Publications by authors named "Leslie van der Fits"

28 Publications

  • Page 1 of 1

Low-dose Ad26.COV2.S protection against SARS-CoV-2 challenge in rhesus macaques.

Cell 2021 06 1;184(13):3467-3473.e11. Epub 2021 Jun 1.

Bioqual, Rockville, MD 20852, USA.

We previously reported that a single immunization with an adenovirus serotype 26 (Ad26)-vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. To evaluate reduced doses of Ad26.COV2.S, 30 rhesus macaques were immunized once with 1 × 10, 5 × 10, 1.125 × 10, or 2 × 10 viral particles (vp) Ad26.COV2.S or sham and were challenged with SARS-CoV-2. Vaccine doses as low as 2 × 10 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125 × 10 vp were required for protection in nasal swabs. Activated memory B cells and binding or neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show enhancement of disease. These data demonstrate that a single immunization with relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques, although a higher vaccine dose may be required for protection in the upper respiratory tract.
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http://dx.doi.org/10.1016/j.cell.2021.05.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8166510PMC
June 2021

Ad26.COV2.S protects Syrian hamsters against G614 spike variant SARS-CoV-2 and does not enhance respiratory disease.

NPJ Vaccines 2021 Mar 19;6(1):39. Epub 2021 Mar 19.

Janssen Vaccines & Prevention B.V., Leiden, The Netherlands.

Previously we have shown that a single dose of recombinant adenovirus serotype 26 (Ad26) vaccine expressing a prefusion stabilized SARS-CoV-2 spike antigen (Ad26.COV2.S) is immunogenic and provides protection in Syrian hamster and non-human primate SARS-CoV-2 infection models. Here, we investigated the immunogenicity, protective efficacy, and potential for vaccine-associated enhanced respiratory disease (VAERD) mediated by Ad26.COV2.S in a moderate disease Syrian hamster challenge model, using the currently most prevalent G614 spike SARS-CoV-2 variant. Vaccine doses of 1 × 10 and 1 × 10 VP elicited substantial neutralizing antibodies titers and completely protected over 80% of SARS-CoV-2 inoculated Syrian hamsters from lung infection and pneumonia but not upper respiratory tract infection. A second vaccine dose further increased neutralizing antibody titers that was associated with decreased infectious viral load in the upper respiratory tract after SARS-CoV-2 challenge. Suboptimal non-protective immune responses elicited by low-dose A26.COV2.S vaccination did not exacerbate respiratory disease in SARS-CoV-2-inoculated Syrian hamsters with breakthrough infection. In addition, dosing down the vaccine allowed to establish that binding and neutralizing antibody titers correlate with lower respiratory tract protection probability. Overall, these preclinical data confirm efficacy of a one-dose vaccine regimen with Ad26.COV2.S in this G614 spike SARS-CoV-2 virus variant Syrian hamster model, show the added benefit of a second vaccine dose, and demonstrate that there are no signs of VAERD under conditions of suboptimal immunity.
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http://dx.doi.org/10.1038/s41541-021-00301-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979827PMC
March 2021

A Double-Blind, Randomized, Placebo-Controlled Phase 1 Study of Ad26.ZIKV.001, an Ad26-Vectored Anti-Zika Virus Vaccine.

Ann Intern Med 2021 05 16;174(5):585-594. Epub 2021 Feb 16.

Beth Israel Deaconess Medical Center, Boston, Massachusetts (K.E.S., D.G.K., R.A.L., P.A., J.L., L.P., D.H.B.).

Background: Zika virus (ZIKV) may cause severe congenital disease after maternal-fetal transmission. No vaccine is currently available.

Objective: To assess the safety and immunogenicity of Ad26.ZIKV.001, a prophylactic ZIKV vaccine candidate.

Design: Phase 1 randomized, double-blind, placebo-controlled clinical study. (ClinicalTrials.gov: NCT03356561).

Setting: United States.

Participants: 100 healthy adult volunteers.

Intervention: Ad26.ZIKV.001, an adenovirus serotype 26 vector encoding ZIKV M-Env, administered in 1- or 2-dose regimens of 5 × 10 or 1 × 10 viral particles (vp), or placebo.

Measurements: Local and systemic adverse events; neutralization titers by microneutralization assay (MN50) and T-cell responses by interferon-γ enzyme-linked immunospot and intracellular cytokine staining; and protectivity of vaccine-induced antibodies in a subset of participants through transfer in an exploratory mouse ZIKV challenge model.

Results: All regimens were well tolerated, with no safety concerns identified. In both 2-dose regimens, ZIKV neutralizing titers peaked 14 days after the second vaccination, with geometric mean MN50 titers (GMTs) of 1065.6 (95% CI, 494.9 to 2294.5) for 5 × 10 vp and 956.6 (595.8 to 1535.8) for 1 × 10 vp. Titers persisted for at least 1 year at a GMT of 68.7 (CI, 26.4-178.9) for 5 × 10 vp and 87.0 (CI, 29.3 to 258.6) for 1 × 10 vp. A 1-dose regimen of 1 × 10 vp Ad26.ZIKV.001 induced seroconversion in all participants 56 days after the first vaccination (GMT, 103.4 [CI, 52.7 to 202.9]), with titers persisting for at least 1 year (GMT, 90.2 [CI, 38.4 to 212.2]). Env-specific cellular responses were induced. Protection against ZIKV challenge was observed after antibody transfer from participants into mice, and MN50 titers correlated with protection in this model.

