Publications by authors named "Cor Breukel"

24 Publications

  • Page 1 of 1

Involvement of Virus-Induced Interferon Production in IgG Autoantibody-Mediated Anemia.

Int J Mol Sci 2021 Aug 21;22(16). Epub 2021 Aug 21.

Unit of Experimental Medicine, de Duve Institute, Université Catholique de Louvain, 1200 Bruxelles, Belgium.

Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fcγ receptors (FcγR) I, III, and IV. Whereas LDV infection decreases FcγR III expression, it enhances FcγR I and IV expression in wild-type animals. The LDV-associated increase in the expression of FcγR I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of FcγR I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes.
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http://dx.doi.org/10.3390/ijms22169027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8396558PMC
August 2021

Focal and generalized seizure activity after local hippocampal or cortical ablation of Na 1.1 channels in mice.

Epilepsia 2020 04 19;61(4):e30-e36. Epub 2020 Mar 19.

Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Early onset seizures are a hallmark of Dravet syndrome. Previous studies in rodent models have shown that the epileptic phenotype is caused by loss-of-function of voltage-gated Na 1.1 sodium channels, which are chiefly expressed in γ-aminobutyric acid (GABA)ergic neurons. Recently, a possibly critical role has been attributed to the hippocampus in the seizure phenotype, as local hippocampal ablation of Na 1.1 channels decreased the threshold for hyperthermia-induced seizures. However, the effect of ablation of Na 1.1 channels restricted to cortical sites has not been tested. Here we studied local field potential (LFP) and behavior in mice following local hippocampal and cortical ablation of Scn1a, a gene encoding the α1 subunit of Na 1.1 channels, and we compared seizure characteristics with those of heterozygous global knockout Scn1 mice. We found a high incidence of spontaneous seizures following either local hippocampal or cortical ablation, notably during a transient time window, similar to Scn1a mice. Nonconvulsive seizure activity in the injected area was common and preceded generalized seizures. Moreover, mice were susceptible to hyperthermia-induced seizures. In conclusion, local ablation of Na 1.1 channels in the hippocampus and cortex results in focal seizure activity that can generalize. These data indicate that spontaneous epileptic activity may initiate in multiple brain regions in Dravet syndrome.
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http://dx.doi.org/10.1111/epi.16482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216883PMC
April 2020

Immunogenicity of rat-neu mouse mammary tumours determines the T cell-dependent therapeutic efficacy of anti-neu monoclonal antibody treatment.

Sci Rep 2020 03 3;10(1):3933. Epub 2020 Mar 3.

Department of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands.

The use of Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2/neu, results in an increased median survival in Her2 breast cancer patients. The tumour mutational burden and the presence of tumour infiltrating lymphocytes (TILs) clearly correlate with response to trastuzumab. Here, we investigated if the immunogenicity of the transplantable rat-neu tumour cell line (TUBO) derived from a BALB/c-NeuT primary tumour is associated with the response to anti-neu mAb therapy. We compared the TUBO tumour outgrowth and tumour infiltrating T cells in isogenic (BALB/c-NeuT) and non-isogenic (WT BALB/c) recipient mice. Furthermore, therapeutic efficacy of anti-neu mAb and the contribution of T cells were examined in both mouse strains. The outgrowth of untreated tumours was significantly better in BALB/c-NeuT than WT BALB/c mice. Moreover, tumour infiltrating T cells were more abundantly present in WT BALB/c than BALB/c-NeuT mice, showing that the TUBO tumour was more immunogenic in WT BALB/c mice. In TUBO tumour bearing WT BALB/c mice, anti-neu mAb therapy resulted in an increase of tumour infiltrating T cells and long-term survival. When T cells were depleted, this strong anti-tumour effect was reduced to an outgrowth delay. In contrast, in TUBO tumour bearing BALB/c-NeuT mice, treatment with anti-neu mAb resulted only in tumour outgrowth delay, both in the presence and absence of T cells. We concluded that in immunogenic tumours the response to anti-neu mAb therapy is enhanced by additional T cell involvement compared to the response to anti-neu mAb in non-immunogenic tumours.
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http://dx.doi.org/10.1038/s41598-020-60893-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054273PMC
March 2020

First FHM3 mouse model shows spontaneous cortical spreading depolarizations.

