In contrast, SIRL-1 was not expressed by monocyte-derived cells in skin and colon, whereas it was highly expressed by lung classical monocytes. Lung monocytes from individuals with a rs612529C allele had decreased SIRL-1 expression, consistent with the genotype association in blood. Within the different monocyte subsets in blood and lung, SIRL-1 expression was highest in classical monocytes and lowest in nonclassical monocytes. SIRL-1 was not expressed by dendritic cells in blood and barrier tissues. Together, these results indicate that SIRL-1 is differentially expressed on phagocyte subsets in blood and barrier tissues, and that its expression on monocytes is genotype- and tissue-specific. Immune regulation of monocytes by SIRL-1 may be of particular importance in the lung.
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http://dx.doi.org/10.1016/j.cellimm.2020.104199DOI Listing
November 2020

Confirmation of multiple endotypes in atopic dermatitis based on serum biomarkers.

J Allergy Clin Immunol 2021 Jan 8;147(1):189-198. Epub 2020 Jun 8.

National Expertise Center for Atopic Dermatitis, Department of Dermatology and Allergology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Atopic dermatitis (AD) is a highly heterogeneous disease, both clinically and biologically, whereas patients are still being treated according to a "one-size-fits-all" approach. Stratification of patients into biomarker-based endotypes is important for future development of personalized therapies.

Objective: Our aim was to confirm previously defined serum biomarker-based patient clusters in a new cohort of patients with AD.

Methods: A panel of 143 biomarkers was measured by using Luminex technology in serum samples from 146 patients with severe AD (median Eczema Area and Severity Index = 28.3; interquartile range = 25.2-35.3). Principal components analysis followed by unsupervised k-means cluster analysis of the biomarker data was used to identify patient clusters. A prediction model was built on the basis of a previous cohort to predict the 1 of the 4 previously identified clusters to which the patients of our new cohort would belong.

Results: Cluster analysis identified 4 serum biomarker-based clusters, 3 of which (clusters B, C, and D) were comparable to the previously identified clusters. Cluster A (33.6%) could be distinguished from the other clusters as being a "skin-homing chemokines/IL-1R1-dominant" cluster, whereas cluster B (18.5%) was a "T1/T2/T17-dominant" cluster, cluster C (18.5%) was a "T2/T22/PARC-dominant" cluster, and cluster D (29.5%) was a "T2/eosinophil-inferior" cluster. Additionally, by using a prediction model based on our previous cohort we accurately assigned the new cohort to the 4 previously identified clusters by including only 10 selected serum biomarkers.

Conclusion: We confirmed that AD is heterogeneous at the immunopathologic level and identified 4 distinct biomarker-based clusters, 3 of which were comparable with previously identified clusters. Cluster membership could be predicted with a model including 10 serum biomarkers.
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http://dx.doi.org/10.1016/j.jaci.2020.04.062DOI Listing
January 2021

Improved lipid productivity in in nitrogen-replete conditions by selection of pale green mutants.

Biotechnol Biofuels 2020 21;13:78. Epub 2020 Apr 21.

1Dipartimento di Biotecnologie, Università degli Studi di Verona, Strada le Grazie 15, 37134 Verona, Italy.

Background: is a photosynthetic unicellular microalgae considered one of the most interesting marine algae to produce biofuels and food additive due to its rapid growth rate and high lipid accumulation. Although microalgae are attractive platforms for solar energy bioconversion, the overall efficiency of photosynthesis is reduced due to the steep light gradient in photobioreactors. Moreover, accumulation of lipids in microalgae for biofuels production is usually induced in a two-phase cultivation process by nutrient starvation, with additional time and costs associated. In this work, a biotechnological approach was directed for the isolation of strains with improved light penetration in photobioreactor combined with increased lipids productivity.