Limitation: The study was conducted in a nonendemic area, so it did not assess safety and immunogenicity in a flavivirus-exposed population.

Conclusion: The safety and immunogenicity profile makes Ad26.ZIKV.001 a promising candidate for further development if the need reemerges.

Primary Funding Source: Janssen Vaccines and Infectious Diseases.
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http://dx.doi.org/10.7326/M20-5306DOI Listing
May 2021

Low-Dose Ad26.COV2.S Protection Against SARS-CoV-2 Challenge in Rhesus Macaques.

bioRxiv 2021 Jan 27. Epub 2021 Jan 27.

We previously reported that a single immunization with an adenovirus serotype 26 (Ad26) vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. In this study, we evaluated the immunogenicity and protective efficacy of reduced doses of Ad26.COV2.S. 30 rhesus macaques were immunized once with 1×10 , 5×10 , 1.125×10 , or 2×10 vp Ad26.COV2.S or sham and were challenged with SARS-CoV-2 by the intranasal and intratracheal routes. Vaccine doses as low as 2×10 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125×10 vp were required for protection in nasal swabs. Activated memory B cells as well as binding and neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show evidence of virologic, immunologic, histopathologic, or clinical enhancement of disease compared with sham controls. These data demonstrate that a single immunization with a relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques. Moreover, our findings show that a higher vaccine dose may be required for protection in the upper respiratory tract compared with the lower respiratory tract.
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http://dx.doi.org/10.1101/2021.01.27.428380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852276PMC
January 2021

Adenovector 26 encoded prefusion conformation stabilized RSV-F protein induces long-lasting Th1-biased immunity in neonatal mice.

NPJ Vaccines 2020 12;5(1):49. Epub 2020 Jun 12.

Janssen Vaccines & Prevention B.V., Leiden, The Netherlands.

While RSV is a major cause of respiratory morbidity in infants, vaccine development is hindered by the immaturity and Th2-bias of the infant immune system and the legacy of enhanced respiratory disease (ERD) after RSV infection following immunization with formalin inactivated (FI)-RSV vaccine in earlier clinical trials. Preclinical studies have demonstrated that an adenoviral vector-based RSV F vaccine candidate (Ad26.RSV.FA2) induces Th1-biased protective immune responses, without signs of ERD upon subsequent RSV challenge. We here developed an Ad26 vector encoding the RSV F protein stabilized in its prefusion conformation (Ad26.RSV.preF). In adult mice, Ad26.RSV.preF induced superior, Th1-biased IgG2a-dominated humoral responses as compared to Ad26.RSV.FA2, while maintaining the strong Th1-biased cellular responses. Similar to adult mice, Ad26.RSV.preF induced robust and durable humoral immunity in neonatal mice, again characterized by IgG2a-dominated RSV F-binding antibodies, and high and stable virus-neutralizing titers. In addition, vaccine-elicited cellular immune responses were durable and characterized by IFN-γ-producing CD4+ and CD8+ T cells, with a profound Th1 bias. In contrast, immunization of neonatal mice with FI-RSV resulted in IgG1 RSV F-binding antibodies associated with a Th2 phenotype, no detectable virus-neutralizing antibodies, and a Th2-biased cellular response. These results are supportive for the clinical development of Ad26.RSV.preF for use in infants.
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http://dx.doi.org/10.1038/s41541-020-0200-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293210PMC
June 2020

Adenoviral vector type 26 encoding Zika virus (ZIKV) M-Env antigen induces humoral and cellular immune responses and protects mice and nonhuman primates against ZIKV challenge.

PLoS One 2018 24;13(8):e0202820. Epub 2018 Aug 24.

Janssen Vaccines & Prevention B.V., Leiden, The Netherlands.

In 2015, there was a large outbreak of Zika virus (ZIKV) in Brazil. Despite its relatively mild impact on healthy adults, ZIKV infection during pregnancy has been associated with severe birth defects. Currently, there is no ZIKV vaccine available, but several vaccine candidates based on the ZIKV membrane (M) and envelope (Env) structural proteins showed promising results in preclinical and clinical studies. Here, the immunogenicity and protective efficacy of a non-replicating adenoviral vector type 26 (Ad26) that encodes the ZIKV M-Env antigens (Ad26.ZIKV.M-Env) was evaluated in mice and non-human primates (NHP). Ad26.ZIKV.M-Env induced strong and durable cellular and humoral immune responses in preclinical models. Humoral responses were characterized by Env-binding and ZIKV neutralizing antibody responses while cellular responses were characterized by ZIKV reactive CD4+ and CD8+ T cells. Importantly, a single immunization with a very low dose of 4x107 vp of Ad26.ZIKV.M-Env protected mice from ZIKV challenge. In NHP, a single immunization with a typical human dose of 1x1011 vp of Ad26.ZIKV.M-Env also induced Env-binding and ZIKV neutralizing antibodies and Env and M specific cellular immune responses that associated with complete protection against viremia from ZIKV challenge as measured in plasma and other body fluids. Together these data provide the rationale to progress the Ad26.ZIKV.M-Env candidate vaccine to clinical testing.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202820PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6108497PMC
February 2019

Evaluation of Immunophenotypic and Molecular Biomarkers for Sézary Syndrome Using Standard Operating Procedures: A Multicenter Study of 59 Patients.