Ann Clin Transl Neurol 2020 01 27;7(1):132-138. Epub 2019 Dec 27.

Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Here we show, for the first time, spontaneous cortical spreading depolarization (CSD) events - the electrophysiological correlate of the migraine aura - in animals by using the first generated familial hemiplegic migraine type 3 (FHM3) transgenic mouse model. The mutant mice express L263V-mutated α1 subunits in voltage-gated Na 1.1 sodium channels (Scn1a ). CSDs consistently propagated from visual to motor cortex, recapitulating what has been shown in patients with migraine with aura. This model may be valuable for the preclinical study of migraine with aura and other diseases in which spreading depolarization is a prominent feature.
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http://dx.doi.org/10.1002/acn3.50971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952313PMC
January 2020

High FcγR Expression on Intratumoral Macrophages Enhances Tumor-Targeting Antibody Therapy.

J Immunol 2018 12 5;201(12):3741-3749. Epub 2018 Nov 5.

Department of Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands;

Therapy with tumor-specific Abs is common in the clinic but has limited success against solid malignancies. We aimed at improving the efficacy of this therapy by combining a tumor-specific Ab with immune-activating compounds. In this study, we demonstrate in the aggressive B16F10 mouse melanoma model that concomitant application of the anti-TRP1 Ab (clone TA99) with TLR3-7/8 or -9 ligands, and IL-2 strongly enhanced tumor control in a therapeutic setting. Depletion of NK cells, macrophages, or CD8 T cells all mitigated the therapeutic response, showing a coordinated immune rejection by innate and adaptive immune cells. FcγRs were essential for the therapeutic effect, with a dominant role for FcγRI and a minor role for FcγRIII and FcγRIV. FcγR expression on NK cells and granulocytes was dispensable, indicating that other tumoricidal functions of NK cells were involved and implicating that FcγRI, -III, and -IV exerted their activity on macrophages. Indeed, F4/80Ly-6C inflammatory macrophages in the tumor microenvironment displayed high levels of these receptors. Whereas administration of the anti-TRP1 Ab alone reduced the frequency of these macrophages, the combination with a TLR agonist retained these cells in the tumor microenvironment. Thus, the addition of innate stimulatory compounds, such as TLR ligands, to tumor-specific Ab therapy could greatly enhance its efficacy in solid cancers via optimal exploitation of FcγRs.
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http://dx.doi.org/10.4049/jimmunol.1800700DOI Listing
December 2018

FcγR interaction is not required for effective anti-PD-L1 immunotherapy but can add additional benefit depending on the tumor model.

Int J Cancer 2019 01 12;144(2):345-354. Epub 2018 Nov 12.

Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands.

Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models.
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http://dx.doi.org/10.1002/ijc.31899DOI Listing
January 2019

FcγRI expression on macrophages is required for antibody-mediated tumor protection by cytomegalovirus-based vaccines.

Oncotarget 2018 Jun 29;9(50):29392-29402. Epub 2018 Jun 29.

Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Cytomegalovirus (CMV)-based vaccine vectors are promising vaccine platforms because they induce strong and long-lasting immune responses. Recently it has been shown that vaccination with a mouse CMV (MCMV) vector expressing the melanoma-specific antigen TRP2 (MCMV-TRP2) protects mice against outgrowth of TRP2-positive B16 melanoma tumors, and this protection was dependent on the induction of IgG antibodies. Here we demonstrate that, although mice lacking all receptors for the Fc part of IgG (FcγRs) develop normal IgG responses after MCMV-TRP2 vaccination, the protection against B16 melanoma was completely abrogated, indicating that FcγRs are indispensable in the downstream effector pathway of the polyclonal anti-TRP2 antibody response. By investigating compound FcγR-deficient mouse strains and by using immune cell type-specific cell ablation we show that the IgG antibody-mediated tumor protection elicited by MCMV-TRP2 mainly depends on FcγRI expression on macrophages, whereas FcγRIV plays only a modest role. Thus, tumor-specific antibody therapy might benefit from combination therapy that recruits FcγRI-expressing pro-inflammatory macrophages to the tumor micro-environment.
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http://dx.doi.org/10.18632/oncotarget.25630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047664PMC
June 2018

A Restricted Role for FcγR in the Regulation of Adaptive Immunity.

J Immunol 2018 04 9;200(8):2615-2626. Epub 2018 Mar 9.