Results: Mutants of were obtained by chemical mutagenesis and screened for having both a reduced chlorophyll content per cell and increased affinity for Nile red, a fluorescent dye which binds to cellular lipid fraction. Accordingly, one mutant, called , was selected and characterized for having a 30% reduction of chlorophyll content per cell and an almost 80% increase of lipid productivity compared to WT in nutrient-replete conditions, with C16:0 and C18:0 fatty acids being more than doubled in the mutant. Whole-genome sequencing revealed mutations in 234 genes in mutant among which there is a non-conservative mutation in the synthase gene. This gene encodes for an enzyme involved in the biosynthesis of DGDG, one of the major lipids found in the thylakoid membrane and it is thus involved in chloroplast biogenesis. Lipid biosynthesis is strongly influenced by light availability in several microalgae species, including : reduced chlorophyll content per cell and more homogenous irradiance in photobioreactor is at the base for the increased lipid productivity observed in the mutant.

Conclusions: The results herein obtained presents a promising strategy to produce algal biomass enriched in lipid fraction to be used for biofuel and biodiesel production in a single cultivation process, without the additional complexity of the nutrient starvation phase. Genome sequencing and identification of the mutations introduced in mutant suggest possible genes responsible for the observed phenotypes, identifying putative target for future complementation and biotechnological application.
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http://dx.doi.org/10.1186/s13068-020-01718-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175523PMC
April 2020

Extracellular SPARC cooperates with TGF-β signalling to induce pro-fibrotic activation of systemic sclerosis patient dermal fibroblasts.

Rheumatology (Oxford) 2020 09;59(9):2258-2263

Department of Rheumatology and Clinical Immunology.

Objectives: SSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc.

Methods: Dermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-β1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot.

Results: Exogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-β and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-β signalling.

Conclusion: These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-β dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.
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http://dx.doi.org/10.1093/rheumatology/kez583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449812PMC
September 2020

Dupilumab is very effective in a large cohort of difficult-to-treat adult atopic dermatitis patients: First clinical and biomarker results from the BioDay registry.

Allergy 2020 01 31;75(1):116-126. Epub 2019 Oct 31.

Department of Dermatology and Allergology, National Expertise Center for Atopic Dermatitis, University Medical Center Utrecht, Utrecht, The Netherlands.

Introduction: Dupilumab has recently been approved for the treatment of moderate to severe atopic dermatitis (AD) in adults. Daily practice data on dupilumab treatment are scarce.

Objective: To study the effect of 16-week treatment with dupilumab on clinical response and serum biomarkers in adult patients with moderate-severe AD in daily practice.

Methods: Data were extracted from the BioDay registry, a prospective multicenter registry. Sixteen-week clinical effectiveness of dupilumab was expressed as number of patients achieving EASI-50 (Eczema Area and Severity Index) or EASI-75, as well as patient-reported outcomes measures (Patient-Oriented Eczema Measure, Dermatology Life Quality Index, Numeric Rating Scale pruritus). Twenty-one biomarkers were measured in patients treated with dupilumab without concomitant use of oral immunosuppressive drugs at five different time points (baseline, 4, 8, 12, and 16 weeks).

Results: In total, 138 patients treated with dupilumab in daily practice were included. This cohort consisted of patients with very difficult-to-treat AD, including 84 (61%) patients who failed treatment on ≥2 immunosuppressive drugs. At week 16, the mean percent change in EASI score was 73%. The EASI-50 and EASI-75 were achieved by 114 (86%) and 82 (62%) patients after 16 weeks of treatment. The most reported side effect was conjunctivitis, occurring in 47 (34%) patients. During dupilumab treatment, disease severity-related serum biomarkers (TARC, PARC, periostin, and IL-22), eotaxin-1, and eotaxin-3 significantly decreased.

Conclusion: Treatment with dupilumab significantly improved disease severity and decreased severity-related serum biomarkers in patients with very difficult-to-treat AD in a daily practice setting.
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http://dx.doi.org/10.1111/all.14080DOI Listing
January 2020

Early identification of atopic dermatitis patients in need of systemic immunosuppressive treatment.

Clin Exp Allergy 2019 12 30;49(12):1641-1644. Epub 2019 Sep 30.

Department of Dermatology and Allergology, National Expertise Center for Atopic Dermatitis, University Medical Center Utrecht, Utrecht, The Netherlands.

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http://dx.doi.org/10.1111/cea.13495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6973172PMC
December 2019

Biomarkers detected in dried blood spots from atopic dermatitis patients strongly correlate with disease severity.

Allergy 2019 11 5;74(11):2240-2243. Epub 2019 Aug 5.

Department of Dermatology and Allergology, University Medical Center Utrecht, Utrecht, The Netherlands.