J Invest Dermatol 2016 07 28;136(7):1364-1372. Epub 2016 Feb 28.

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

Differentiation between Sézary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sézary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sézary syndrome were investigated. Peripheral blood CD4(+) T cells from 59 patients with Sézary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sézary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (≥80% CD4(+) T cells) and/or CD7 (≥40% CD4(+) T cells) and combination of altered expression of STAT4, TWIST1, and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sézary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sézary syndrome versus erythrodermic inflammatory dermatoses.
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http://dx.doi.org/10.1016/j.jid.2016.01.038DOI Listing
July 2016

EPHA4 is overexpressed but not functionally active in Sézary syndrome.

Oncotarget 2015 Oct;6(31):31868-76

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

EPHA4 belongs to the largest subfamily of receptor tyrosine kinases. In addition to its function during development, overexpression of EPHA4 in tumors has been correlated with increased proliferation, migration and poor survival. Several genome-wide transcription profiling studies have demonstrated high EPHA4 expression in Sézary syndrome (SS), a leukemic variant of cutaneous CD4+ T-cell lymphoma (CTCL) with an aggressive clinical course and poor prognosis. In this study we set out to explore the functional role of EPHA4 in SS. Both high EPHA4 mRNA and protein expression was found in circulating SS-cells of patients compared to healthy CD4+ T-cells. However, using a phosphospecific EPHA4 antibody, phosphorylation of the EPHA4 kinase domain was not detected in either circulating or skin residing SS cells. Moreover, treatment with the phosphatase inhibitor pervanadate did not result in detectable phosphorylation of the EPHA4 kinase domain, in either SS cells or in healthy CD4+ T-cells. Thus, the results from our study confirm high EPHA4 expression in SS cells both on the mRNA and protein levels, making EPHA4 a good diagnostic marker. However, the overexpressed EPHA4 does not appear to be functionally active and its overexpression might be secondary to other oncogenic drivers in SS, like STAT3 and TWIST1.
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http://dx.doi.org/10.18632/oncotarget.5573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741646PMC
October 2015

Mutation in PIGA results in a CD52-negative escape variant in a Sézary syndrome patient during alemtuzumab treatment.

J Invest Dermatol 2015 Apr 28;135(4):1199-1202. Epub 2014 Nov 28.

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

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http://dx.doi.org/10.1038/jid.2014.501DOI Listing
April 2015

Exploring the IL-21-STAT3 axis as therapeutic target for Sézary syndrome.

J Invest Dermatol 2014 Oct 22;134(10):2639-2647. Epub 2014 Apr 22.

Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands. Electronic address:

Sézary syndrome is an aggressive cutaneous T-cell lymphoma. The malignant cells (Sézary cells) are present in skin, lymph nodes, and blood, and express constitutively activated signal transducer and activator of transcription (STAT)3. STAT3 can be activated by IL-21 in vitro and the IL-21 gene itself is a STAT3 target gene, thereby creating an autocrine positive feedback loop that might serve as a therapeutic target. Sézary cells underwent apoptosis when incubated with Stattic, a selective STAT3 inhibitor. STAT3 activation in Sézary cells did not affect expression of the supposed anti-apoptotic STAT3 target genes BCL2, BCL-xL, and SURVIVIN, whereas expression of (proto)oncogenes miR-21, TWIST1, MYC, and PIM1 was significantly increased. CD3/CD28-mediated activation of Sézary cells induced IL-21 expression, accompanied by STAT3 activation and increased proliferation. Blocking IL-21 in CD3/CD28-activated cells had no effects, whereas Stattic abrogated IL-21 expression and cell proliferation. Thus, specific inhibition of STAT3 is highly efficient in the induction of apoptosis of Sézary cells, likely mediated via the regulation of (proto)oncogenes. In contrast, blocking IL-21 alone seems insufficient to affect STAT3 activation, cell proliferation, or apoptosis. These data provide further insights into the pathogenic role of STAT3 in Sézary syndrome and strengthen the notion that STAT3 represents a promising therapeutic target in this disease.
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http://dx.doi.org/10.1038/jid.2014.199DOI Listing
October 2014

Caspase-14-deficient mice are more prone to the development of parakeratosis.

J Invest Dermatol 2013 Mar 27;133(3):742-750. Epub 2012 Sep 27.

Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. Electronic address:

Caspase-14 is an important protease in the proper formation of a fully functional skin barrier. Newborn mice that are deficient in caspase-14 exhibit increased transepidermal water loss and are highly sensitive to UVB-induced photodamage. Decreased caspase-14 expression and incomplete caspase-14 processing in lesional psoriatic parakeratotic stratum corneum has been reported previously. In this study, we show that caspase-14-deficient skin frequently displays incompletely cornified cells in the transitional zone between the granular and the cornified layers, pointing to a delay in cornification. We also demonstrate that after challenge of epidermal permeability barrier function by repetitive acetone treatment, a higher incidence of large parakeratotic plaques was observed in caspase-14-deficient skin. Furthermore, caspase-14-deficient mice are more prone than control mice to the development of parakeratosis upon induction of psoriasis-like dermatitis by imiquimod treatment. These results show that lack of caspase-14 expression predisposes to the development of parakeratosis and that caspase-14 has an important role in keratinocyte terminal differentiation and the maintenance of normal stratum corneum, especially in conditions causing epidermal hyperproliferation.
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http://dx.doi.org/10.1038/jid.2012.350DOI Listing
March 2013

A novel mouse model for Sézary syndrome using xenotransplantation of Sézary cells into immunodeficient RAG2(-/-) γc(-/-) mice.

Exp Dermatol 2012 Sep;21(9):706-9

Department of Dermatology, Leiden University Medical Center, Leiden, the Netherlands.

Sézary syndrome (SS) is an aggressive cutaneous T-cell lymphoma with CD4+ tumor cells localized in the skin, lymph nodes and peripheral blood. Characteristic molecular aberrancies in SS have been identified; however, paucity of functional models severely hampered the translation of these observations into pathogenic mechanisms, and subsequent validation of novel therapeutic targets. We therefore developed a mouse model for SS using intrahepatic injection of SS cells in newborn immunodeficient RAG2(-/-) γc(-/-) mice that are completely devoid of T-, B- and NK-cell activity. Injection of the SS cell line SeAx led to long-term and reproducible systemic repopulation of the mice. Injection of mice with the SS cell line HuT-78 led to the death of the mice owing to massive growth of internal tumors. Four weeks after injection of primary SS cells, human CD3+ T cells could be tracked back in the liver, peripheral blood, lymph nodes, spleen and skin of the mice, although the engraftment rate varied when using cells from different patients. In conclusion, we demonstrate that injection of SS cell lines or primary cells in newborn RAG2(-/-) γc(-/-) mice results in long-term systemic repopulation of the mice, thereby providing a novel mouse model for Sézary syndrome.
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http://dx.doi.org/10.1111/j.1600-0625.2012.01556.xDOI Listing
September 2012

NOTCH1 signaling as a therapeutic target in Sézary syndrome.

J Invest Dermatol 2012 Dec 21;132(12):2810-7. Epub 2012 Jun 21.

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

NOTCH signaling is important for development and tissue homeostasis and is activated in many human cancers. We investigated a role for NOTCH1 signaling in Sézary syndrome (SS), a cutaneous T-cell lymphoma in which CD4+ tumor cells (Sézary cells) are present in the skin, lymph nodes, and peripheral blood. We show consistent expression of activated NOTCH1 by Sézary cells isolated from peripheral blood of SS patients, as well as the SS-derived cell lines SeAx and HuT78. In addition, immunohistochemical stainings of skin biopsies from SS patients showed consistent expression of nuclear NOTCH1 and its downstream target hairy/enhancer of split-1 (HES1) by Sézary cells. We demonstrate that this persistent NOTCH1 activation is not caused by mutations in the coding regions of NOTCH1 and F-box and WD40 domain protein 7 (FBWX7) genes. Inhibition of NOTCH1 signaling by gamma secretase inhibitors decreased cellular viability and induced apoptosis of Sézary cells. These observations argue that NOTCH1 signaling is functionally involved in the pathogenesis of SS, and inhibition of NOTCH1 signaling represents a new therapeutic target for the treatment of SS.
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http://dx.doi.org/10.1038/jid.2012.203DOI Listing
December 2012

Autocrine IL-21 stimulation is involved in the maintenance of constitutive STAT3 activation in Sézary syndrome.

J Invest Dermatol 2012 Feb 22;132(2):440-7. Epub 2011 Sep 22.

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

Sézary syndrome (SS) is a cutaneous T-cell lymphoma (CTCL) with malignant CD4+ T cells (SS cells) in skin, lymph nodes, and blood. Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in SS cells, whereas this activation is lost upon in vitro culturing, indicating that STAT3 activation observed in vivo is the result of activating factors in the micromilieu of the malignant cells. We investigated which factors are involved in STAT3 activation in SS, focusing on cytokines of the common γ-chain family because of their crucial role in T-cell activation. Furthermore, downstream effects of STAT3 signaling in SS cells were assayed. In SS cells, STAT3 was strongly activated by IL-21, and increased expression of IL-21 and its receptor chains was observed in peripheral blood SS cells. IL-21 and IL-21R protein expression was detectable on neoplastic cells in SS skin biopsies. Using short-term culturing experiments, we demonstrate that IL-21 itself and the α-chain of the IL-2 receptor are STAT3 target genes in SS cells, thereby rendering cells more sensitive to stimulation with the T-cell proliferation and activating cytokine IL-2. Combined, our data point toward a pivotal role for an autocrine positive feedback loop involving IL-21 and consequent persistent STAT3 activation in the pathogenesis of SS, thereby indicating IL-21 and IL-21R as new therapeutical targets.
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http://dx.doi.org/10.1038/jid.2011.293DOI Listing
February 2012

GATA3 expression is decreased in psoriasis and during epidermal regeneration; induction by narrow-band UVB and IL-4.

PLoS One 2011 17;6(5):e19806. Epub 2011 May 17.