Department of Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands;

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity.
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http://dx.doi.org/10.4049/jimmunol.1700429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896742PMC
April 2018

A dystrophic Duchenne mouse model for testing human antisense oligonucleotides.

PLoS One 2018 21;13(2):e0193289. Epub 2018 Feb 21.

Department of Human Genetics, Leiden University Medical Center, Leiden, RC, the Netherlands.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193289PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821388PMC
May 2018

Axin2-mTurquoise2: A novel reporter mouse model for the detection of canonical Wnt signalling.

Genesis 2017 10 19;55(10). Epub 2017 Sep 19.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, The Netherlands.

The canonical Wnt signalling pathway has been implicated in organogenesis and self-renewal of essentially all stem cell systems. In vivo reporter systems are crucial to assess the role of Wnt signalling in the biology and pathology of stem cell systems. We set out to develop a Turquoise (TQ) fluorescent protein based Wnt reporter. We used a CRISPR-Cas9 approach to insert a TQ fluorescent protein encoding gene into the general Wnt target gene Axin2, thereby establishing a Wnt reporter mouse similar to previously generated Wnt reporter mice but with the mTurquoise2 gene instead of E. coli-β-galactosidase (LacZ). The use of mTurquoise2 is especially important in organ systems in which cells need to a be alive for further experimentation such as in vitro activation or transplantation studies. We here report successful generation of Axin2-TQ mice and show that cells from these mice faithfully respond to Wnt signals. High Wnt signals were detected in the intestinal crypts, a classical Wnt signalling site in vivo, and by flow cytometry in the thymus. These mice are an improved tool to further elucidate the role of Wnt signalling in vivo.
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http://dx.doi.org/10.1002/dvg.23068DOI Listing
October 2017

The NOTCH3 score: a pre-clinical CADASIL biomarker in a novel human genomic NOTCH3 transgenic mouse model with early progressive vascular NOTCH3 accumulation.

Acta Neuropathol Commun 2015 Dec 29;3:89. Epub 2015 Dec 29.

Department of Clinical Genetics, K5-R, Leiden University Medical Center, PO Box 9600, 2300, RC, Leiden, The Netherlands.

Introduction: CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a hereditary small vessel disease caused by mutations in the NOTCH3 gene, leading to toxic NOTCH3 protein accumulation in the small- to medium sized arterioles. The accumulation is systemic but most pronounced in the brain vasculature where it leads to clinical symptoms of recurrent stroke and dementia. There is no therapy for CADASIL, and therapeutic development is hampered by a lack of feasible clinical outcome measures and biomarkers, both in mouse models and in CADASIL patients. To facilitate pre-clinical therapeutic interventions for CADASIL, we aimed to develop a novel, translational CADASIL mouse model.

Results: We generated transgenic mice in which we overexpressed the full length human NOTCH3 gene from a genomic construct with the archetypal c.544C > T, p.Arg182Cys mutation. The four mutant strains we generated have respective human NOTCH3 RNA expression levels of 100, 150, 200 and 350 % relative to endogenous mouse Notch3 RNA expression. Immunohistochemistry on brain sections shows characteristic vascular human NOTCH3 accumulation in all four mutant strains, with human NOTCH3 RNA expression levels correlating with age at onset and progression of NOTCH3 accumulation. This finding was the basis for developing the 'NOTCH3 score', a quantitative measure for the NOTCH3 accumulation load. This score proved to be a robust and sensitive method to assess the progression of NOTCH3 accumulation, and a feasible biomarker for pre-clinical therapeutic testing.

Conclusions: This novel, translational CADASIL mouse model is a suitable model for pre-clinical testing of therapeutic strategies aimed at delaying or reversing NOTCH3 accumulation, using the NOTCH3 score as a biomarker.
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http://dx.doi.org/10.1186/s40478-015-0268-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696336PMC
December 2015

Involvement of Fcα/μ Receptor in IgM Anti-Platelet, but Not Anti-Red Blood Cell Autoantibody Pathogenicity in Mice.

J Immunol 2015 Nov 18;195(9):4171-5. Epub 2015 Sep 18.