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http://dx.doi.org/10.1111/all.13839DOI Listing
November 2019

Induction of Inflammation and Fibrosis by Semaphorin 4A in Systemic Sclerosis.

Arthritis Rheumatol 2019 10 27;71(10):1711-1722. Epub 2019 Aug 27.

University Medical Center Utrecht, University of Utrecht, Utrecht, The Netherlands.

Objective: To analyze the potential role of semaphorin 4A (Sema4A) in inflammatory and fibrotic processes involved in the pathology of systemic sclerosis (SSc).

Methods: Sema4A levels in the plasma of healthy controls (n = 11) and SSc patients (n = 20) were determined by enzyme-linked immunosorbent assay (ELISA). The expression of Sema4A and its receptors in monocytes and CD4+ T cells from healthy controls and SSc patients (n = 6-7 per group) was determined by ELISA and flow cytometry. Th17 cytokine production by CD4+ T cells (n = 5-7) was analyzed by ELISA and flow cytometry. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblast cells (n = 6) was analyzed by quantitative polymerase chain reaction, ELISA, Western blotting, confocal microscopy, and ECM deposition assay.

Results: Plasma levels of Sema4A, and Sema4A expression by circulating monocytes and CD4+ T cells, were significantly higher in SSc patients than in healthy controls (P < 0.05). Inflammatory mediators significantly up-regulated the secretion of Sema4A by monocytes and CD4+ T cells from SSc patients (P < 0.05 versus unstimulated SSc cells). Functional assays showed that Sema4A significantly enhanced the expression of Th17 cytokines induced by CD3/CD28 in total CD4+ T cells as well in different CD4+ T cell subsets (P < 0.05 versus unstimulated SSc cells). Finally, Sema4A induced a profibrotic phenotype in dermal fibroblasts from both healthy controls and SSc patients, which was abrogated by blocking or silencing the expression of Sema4A receptors.

Conclusion: Our findings indicate that Sema4A plays direct and dual roles in promoting inflammation and fibrosis, 2 main features of SSc, suggesting that Sema4A might be a novel therapeutic target in SSc.
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http://dx.doi.org/10.1002/art.40915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790618PMC
October 2019

EASI p-EASI: Predicting disease severity in atopic dermatitis patients treated with cyclosporin A.

Allergy 2019 03 16;74(3):613-617. Epub 2018 Dec 16.

Department of Dermatology and Allergology, University Medical Center Utrecht, Utrecht, The Netherlands.

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http://dx.doi.org/10.1111/all.13651DOI Listing
March 2019

PD-1+CD8+ T cells are clonally expanding effectors in human chronic inflammation.

J Clin Invest 2018 10 2;128(10):4669-4681. Epub 2018 Aug 2.

Department of Pediatrics, Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, Netherlands.

Chronic inflammatory diseases are characterized by recurrent inflammatory attacks in the tissues mediated by autoreactive T cells. Identity and functional programming of CD8+ T cells at the target site of inflammation still remain elusive. One key question is whether, in these antigen-rich environments, chronic stimulation leads to CD8+ T cell exhaustion comparable to what is observed in infectious disease contexts. In the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients, a model of chronic inflammation, an overrepresentation of PD-1+CD8+ T cells was found. Gene expression profiling, gene set enrichment analysis, functional studies, and extracellular flux analysis identified PD-1+CD8+ T cells as metabolically active effectors, with no sign of exhaustion. Furthermore, PD-1+CD8+ T cells were enriched for a tissue-resident memory (Trm) cell transcriptional profile and demonstrated increased clonal expansion compared with the PD-1- counterpart, suggesting antigen-driven expansion of locally adapted cells. Interestingly, this subset was also found increased in target tissues in other human chronic inflammatory diseases. These data indicate that local chronic inflammation drives the induction and expansion of CD8+ T cells endowed with potential detrimental properties. Together, these findings lay the basis for investigation of PD-1-expressing CD8+ T cell targeting strategies in human chronic inflammatory diseases.
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http://dx.doi.org/10.1172/JCI96107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159979PMC
October 2018

Serum biomarker profiles suggest that atopic dermatitis is a systemic disease.