Department of Dermatology, Erasmus University Medical Center, Rotterdam, The Netherlands.

Psoriasis is characterized by hyperproliferation of keratinocytes and by infiltration of activated Th1 and Th17 cells in the (epi)dermis. By expression microarray, we previously found the GATA3 transcription factor significantly downregulated in lesional psoriatic skin. Since GATA3 serves as a key switch in both epidermal and T helper cell differentiation, we investigated its function in psoriasis. Because psoriatic skin inflammation shares many characteristics of epidermal regeneration during wound healing, we also studied GATA3 expression under such conditions.Psoriatic lesional skin showed decreased GATA3 mRNA and protein expression compared to non-lesional skin. GATA3 expression was also markedly decreased in inflamed skin of mice with a psoriasiform dermatitis induced with imiquimod. Tape-stripping of non-lesional skin of patients with psoriasis, a standardized psoriasis-triggering and skin regeneration-inducing technique, reduced the expression of GATA3. In wounded skin of mice, low GATA3 mRNA and protein expression was detected. Taken together, GATA3 expression is downregulated under regenerative and inflammatory hyperproliferative skin conditions. GATA3 expression could be re-induced by successful narrow-band UVB treatment of both human psoriasis and imiquimod-induced psoriasiform dermatitis in mice. The prototypic Th2 cytokine IL-4 was the only cytokine capable of inducing GATA3 in skin explants from healthy donors. Based on these findings we argue that GATA3 serves as a key regulator in psoriatic inflammation, keratinocyte hyperproliferation and skin barrier dysfunction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019806PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096641PMC
September 2011

Effective treatment of psoriasis with narrow-band UVB phototherapy is linked to suppression of the IFN and Th17 pathways.

J Invest Dermatol 2011 Jul 17;131(7):1547-58. Epub 2011 Mar 17.

Department of Dermatology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands.

Narrow-band ultraviolet-B (NB-UVB) phototherapy is an effective treatment for psoriasis. The molecular mechanisms underlying its efficacy are incompletely understood. To identify NB-UVB-induced molecular pathways that may account for its anti-inflammatory efficacy, gene expression profiling was performed using epidermal RNA from lesional and nonlesional skin from patients with psoriasis undergoing NB-UVB therapy. Downregulation of Th17 signaling pathway was observed during NB-UVB therapy in psoriatic epidermis. Strong inhibition of the Th17 pathway by UVB was confirmed in an ex vivo organ culture system by demonstrating reduced signal transducer and activator of transcription 3 (STAT3) phosphorylation and β-defensin-2 production. These results were further substantiated by demonstrating that NB-UVB inhibited the Th17-dependent psoriasis-like dermatitis in mice. Other pathways affected by NB-UVB therapy include the IFN signaling pathway, epidermal differentiation, and other well-known therapeutic targets in psoriasis, such as the glucocorticoid, vitamin D, peroxisome proliferator-activated receptor, and IL-4 signaling pathways. In conclusion, clinical improvement of psoriasis by NB-UVB is linked to suppression of Th17 and type I and type II IFN signaling pathways, which are critical in the pathogenesis of the disease. Our results show that clinically effective NB-UVB therapy is based on suppression of a broad range of important molecular pathways in psoriatic skin.
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http://dx.doi.org/10.1038/jid.2011.53DOI Listing
July 2011

miRNA expression profiling of mycosis fungoides.

Mol Oncol 2011 Jun 24;5(3):273-80. Epub 2011 Feb 24.

Department of Dermatology, LUMC, Leiden, The Netherlands.

MicroRNAs (miRNAs) are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many malignancies including lymphoma. However, the role of miRNAs in the pathogenesis of T-cell lymphoid malignancies is poorly understood. Previously we examined the miRNA profile of Sézary syndrome (Sz), a leukemia of skin-homing memory T cells. In this study we determined the complete miRNome of mycosis fungoides (MF), the most common type of cutaneous T cell lymphoma. The miRNA profile of skin biopsies from 19 patients with tumor stage MF and 12 patients with benign inflammatory dermatoses (eczema and lichen planus) were compared by microarray analysis. We identified 49 miRNAs that are differentially expressed in tumor stage MF compared to benign inflammatory dermatoses using ANOVA analysis (P < 0.05, Benjamini-Hochberg corrected). The majority of the differentially expressed miRNAs (30/49) were up-regulated in tumor stage MF. The most significant differentially expressed were miR-155 and miR-92a (both up-regulated in tumor stage MF), while miR-93 showed the highest up-regulation in tumor stage MF with a fold difference of 5.8. Differential expression of a selection of these miRNAs was validated by miRNA-Q-PCR on additional test groups (tumors and controls). None of the miRNAs up-regulated in tumor stage MF was previously shown to be up-regulated in Sz, and only 2 of the 19 miRNAs down-regulated in tumor stage MF were also down-regulated in Sz. Taken together this report is the first describing the miRNA signature of tumor stage MF.
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http://dx.doi.org/10.1016/j.molonc.2011.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528293PMC
June 2011

MicroRNA-21 expression in CD4+ T cells is regulated by STAT3 and is pathologically involved in Sézary syndrome.