Unité de Médecine Experimentale, Institut de Pathologie Cellulaire, Université Catholique de Louvain, 1200 Bruxelles, Belgium;

IgM anti-mouse platelet autoantibodies cause thrombocytopenia by mediating uptake of opsonized thrombocytes, whereas IgM anti-erythrocyte autoantibodies induce anemia through a phagocytosis-independent cell destruction. In this article, we show that infection with lactate dehydrogenase-elevating virus, a benign mouse arterivirus, exacerbates the pathogenicity of IgM anti-platelet, but not anti-erythrocyte autoantibodies. To define the role of Fcα/μ receptor (Fcα/μR) in IgM-mediated thrombocytopenia and anemia, we generated mice deficient for this receptor. These animals were resistant to IgM autoantibody-mediated thrombocytopenia, but not anemia. However, the lactate dehydrogenase-elevating virus-induced exacerbation of thrombocytopenia was not associated with enhanced Fcα/μR expression on macrophages. These results indicate that Fcα/μR is required for the pathogenicity of IgM anti-platelet autoantibodies but is not sufficient to explain the full extent of the disease in virally infected animals.
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http://dx.doi.org/10.4049/jimmunol.1500798DOI Listing
November 2015

FcγRIIb on myeloid cells rather than on B cells protects from collagen-induced arthritis.

J Immunol 2014 Jun 19;192(12):5540-7. Epub 2014 May 19.

Department of Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands;

Extensive analysis of a variety of arthritis models in germline KO mice has revealed that all four receptors for the Fc part of IgG (FcγR) play a role in the disease process. However, their precise cell type-specific contribution is still unclear. In this study, we analyzed the specific role of the inhibiting FcγRIIb on B lymphocytes (using CD19Cre mice) and in the myeloid cell compartment (using C/EBPαCre mice) in the development of arthritis induced by immunization with either bovine or chicken collagen type II. Despite their comparable anti-mouse collagen autoantibody titers, full FcγRIIb knockout (KO), but not B cell-specific FcγRIIb KO, mice showed a significantly increased incidence and severity of disease compared with wild-type control mice when immunized with bovine collagen. When immunized with chicken collagen, disease incidence was significantly increased in pan-myeloid and full FcγRIIb KO mice, but not in B cell-specific KO mice, whereas disease severity was only significantly increased in full FcγRIIb KO mice compared with incidence and severity in wild-type control mice. We conclude that, although anti-mouse collagen autoantibodies are a prerequisite for the development of collagen-induced arthritis, their presence is insufficient for disease development. FcγRIIb on myeloid effector cells, as a modulator of the threshold for downstream Ab effector pathways, plays a dominant role in the susceptibility to collagen-induced arthritis, whereas FcγRIIb on B cells, as a regulator of Ab production, has a minor effect on disease susceptibility.
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http://dx.doi.org/10.4049/jimmunol.1303272DOI Listing
June 2014

TSSV: a tool for characterization of complex allelic variants in pure and mixed genomes.

Bioinformatics 2014 Jun 13;30(12):1651-9. Epub 2014 Feb 13.

Department of Human Genetic, Leiden Genome Technology Center, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands and Netherlands Bioinformatics Centre, Leiden, The NetherlandsDepartment of Human Genetic, Leiden Genome Technology Center, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands and Netherlands Bioinformatics Centre, Leiden, The NetherlandsDepartment of Human Genetic, Leiden Genome Technology Center, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands and Netherlands Bioinformatics Centre, Leiden, The Netherlands.

Motivation: Advances in sequencing technologies and computational algorithms have enabled the study of genomic variants to dissect their functional consequence. Despite this unprecedented progress, current tools fail to reliably detect and characterize more complex allelic variants, such as short tandem repeats (STRs). We developed TSSV as an efficient and sensitive tool to specifically profile all allelic variants present in targeted loci. Based on its design, requiring only two short flanking sequences, TSSV can work without the use of a complete reference sequence to reliably profile highly polymorphic, repetitive or uncharacterized regions.

Results: We show that TSSV can accurately determine allelic STR structures in mixtures with 10% representation of minor alleles or complex mixtures in which a single STR allele is shared. Furthermore, we show the universal utility of TSSV in two other independent studies: characterizing de novo mutations introduced by transcription activator-like effector nucleases (TALENs) and profiling the noise and systematic errors in an IonTorrent sequencing experiment. TSSV complements the existing tools by aiding the study of highly polymorphic and complex regions and provides a high-resolution map that can be used in a wide range of applications, from personal genomics to forensic analysis and clinical diagnostics.