J Allergy Clin Immunol 2018 04 2;141(4):1523-1526. Epub 2018 Feb 2.

Department of Dermatology and Allergology, University Medical Center Utrecht, Utrecht, The Netherlands; Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2017.12.991DOI Listing
April 2018

Serum microRNA screening and functional studies reveal miR-483-5p as a potential driver of fibrosis in systemic sclerosis.

J Autoimmun 2018 05 20;89:162-170. Epub 2018 Jan 20.

Department of Rheumatology & Clinical Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands; Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands. Electronic address:

Objective: MicroRNAs (miRNAs) are regulatory molecules, which have been addressed as potential biomarkers and therapeutic targets in rheumatic diseases. Here, we investigated the miRNA signature in the serum of systemic sclerosis (SSc) patients and we further assessed their expression in early stages of the disease.

Methods: The levels of 758 miRNAs were evaluated in the serum of 26 SSc patients as compared to 9 healthy controls by using an Openarray platform. Three miRNAs were examined in an additional cohort of 107 SSc patients and 24 healthy donors by single qPCR. MiR-483-5p expression was further analysed in the serum of patients with localized scleroderma (LoS) (n = 22), systemic lupus erythematosus (SLE) (n = 33) and primary Sjögren's syndrome (pSS) (n = 23). The function of miR-483-5p was examined by transfecting miR-483-5p into primary human dermal fibroblasts and pulmonary endothelial cells.

Results: 30 miRNAs were significantly increased in patients with SSc. Of these, miR-483-5p showed reproducibly higher levels in an independent SSc cohort and was also elevated in patients with preclinical-SSc symptoms (early SSc). Notably, miR-483-5p was not differentially expressed in patients with SLE or pSS, whereas it was up-regulated in LoS, indicating that this miRNA could be involved in the development of skin fibrosis. Consistently, miR-483-5p overexpression in fibroblasts and endothelial cells modulated the expression of fibrosis-related genes.

Conclusions: Our findings showed that miR-483-5p is up-regulated in the serum of SSc patients, from the early stages of the disease onwards, and indicated its potential function as a fine regulator of fibrosis in SSc.
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http://dx.doi.org/10.1016/j.jaut.2017.12.015DOI Listing
May 2018

Effect of anticoagulants on 162 circulating immune related proteins in healthy subjects.

Cytokine 2018 06 28;106:114-124. Epub 2017 Oct 28.

Department of Pediatric Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; Multiplex Core Facility, Laboratory of Translational Immunology, University Medical center Utrecht, Utrecht, The Netherlands. Electronic address:

Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune-monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal. We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze-thaw cycles are performed of this in-house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants. Intra-assay variance of each mediator was <10%. Inter-assay variance ranged between 1.6 and 37% with an average of 12.2%. Recoveries were similar for all mediators (mean 99.8 ± 2.6%) with a range between 89-107%. Next we measured all mediators in serum, EDTA plasma and sodium heparin plasma of 43 healthy control donors. Of these markers only 19 showed similar expression profiles in the 3 different matrixes. Only 5 mediators were effected by multiple freeze-thawing cycles. Principal component analysis revealed different coagulants cluster separately and that sodium heparin shows the most consistent profile.
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http://dx.doi.org/10.1016/j.cyto.2017.10.021DOI Listing
June 2018

Endoplasmic reticulum stress cooperates with Toll-like receptor ligation in driving activation of rheumatoid arthritis fibroblast-like synoviocytes.

Arthritis Res Ther 2017 09 18;19(1):207. Epub 2017 Sep 18.

Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Endoplasmic reticulum (ER) stress has proinflammatory properties, and transgenic animal studies of rheumatoid arthritis (RA) indicate its relevance in the process of joint destruction. Because currently available studies are focused primarily on myeloid cells, we assessed how ER stress might affect the inflammatory responses of stromal cells in RA.

Methods: ER stress was induced in RA fibroblast-like synoviocytes (FLS), dermal fibroblasts, and macrophages with thapsigargin or tunicamycin alone or in combination with Toll-like receptor (TLR) ligands, and gene expression and messenger RNA (mRNA) stability was measured by quantitative polymerase chain reaction. Cellular viability was measured using cell death enzyme-linked immunosorbent assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and signaling pathway activation was analyzed by immunoblotting.