J Invest Dermatol 2011 Mar 18;131(3):762-8. Epub 2010 Nov 18.

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

MicroRNAs (miRNAs) are small RNAs that control gene expression, and are involved in the regulation of fundamental biological processes including development, cell differentiation, proliferation, and apoptosis. miRNAs regulate gene expression in normal hematopoiesis, and aberrant miRNA expression might contribute to leukomogenesis. Specifically, miR-21 is abundantly expressed in various tumors including leukemia and lymphoma, and is functionally involved in oncogenic processes. We investigated a role for miR-21 in Sézary Syndrome (SS), a cutaneous T-cell lymphoma (CTCL) with CD4+ tumor cells (Sézary cells) present in the skin, lymph nodes, and peripheral blood. It was shown previously that SS is characterized by constitutively activated signal transducer and activator of transcription 3 (STAT3) signaling. In this study we show by chromatin immunoprecipitation that miR-21 is a direct STAT3 target in Sézary cells. Stimulation of Sézary cells or healthy CD4+ T cells with the common-γ chain cytokine IL-21 results in a strong activation of STAT3, and subsequent upregulation of miR-21 expression. Both pri- and mature miR-21 expression are increased in Sézary cells when compared with CD4+ T cells from healthy donors. Silencing of miR-21 in Sézary cells results in increased apoptosis, suggesting a functional role for miR-21 in the leukomogenic process. Consequently, miR-21 might represent a therapeutic target for the treatment of SS.
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http://dx.doi.org/10.1038/jid.2010.349DOI Listing
March 2011

Cellular and molecular effects of pulsed dye laser and local narrow-band UVB therapy in psoriasis.

Lasers Surg Med 2010 Mar;42(3):201-10

Department of Dermatology, Erasmus University Medical Center, 3015 GE Rotterdam, the Netherlands. [email protected]

Background And Objectives: Pulsed dye laser (PDL) therapy is effective in clearing psoriasis plaques, but the mechanism of action is only partially understood. Local narrow-band ultraviolet B (NB-UVB), which has a better-defined mode of action, is an effective standard treatment for psoriasis. Our aim was to evaluate the cellular and molecular effects of PDL and to compare them with those of local NB-UVB in order to gain further insight into their mechanisms of action in psoriasis.

Study Design/patients And Methods: Nineteen patients with stable plaque-type psoriasis were treated either with PDL or NB-UVB. Lesional punch biopsies were obtained from all patients before treatment. Additional biopsies were obtained at 3 and 24 hours after PDL treatment in five of these patients. In 14 patients additional biopsies were taken after 7 and 13 weeks of treatment. Samples were histopathologically examined for the level of dermal T cell infiltrate, and the expression of epidermal beta-defensin 2, immune cell-derived tumor necrosis factor (TNF)-alpha, endothelial E-selectin, vascular endothelial growth factor receptor (VEGFR) 2 and 3, and the expression of interleukin (IL)-23 before and after treatment.

Results: The expression of VEGFR2, VEGFR3, and E-selectin was decreased in clinically high responders within 24 hours after PDL treatment. The expression of IL-23, TNF-alpha mRNA, and E-selectin protein were significantly reduced after two PDL treatments, whereas the expression of all epidermal markers and dermal T cell infiltrates had normalized after four treatments. The expression of epidermal activation markers and E-selectin were significantly reduced after 13 weeks of NB-UVB treatment.

Conclusions: The expression of epidermal activation markers and the dermal T cell infiltrates were decreased after both treatments. The decreased expression of VEGFR2 and VEGFR3 followed by the down-regulation of TNF-alpha and IL-23p19 may be contributory factors in the efficacy of PDL in stable plaque-type psoriasis.
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http://dx.doi.org/10.1002/lsm.20898DOI Listing
March 2010

Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.

J Immunol 2009 May;182(9):5836-45

Department of Dermatology, Erasmus Medical Center, Rotterdam, The Netherlands.

Topical application of imiquimod (IMQ), a TLR7/8 ligand and potent immune activator, can induce and exacerbate psoriasis, a chronic inflammatory skin disorder. Recently, a crucial role was proposed for the IL-23/IL-17 axis in psoriasis. We hypothesized that IMQ-induced dermatitis in mice can serve as a model for the analysis of pathogenic mechanisms in psoriasis-like dermatitis and assessed its IL-23/IL-17 axis dependency. Daily application of IMQ on mouse back skin induced inflamed scaly skin lesions resembling plaque type psoriasis. These lesions showed increased epidermal proliferation, abnormal differentiation, epidermal accumulation of neutrophils in microabcesses, neoangiogenesis, and infiltrates consisting of CD4(+) T cells, CD11c(+) dendritic cells, and plasmacytoid dendritic cells. IMQ induced epidermal expression of IL-23, IL-17A, and IL-17F, as well as an increase in splenic Th17 cells. IMQ-induced dermatitis was partially dependent on the presence of T cells, whereas disease development was almost completely blocked in mice deficient for IL-23 or the IL-17 receptor, demonstrating a pivotal role of the IL-23/IL-17 axis. In conclusion, the sole application of the innate TLR7/8 ligand IMQ rapidly induces a dermatitis closely resembling human psoriasis, critically dependent on the IL-23/IL-17 axis. This rapid and convenient model allows further elucidation of pathogenic mechanisms and evaluation of new therapies in psoriasis.
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http://dx.doi.org/10.4049/jimmunol.0802999DOI Listing
May 2009

Polymorphisms in the interferon regulatory factor-1 promoter are not associated with psoriasis and do not influence IFN-alpha-induced Th1 polarization.