Availability And Implementation: We have implemented TSSV as a Python package that can be installed through the command-line using pip install TSSV command. Its source code and documentation are available at https://pypi.python.org/pypi/tssv and http://www.lgtc.nl/tssv.
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http://dx.doi.org/10.1093/bioinformatics/btu068DOI Listing
June 2014

Inhibition of IL-1 Signaling by Antisense Oligonucleotide-mediated Exon Skipping of IL-1 Receptor Accessory Protein (IL-1RAcP).

Mol Ther Nucleic Acids 2013 Jan 22;2:e66. Epub 2013 Jan 22.

Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

The cytokine interleukin 1(IL-1) initiates a wide range of proinflammatory cascades and its inhibition has been shown to decrease inflammation in a variety of diseases. IL-1 receptor accessory protein (IL-1RAcP) is an indispensible part of the IL-1R complex that stabilizes IL-1/IL-1R interaction and plays an important role in the signal transduction of the receptor complex. The soluble form of IL-1RAcP (sIL-1RAcP) contains only the extracellular domain and serves as a natural inhibitor of IL-1 signaling. Therefore, increasing sIL-1RAcP levels might be an attractive therapeutic strategy to inhibit IL-1-driven inflammation. To achieve this we designed specific antisense oligonucleotides (AON), to redirect pre-mRNA IL-1RAcP splicing by skipping of the transmembrane domain encoding exon 9. This would give rise to a novel Δ9IL-1RAcP mRNA encoding a soluble, secreted form of IL-1RAcP, which might have similar activity as natural sIL-1RAcP. AON treatment resulted in exon 9 skipping both in vitro and in vivo. A single dose injection of 10 mg AON/kg body weight induced 90% skipping in mouse liver during at least 5 days. The truncated mRNA encoded for a secreted, soluble Δ9IL-1RAcP protein. IL-1RAcP skipping resulted in a substantial inhibition of IL-1 signaling in vitro. These results indicate that skipping of the transmembrane encoding exon 9 of IL-1RAcP using specific AONs might be a promising therapeutic strategy in a variety of chronic inflammatory diseases.Molecular Therapy - Nucleic Acids (2013) 2, e66; doi:10.1038/mtna.2012.58; published online 22 January 2013.
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http://dx.doi.org/10.1038/mtna.2012.58DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564974PMC
January 2013

The inhibiting Fc receptor for IgG, FcγRIIB, is a modifier of autoimmune susceptibility.

J Immunol 2011 Aug 1;187(3):1304-13. Epub 2011 Jul 1.

Department of Human Genetics, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands.

FcγRIIB-deficient mice generated in 129 background (FcγRIIB(129)(-/-)) if back-crossed into C57BL/6 background exhibit a hyperactive phenotype and develop lethal lupus. Both in mice and humans, the Fcγr2b gene is located within a genomic interval on chromosome 1 associated with lupus susceptibility. In mice, the 129-derived haplotype of this interval, named Sle16, causes loss of self-tolerance in the context of the B6 genome, hampering the analysis of the specific contribution of FcγRIIB deficiency to the development of lupus in FcγRIIB(129)(-/-) mice. Moreover, in humans genetic linkage studies revealed contradictory results regarding the association of "loss of function" mutations in the Fcγr2b gene and susceptibility to systemic lupus erythematosis. In this study, we demonstrate that FcγRIIB(-/-) mice generated by gene targeting in B6-derived ES cells (FcγRIIB(B6)(-/-)), lacking the 129-derived flanking Sle16 region, exhibit a hyperactive phenotype but fail to develop lupus indicating that in FcγRIIB(129)(-/-) mice, not FcγRIIB deficiency but epistatic interactions between the C57BL/6 genome and the 129-derived Fcγr2b flanking region cause loss of tolerance. The contribution to the development of autoimmune disease by the resulting autoreactive B cells is amplified by the absence of FcγRIIB, culminating in lethal lupus. In the presence of the Yaa lupus-susceptibility locus, FcγRIIB(B6)(-/-) mice do develop lethal lupus, confirming that FcγRIIB deficiency only amplifies spontaneous autoimmunity determined by other loci.
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http://dx.doi.org/10.4049/jimmunol.1101194DOI Listing
August 2011

A new conditional Apc-mutant mouse model for colorectal cancer.