Results: No cytotoxicity was observed in FLS exposed to thapsigargin, despite significant induction of ER stress markers. Screening of 84 proinflammatory genes revealed minor changes in their expression (fold change 90th percentile range 2.8-8.3) by thapsigargin alone, but the vast majority were hyperinduced during combined stimulation with thapsigargin and TLR ligands (35% greater than fivefold vs lipopolysaccharide alone). The synergistic response could not be explained by quantitative effects on nuclear factor-κB and mitogen-activated protein kinase pathways alone, but it was dependent on increased mRNA stability. mRNA stabilization was similarly enhanced by ER stress in dermal fibroblasts but not in macrophages, correlating with minimal cooperative effects on gene induction in macrophages.

Conclusions: RA FLS are resistant to apoptosis induced by ER stress, but ER stress potentiates their activation by multiple TLR ligands. Interfering with downstream signaling pathway components of ER stress may be of therapeutic potential in the treatment of RA.
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http://dx.doi.org/10.1186/s13075-017-1386-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604427PMC
September 2017

EASI p-EASI: Utilizing a combination of serum biomarkers offers an objective measurement tool for disease severity in atopic dermatitis patients.

J Allergy Clin Immunol 2017 12 16;140(6):1703-1705. Epub 2017 Aug 16.

Department of Dermatology and Allergology, University Medical Center, Utrecht, The Netherlands; Laboratory of Translational Immunology, Utrecht, The Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2017.06.046DOI Listing
December 2017

Moving toward endotypes in atopic dermatitis: Identification of patient clusters based on serum biomarker analysis.

J Allergy Clin Immunol 2017 Sep 13;140(3):730-737. Epub 2017 Apr 13.

Department of Dermatology and Allergology, University Medical Center Utrecht, Utrecht, The Netherlands; Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address:

Background: Atopic dermatitis (AD) is a complex, chronic, inflammatory skin disease with a diverse clinical presentation. However, it is unclear whether this diversity exists at a biological level.

Objective: We sought to test the hypothesis that AD is heterogeneous at the biological level of individual inflammatory mediators.

Methods: Sera from 193 adult patients with moderate-to-severe AD (six area, six sign atopic dermatitis [SASSAD] score: geometric mean, 22.3 [95% CI, 21.3-23.3] and 39.1 [95% CI, 37.5-40.9], respectively) and 30 healthy control subjects without AD were analyzed for 147 serum mediators, total IgE levels, and 130 allergen-specific IgE levels. Population heterogeneity was assessed by using principal component analysis, followed by unsupervised k-means cluster analysis of the principal components.

Results: Patients with AD showed pronounced evidence of inflammation compared with healthy control subjects. Principal component analysis of data on sera from patients with AD revealed the presence of 4 potential clusters. Fifty-seven principal components described approximately 90% of the variance. Unsupervised k-means cluster analysis of the 57 largest principal components delivered 4 distinct clusters of patients with AD. Cluster 1 had high SASSAD scores and body surface areas with the highest levels of pulmonary and activation-regulated chemokine, tissue inhibitor of metalloproteinases 1, and soluble CD14. Cluster 2 had low SASSAD scores with the lowest levels of IFN-α, tissue inhibitor of metalloproteinases 1, and vascular endothelial growth factor. Cluster 3 had high SASSAD scores with the lowest levels of IFN-β, IL-1, and epithelial cytokines. Cluster 4 had low SASSAD scores but the highest levels of the inflammatory markers IL-1, IL-4, IL-13, and thymic stromal lymphopoietin.

Conclusion: AD is a heterogeneous disease both clinically and biologically. Four distinct clusters of patients with AD have been identified that could represent endotypes with unique biological mechanisms. Elucidation of these endotypes warrants further investigation and will require future intervention trials with specific agents, such as biologics.
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http://dx.doi.org/10.1016/j.jaci.2017.03.023DOI Listing
September 2017

Size matters: decreased glandular levels of anti-inflammatory short thymic stromal lymphopoietin in primary Sjögren's syndrome.

Clin Exp Rheumatol 2016 Sep-Oct;34(5):959-960. Epub 2016 Aug 2.

Department of Rheumatology and Clinical Immunology; and Laboratory of Translational Immunology, University Medical Centre, Utrecht, The Netherlands.