J Interferon Cytokine Res 2007 Oct;27(10):841-6

Department of Immunology, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Psoriasis is a chronic inflammatory skin disease, characterized by a Th1 cytokine profile. We previously demonstrated that type I interferon (IFN-alpha/beta) signaling is activated in psoriatic skin. Type I IFNs regulate the expression of many proinflammatory and anti-inflammatory cytokines, resulting in Th1 polarization. We assessed whether peripheral blood mononuclear cells (PBMC) from psoriatic patients show aberrant IFN-alpha responses. IFN-alpha stimulation caused a similar enhancement of IFN-gamma and interleukin-10 (IL-10) secretion in psoriasis patients and controls, although the level of induction was variable. It was previously suggested that IFN-alpha-induced Th1 polarization is influenced by single nucleotide polymorphisms (SNPs) in the promoter of IFN regulatory factor-1 (IRF-1), a transcription factor involved in IFN signaling, providing a putative explanation for the observed variation. However, sequence analysis revealed no correlation between SNPs in the IRF-1 promoter and induction of IFN-gamma or IL-10 expression. Furthermore, the frequency of the SNPs and psoriasis were not linked. Our data demonstrate that the described IRF-1 promoter SNPs do not play a role in the pathogenesis of psoriasis or in influencing IFN-alpha-induced Th1 polarization. We further demonstrate that psoriatic PBMCs do not respond aberrantly to IFN-alpha with respect to the production of the proinflammatory IFN-gamma and the anti-inflammatory IL-10.
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http://dx.doi.org/10.1089/jir.2007.0013DOI Listing
October 2007

IFN-alpha enhances poly-IC responses in human keratinocytes by inducing expression of cytosolic innate RNA receptors: relevance for psoriasis.

J Invest Dermatol 2008 Apr 11;128(4):932-8. Epub 2007 Oct 11.

Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Keratinocytes play a key role in innate immune responses of the skin to bacterial and viral pathogens. Viral double-stranded RNA and its synthetic analogue polyriboinosinic-polyribocytidylic acid (poly-IC) are recognized via multiple pathways involving the receptors Toll-like receptor 3 (TLR3), protein kinase R (PKR), and the recently described cytosolic RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). We show that preincubation of human keratinocytes with IFN-alpha enhances the proinflammatory responses to poly-IC. Kinetic studies suggest that this is mediated via upregulation of the receptors TLR3, PKR, RIG-I, and MDA5. Interestingly, expression of RIG-I, MDA5, and PKR was significantly increased in lesional skin from patients with psoriasis, a chronic inflammatory skin disease that is characterized by high IFN-alpha levels. These results suggest that psoriatic keratinocytes show increased sensitivity to viral RNA intermediates, thereby leading to excessive proinflammatory responses and maintenance of the inflammatory skin phenotype. Here, we provide early evidence that point toward a role for the recently described cytosolic innate RNA receptors in non-viral chronic inflammatory diseases.
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http://dx.doi.org/10.1038/sj.jid.5701087DOI Listing
April 2008

Identification of serum N-acetylmuramoyl-l-alanine amidase as liver peptidoglycan recognition protein 2.

Biochim Biophys Acta 2005 Aug;1752(1):34-46

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

N-acetylmuramoyl-l-alanine amidase (NAMLAA) hydrolyzes bacterial peptidoglycan and is present in human serum. A peptidoglycan-recognition protein 2 (PGLYRP2) is expressed in human liver and has N-acetylmuramoyl-l-alanine amidase activity. Here, we determined the amino acid sequences of human serum NAMLAA and liver PGLYRP2 and tested the hypothesis that serum NAMLAA and PGLYRP2 are the same protein. Liver PGLYRP2 and serum NAMLAA had the same mass determined by mass spectrometry and polyacrylamide gel electrophoresis, and both proteins and recombinant PGLYRP2 reacted with polyclonal anti-NAMLAA and anti-PGLYRP2 antibodies, and with monoclonal anti-NAMLAA antibodies. Digestion of serum NAMLAA with trypsin, chymotrypsin, or trypsin plus V8 protease, or with CNBr yielded, respectively, 37, 40, and 3 overlapping peptides that matched 100% and covered 81% of the deduced amino acid sequence of mature PGLYRP2. These peptides overlapped all exon-intron junctions indicating no alternative splice forms. Digestion of liver PGLYRP2 with trypsin yielded 23 peptides that matched 100% and covered 44% of the deduced amino acid sequence of mature PGLYRP2. Serum NAMLAA had a C398-C404 disulfide, partial phosphorylation of S218, and deamidation of N253 and N301. These results indicate that serum NAMLAA and liver PGLYRP2 are the same protein encoded by the pglyrp2 gene.
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http://dx.doi.org/10.1016/j.bbapap.2005.07.001DOI Listing
August 2005

Elevated interleukin-18 protein expression in early active and progressive plaque-type psoriatic lesions.