Carcinogenesis 2010 May 22;31(5):946-52. Epub 2010 Feb 22.

Department of Human Genetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.

Mutations of the adenomatous polyposis coli (APC) gene predispose individuals to familial adenomatous polyposis (FAP), characterized by multiple tumours in the large intestine. Most mouse models heterozygous for truncating mutant Apc alleles mimic FAP, however, the intestinal tumours occur mainly in the small intestine. To model large intestinal tumours, we generated a new conditional Apc-mutant allele, Apc(15lox), with exon 15 flanked by loxP sites. Similar survival of Apc(1638N/15lox) and Apc(1638N/+) mice indicated that the normal function of Apc was not impaired by the loxP sites. Deletion of exon 15, encoding nearly all functional Apc domains and containing the polyadenylation signal, resulted in a mutant allele expressing low levels of a 74 kDa truncated Apc protein. Germ line Cre-mediated deletion of exon 15 resulted in Apc(Delta15/+) mice, showing a severe Apc(Min/+)-like phenotype characterized by multiple tumours in the small intestine and early lethality. In contrast, conditional Cre-mediated deletion of exon 15 specifically directed to the epithelia of distal small and large intestine of FabplCre;Apc(15lox/+) mice led to longer survival and to tumours that developed predominantly in the large intestine, mimicking human FAP-associated colorectal cancer and sporadic colorectal cancer. We conclude that the FabplCre;Apc(15lox/+) mouse should be an attractive model for studies on prevention and treatment of colorectal cancer.
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http://dx.doi.org/10.1093/carcin/bgq046DOI Listing
May 2010

Highly B lymphocyte-specific tamoxifen inducible transgene expression of CreER T2 by using the LC-1 locus BAC vector.

Genesis 2009 Nov;47(11):729-35

Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

The generation of cell type specific inducible Cre transgenic mice is the most challenging and limiting part in the development of spatio-temporally controlled knockout mouse models. Here we report the generation and characterization of a B lymphocyte-specific tamoxifen-inducible Cre transgenic mouse strain, LC-1-hCD19-CreER(T2). We utilized the human CD19 promoter for expression of the tamoxifen-inducible Cre recombinase (CreER(T2)) gene, embedded in genomic sequences previously reported to give minimal position effects after transgenesis. Cre recombinase activity was evaluated by cross-breeding the LC-1-hCD19-CreER(T2) strain with a strain containing a floxed gene widely expressed in the hematopoietic system. Cre activity was only detected in the presence of tamoxifen and was restricted to B lymphocytes. The efficacy of recombination ranged from 27 to 61% in the hemizygous and homozygous mice, respectively. In conclusion, the LC-1-hCD19-CreER(T2) strain is a powerful tool to study gene function specifically in B lymphocytes at any chosen time point in the lifecycle of the mouse.
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http://dx.doi.org/10.1002/dvg.20549DOI Listing
November 2009

A targeted constitutive mutation in the APC tumor suppressor gene underlies mammary but not intestinal tumorigenesis.

PLoS Genet 2009 Jul 3;5(7):e1000547. Epub 2009 Jul 3.

Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/beta-catenin signaling. Notably, genotype-phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/beta-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/beta-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc(+/1572T) mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc(+/1572T) mice suggests that specific dosages of Wnt/beta-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.
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http://dx.doi.org/10.1371/journal.pgen.1000547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2697381PMC
July 2009

APC and oncogenic KRAS are synergistic in enhancing Wnt signaling in intestinal tumor formation and progression.

Gastroenterology 2006 Oct 16;131(4):1096-109. Epub 2006 Aug 16.

UMR144/Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France.

Background & Aims: Synchronous activation of the Wnt signaling pathway, mostly because of loss of function of the APC tumor suppressor, and of the oncogenic KRAS-signaling pathway is very frequent in colorectal cancer and is associated with poor prognosis.

Methods: We have generated a compound transgenic mouse model, KRAS(V12G)/Apc(+/1638N), to recapitulate the human disease and compared it with single transgenic littermates.