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January 2017

TSLP is differentially regulated by vitamin D3 and cytokines in human skin.

Immun Inflamm Dis 2015 Mar 16;3(1):32-43. Epub 2015 Feb 16.

Department of Dermatology & Allergology, University Medical Center Utrecht Utrecht, the Netherlands ; Department of Immunology, University Medical Center Utrecht Utrecht, the Netherlands.

Thymic stromal lymphopoietin (TSLP) plays an important role in allergic diseases and is highly expressed in keratinocytes in human lesional atopic dermatitis (AD) skin. In nonlesional AD skin TSLP expression can be induced by applying house dust mite allergen onto the skin in the atopy patch test. Several studies have demonstrated that the induction of TSLP expression in mouse skin does not only lead to AD-like inflammation of the skin, but also predisposes to severe inflammation of the airways. In mice, TSLP expression can be induced by application of the 1,25-dihydroxyvitamin D3 (VD3) analogue calcipotriol and results in the development of eczema-like lesions. The objective is to investigate the effect of VD3 (calcitriol) or calcipotriol on TSLP expression in normal human skin and skin from AD patients. Using multiple ex vivo experimental setups, the effects of calci(po)triol on TSLP expression by normal human skin, and skin from AD patients were investigated and compared to effects of calcipotriol on mouse and non-human primates (NHP) skin. No induction of TSLP expression (mRNA or protein) was observed in human keratinocytes, normal human skin, nonlesional AD skin, or NHP skin samples after stimulation with calcipotriol or topical application of calcitriol. The biological activity of calci(po)triol in human skin samples was demonstrated by the increased expression of the VD3-responsive Cyp24a1 gene. TSLP expression was induced by cytokines (IL-4, IL-13, and TNF-α) in skin samples from all three species. In contrast to the findings in human and NHP, a consistent increase in TSLP expression was confirmed in mouse skin biopsies after stimulation with calcipotriol. VD3 failed to induce expression of TSLP in human or monkey skin in contrast to mouse, implicating careful extrapolation of this often-used mouse model to AD patients.
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http://dx.doi.org/10.1002/iid3.48DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4386913PMC
March 2015

CD8(+) T cells in the lesional skin of atopic dermatitis and psoriasis patients are an important source of IFN-γ, IL-13, IL-17, and IL-22.

J Invest Dermatol 2013 Apr 6;133(4):973-9. Epub 2012 Dec 6.

Department of Dermatology, University Medical Center Utrecht, Utrecht, The Netherlands.

Although CD4(+) T cells are known to contribute to the pathology of atopic dermatitis (AD) and psoriasis, the role of CD8(+) T cells in these diseases remains poorly characterized. The aim of this study was to characterize the cytokine production of T cells from AD and psoriasis skin. We found that CD4(+) T cells isolated from AD skin were largely Th2 (T helper type 2) biased, in agreement with prior reports. However, we also observed large numbers of CD8(+) T cells producing IL-13, IFN-γ, and IL-22. We observed increased numbers of CD8(+) T cells isolated from AD skin, and immunohistochemistry studies confirmed the presence of CD8(+) T cells in the dermis and epidermis of AD skin lesions. Surprisingly, T-cell cytokine production was similar in the lesional and nonlesional skin of patients with AD. T cells from psoriatic lesional skin predominantly produced IFN-γ, IL-17, and IL-22, in agreement with prior studies. However, in addition to Th17 cells, we observed high percentages of CD8(+) T cells that produced both IL-22 and IL-17 in psoriatic skin lesions. Our findings demonstrate that CD8(+) T cells are a significant and previously unappreciated source of inflammatory cytokine production in both AD and psoriasis.
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http://dx.doi.org/10.1038/jid.2012.456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835628PMC
April 2013

Assessment of cyclobutane pyrimidine dimers by digital photography in human skin.

J Immunol Methods 2011 Oct 28;373(1-2):240-6. Epub 2011 Jul 28.

University Medical Centre Utrecht, Department of Dermatology & Allergology, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.