Eur Cytokine Netw 2004 Jul-Sep;15(3):210-6

Department of Immunology, Erasmus MC, Rotterdam, The Netherlands.

Psoriasis is a T cell-mediated inflammatory skin disease characterized by an elevated IFN-gamma and IL-12p70 expression in skin lesions. Interleukin-18 (IL-18) synergizes with IL-12 to induce IFN-gamma production and a strong T-helper-1-mediated immune response, or to induce Th2 polarization depending on the immunological context. We have previously shown that keratinocytes in normal skin produce and store large amounts of pro-IL-18. In this study, we hypothesized that the expression of IL-18 in psoriatic lesional skin might be altered compared to normal skin. Therefore, IL-18 expression was assessed in psoriatic, stable, plaque-type lesions and early active and progressive lesions. IL-18 mRNA and protein concentrations were constitutively high, and did not differ between normal and stable, plaque-type epidermis. In active and progressive lesions an elevated expression of total IL-18 protein relative to normal and stable, plaque-type epidermis was detected using ELISA, while on Western blot, the differences in pro- or mature IL-18 were less clear. Our results indicate that the role of IL-18 in the pathogenesis of early phases of psoriasis may be more prominent than in established psoriatic lesions.
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March 2005

Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis genes in Catharanthus roseus.

J Biol Chem 2004 Dec 1;279(51):52940-8. Epub 2004 Oct 1.

Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.

In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus.
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http://dx.doi.org/10.1074/jbc.M404391200DOI Listing
December 2004

In psoriasis lesional skin the type I interferon signaling pathway is activated, whereas interferon-alpha sensitivity is unaltered.

J Invest Dermatol 2004 Jan;122(1):51-60

Department of Immunology, Erasmus MC, University Medical Center Rotterdam, The Netherlands.

The epidermal phenotype as observed in psoriatic skin results from inflammation and abnormal proliferation and terminal differentiation of keratinocytes. Mice deficient for interferon regulatory factor-2, a repressor of interferon signaling, display psoriasis-like skin inflammation. The development of this phenotype is strictly dependent on type I interferon (interferon-alpha/beta) signaling. The aim of this study was to assess the involvement of interferon-alpha/beta in the pathogenesis of human psoriasis. In psoriatic skin, we measured an increased expression of components that play central and crucial roles in interferon-alpha/beta signal transduction. Culturing keratinocytes or healthy skin biopsies with recombinant interferon-alpha stimulated this signaling pathway; however, this did not induce the expression of markers that are generally used to define the psoriasis phenotype. Furthermore, skin from psoriasis patients responded identically to interferon-alpha stimulation, demonstrating that psoriatic skin does not have an aberrant sensitivity to type I interferon. We conclude that in psoriatic lesional skin the type I interferon signaling pathway is activated, despite an unaltered interferon-alpha sensitivity. Our data furthermore show that type I interferon, in contrast to interferon-gamma, does not act directly on keratinocytes to induce a psoriatic phenotype. Thus, if the observed activated type I interferon signaling is indeed functionally involved in the pathogenesis of psoriasis, its contribution might be indirect, putatively involving other cell types besides keratinocytes.
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http://dx.doi.org/10.1046/j.0022-202X.2003.22113.xDOI Listing
January 2004

Psoriatic lesional skin exhibits an aberrant expression pattern of interferon regulatory factor-2 (IRF-2).

J Pathol 2003 Jan;199(1):107-14

Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands.

Psoriasis is a T-cell-mediated inflammatory skin disease. A Th1 cytokine profile with increased levels of interferon-gamma (IFN-gamma) is predominant in skin and peripheral blood mononuclear cells (PBMCs) from psoriasis patients. Furthermore, psoriatic keratinocytes exhibit an aberrant sensitivity and response to IFN-gamma. The transcriptional activator interferon regulatory factor-1 (IRF-1) plays a crucial role in the activation of IFN-gamma-induced gene expression. Recently it was shown that mice deficient in IRF-2, a transcriptional repressor of IFN signalling and thereby acting as an IRF-1 antagonist, display psoriasis-like skin abnormalities. It was therefore hypothesized that a dysbalance between IRF-1 and IRF-2, the activator and repressor of IFN responses, respectively, contributes to the altered IFN-gamma signalling observed in patients with psoriasis. In the epidermis of patients with psoriasis and healthy controls, similar IRF-1 and IRF-2 mRNA expression levels were observed. Furthermore, it was not possible to detect any differences in IRF-1 and IRF-2 protein levels in nuclear extracts from the epidermis of controls and psoriasis patients by electrophoretic mobility shift assay and western blot analysis. Using double immunofluorescence labelling, it was observed that in normal skin IRF-1 was expressed in keratinocytes throughout the epidermis, whereas IRF-2 was restricted to the basal cell layer. In psoriatic skin, IRF-1 expression was comparable to normal skin, whereas IRF-2 was expressed in both basal and suprabasal cell layers. This altered IRF-2 expression in suprabasal cell layers may therefore result in a dysbalance between the activator and repressor of IFN responses in these cell layers, putatively contributing to aberrant responses to IFN-gamma and eventually to the psoriatic skin phenotype.
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http://dx.doi.org/10.1002/path.1263DOI Listing
January 2003
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