UV-mediated DNA damage and repair are important mechanisms in research on UV-induced carcinogenesis. UV-induced DNA-damage and repair can be determined by immunohistochemical staining of photoproduct positive nuclei of keratinocytes in the epidermis. We developed a new method of analysing and quantifying thymine dimer (TT-CPD) positive cells in the epidermis. Normal skin of healthy controls was exposed to UVB ex vivo and in vivo. Skin samples were immunohistochemically stained for TT-CPDs. Digital images of the epidermis were quantified for TT-CPDs both visually and digitally. There was a UVB-dose dependent induction of TT-CPDs present in the ex vivo UVB-irradiated skin samples. The linear measurement range of the digital quantification was increased compared to the manual counting. The average 24-hour repair rate of the initiated TT-CPDs elicited by the UVB irradiation at T=0 of the 8 HCs showed a 34% decrease of TT-CPD photoproducts by the manual counting method and a 51% decrease determined by digital counting. The digital quantification method improves immunohistochemical quantification of DNA photo damage. It is more sensitive in measuring the extent of DNA-damage per nucleus.
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http://dx.doi.org/10.1016/j.jim.2011.07.014DOI Listing
October 2011

GIGYF2 gene disruption in mice results in neurodegeneration and altered insulin-like growth factor signaling.

Hum Mol Genet 2009 Dec 10;18(23):4629-39. Epub 2009 Sep 10.

Division of Endocrinology, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI 02903, USA.

Grb10-Interacting GYF Protein 2 (GIGYF2) was initially identified through its interaction with Grb10, an adapter protein that binds activated IGF-I and insulin receptors. The GIGYF2 gene maps to human chromosome 2q37 within a region linked to familial Parkinson's disease (PARK11 locus), and association of GIGYF2 mutations with Parkinson's disease has been described in some but not other recent publications. This study investigated the consequences of Gigyf2 gene disruption in mice. Gigyf2 null mice undergo apparently normal embryonic development, but fail to feed and die within the first 2 post-natal days. Heterozygous Gigyf2(+/-) mice survive to adulthood with no evident metabolic or growth defects. At 12-15 months of age, the Gigyf2(+/-) mice begin to exhibit motor dysfunction manifested as decreased balance time on a rotating horizontal rod. This is associated with histopathological evidence of neurodegeneration and rare intracytoplasmic Lewy body-like inclusions in spinal anterior horn motor neurons. There are alpha-synuclein positive neuritic plaques in the brainstem and cerebellum, but no abnormalities in the substantia nigra. Primary cultured embryo fibroblasts from Gigyf2 null mice exhibit decreased IGF-I-stimulated IGF-I receptor tyrosine phosphorylation and augmented ERK1/2 phosphorylation. These data provide further evidence for an important role of GIGYF2 in age-related neurodegeneration and IGF pathway signaling.
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http://dx.doi.org/10.1093/hmg/ddp430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773276PMC
December 2009

Mutations in the GIGYF2 (TNRC15) gene at the PARK11 locus in familial Parkinson disease.

Am J Hum Genet 2008 Apr 20;82(4):822-33. Epub 2008 Mar 20.

Division of Endocrinology, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, RI 02903, USA.

The genetic basis for association of the PARK11 region of chromosome 2 with familial Parkinson disease (PD) is unknown. This study examined the GIGYF2 (Grb10-Interacting GYF Protein-2) (TNRC15) gene, which contains the PARK11 microsatellite marker with the highest linkage score (D2S206, LOD 5.14). The 27 coding exons of the GIGYF2 gene were sequenced in 123 Italian and 126 French patients with familial PD, plus 131 Italian and 96 French controls. A total of seven different GIGYF2 missense mutations resulting in single amino acid substitutions were present in 12 unrelated PD index patients (4.8%) and not in controls. Three amino acid insertions or deletions were found in four other index patients and absent in controls. Specific exon sequencing showed that these ten sequence changes were absent from a further 91 controls. In four families with amino acid substitutions in which at least one other PD case was available, the GIGYF2 mutations (Asn56Ser, Thr112Ala, and Asp606Glu) segregated with PD. There were, however, two unaffected carriers in one family, suggesting age-dependent or incomplete penetrance. One index case (PD onset age 33) inherited a GIGYF2 mutation (Ile278Val) from her affected father (PD onset age 66) and a previously described PD-linked mutation in the LRRK2 gene (Ile1371Val) from her affected mother (PD onset age 61). The earlier onset and severe clinical course in the index patient suggest additive effects of the GIGYF2 and LRRK2 mutations. These data strongly support GIGYF2 as a PARK11 gene with a causal role in familial PD.
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http://dx.doi.org/10.1016/j.ajhg.2008.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2427211PMC
April 2008

Distinct Grb10 domain requirements for effects on glucose uptake and insulin signaling.

Mol Cell Endocrinol 2005 Jan;230(1-2):39-50

Endocrinology Division and the Hallett Center for Diabetes and Endocrinology, Rhode Island Hospital, Brown Medical School, One Hoppin Street, Suite 200, Providence, RI 02903, USA.

The adapter protein Grb10 binds to phosphotyrosine residues in insulin receptors via its C-terminal region and regulates insulin signaling. This study investigated Grb10 regulation of glucose uptake and the importance of the Grb10 N-terminal region using 3T3-L1 adipocytes overexpressing full-length (FL-Grb10) or N-terminally truncated Grb10 (BPS-SH2). Overexpression of FL-Grb10 inhibited insulin-stimulated receptor autophosphorylation and glucose uptake. In contrast, the BPS-SH2 fragment of Grb10 had no effect on receptor phosphorylation or glucose uptake. In spite of these differences, both FL-Grb10 and the BPS-SH2 fragment inhibited insulin-stimulated phosphorylation of IRS1, IRS2, Akt/PKB, Shc, ERK1/2, APS, and c-Cbl to a similar extent. Co-precipitation studies demonstrated more sustained binding of the BPS-SH2 fragment than FL-Grb10 to insulin receptors. Although receptor binding domains of Grb10 are sufficient to inhibit insulin effects on proximal post-receptor signaling responses, N-terminal domains of Grb10 are essential for the effects of this adapter protein on receptor phosphorylation and glucose uptake.
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http://dx.doi.org/10.1016/j.mce.2004.11.004DOI Listing
January 2005

Two novel proteins that are linked to insulin-like growth factor (IGF-I) receptors by the Grb10 adapter and modulate IGF-I signaling.

J Biol Chem 2003 Aug 27;278(34):31564-73. Epub 2003 May 27.

Division of Endocrinology and the Hallett Center for Diabetes and Endocrinology, Rhode Island Hospital, Brown Medical School, Providence, Rhode Island 02903, USA.

Grb10 is a protein that binds to the intracellular domains of activated tyrosine kinase receptors, including insulin-like growth factor (IGF-I) and insulin receptors. This occurs through the interaction of two C-terminal Grb10 motifs (BPS and Src homology domains) with receptor phosphotyrosine residues. Published data from transfection/overexpression studies support both positive and negative regulatory effects of Grb10, thus leaving its physiological role unclear. Because Grb10 has the structure of an adapter protein, the objective of this study was to determine whether Grb10 links other proteins to IGF-I receptors and thus modulates IGF-I signaling. Using yeast two-hybrid screening, the N terminus of Grb10 was shown to interact with two novel proteins, designated GIGYF1 (Grb10 interacting GYF protein 1) and GIGYF2. Mutation analysis indicates that a 17-amino acid sequence in GIGYF1 and GIGYF2, homologous to the GYF domain described previously, binds to tandem proline-rich regions in the N terminus of Grb10. In IGF-I receptor-expressing R+ fibroblasts, there is detectable binding of a Myc-tagged fragment of GIGYF1 to Grb10 in the basal state. Stimulation with IGF-I results in increased binding of GIGYF1 to Grb10 and transient binding of both Grb10 and GIGYF1 to IGF-I receptors, presumably via the adapter function of Grb10. At later time points, GIGYF1 dissociates, but Grb10 remains linked to IGF-I receptors. Overexpression of the Grb10 binding fragment of GIGYF1 in R+ cells results in a significant increase in IGF-I-stimulated receptor tyrosine phosphorylation. In conclusion, we have identified two members of a novel protein family, which become transiently linked to IGF-I receptors by the Grb10 adapter protein following IGF-I stimulation. Grb10 and GIGYFs may act cooperatively to regulate receptor signaling.
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http://dx.doi.org/10.1074/jbc.M211572200DOI Listing
August 2003