Results: Compound mutant mice are characterized by a 10-fold increase in tumor multiplicity and by accelerated tumor progression, resulting in strongly enhanced morbidity and mortality. Tumors from compound mutant mice proliferate faster and show decreased levels of apoptosis. Several lines of evidence indicate that the observed increase in tumor multiplicity and malignant transformation is caused by the synergistic activation of Wnt signaling in cells with oncogenic KRAS and loss-of-function Apc mutations. Activated KRAS is known to induce tyrosine phosphorylation of beta-catenin, leading to its release from E-cadherin at the adherens junction. This results in an increased beta-catenin pool in the cytoplasma, its subsequent translocation to the nucleus, and the transcriptional activation of Wnt downstream target genes. Accordingly, intestinal tumors from KRAS(V12G)/Apc(+/1638N) mice show a significant increase in cells with nuclear accumulation of beta-catenin when compared with Apc(+/1638N) animals. Moreover, Apc/KRAS-mutant embryonic stem cells show a significantly enhanced beta-catenin/T-cell factor-mediated transcriptional activation, accompanied by increased beta-catenin nuclear localization.

Conclusions: This KRAS-induced increase in Wnt/beta-catenin signaling may enhance the plasticity and self-renewal capacity of the tumor, thus resulting in the drastically augmented tumor multiplicity and malignant behavior in compound mutant animals.
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http://dx.doi.org/10.1053/j.gastro.2006.08.011DOI Listing
October 2006

Molecular analysis of hereditary nonpolyposis colorectal cancer in the United States: high mutation detection rate among clinically selected families and characterization of an American founder genomic deletion of the MSH2 gene.

Am J Hum Genet 2003 May 25;72(5):1088-100. Epub 2003 Mar 25.

Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

The identification of germline mutations in families with HNPCC is hampered by genetic heterogeneity and clinical variability. In previous studies, MSH2 and MLH1 mutations were found in approximately two-thirds of the Amsterdam-criteria-positive families and in much lower percentages of the Amsterdam-criteria-negative families. Therefore, a considerable proportion of HNPCC seems not to be accounted for by the major mismatch repair (MMR) genes. Does the latter result from a lack of sensitivity of mutation detection techniques, or do additional genes underlie the remaining cases? In this study we address these questions by thoroughly investigating a cohort of clinically selected North American families with HNPCC. We analyzed 59 clinically well-defined U.S. families with HNPCC for MSH2, MLH1, and MSH6 mutations. To maximize mutation detection, different techniques were employed, including denaturing gradient gel electrophoresis, Southern analysis, microsatellite instability, immunohistochemistry, and monoallelic expression analysis. In 45 (92%) of the 49 Amsterdam-criteria-positive families and in 7 (70%) of the 10 Amsterdam-criteria-negative families, a mutation was detected in one of the three analyzed MMR genes. Forty-nine mutations were in MSH2 or MLH1, and only three were in MSH6. A considerable proportion (27%) of the mutations were genomic rearrangements (12 in MSH2 and 2 in MLH1). Notably, a deletion encompassing exons 1-6 of MSH2 was detected in seven apparently unrelated families (12% of the total cohort) and was subsequently proven to be a founder. Screening of a second U.S. cohort with HNPCC from Ohio allowed the identification of two additional kindreds with the identical founder deletion. In the present study, we show that optimal mutation detection in HNPCC is achieved by combining accurate and expert clinical selection with an extensive mutation detection strategy. Notably, we identified a common North American deletion in MSH2, accounting for approximately 10% of our cohort. Genealogical, molecular, and haplotype studies showed that this deletion represents a North American founder mutation that could be traced back to the 19th century.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1180263PMC
http://dx.doi.org/10.1086/373963DOI Listing
May 2003

Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling.

Nat Genet 2002 Dec 11;32(4):594-605. Epub 2002 Nov 11.

Center for Human and Clinical Genetics, Leiden University Medical Center, Sylvius Laboratory, Wassenaarseweg 72, 2333 RA Leiden, The Netherlands.

The Wnt signal-transduction pathway induces the nuclear translocation of membrane-bound beta-catenin (Catnb) and has a key role in cell-fate determination. Tight somatic regulation of this signal is essential, as uncontrolled nuclear accumulation of beta-catenin can cause developmental defects and tumorigenesis in the adult organism. The adenomatous polyposis coli gene (APC) is a major controller of the Wnt pathway and is essential to prevent tumorigenesis in a variety of tissues and organs. Here, we have investigated the effect of different mutations in Apc on the differentiation potential of mouse embryonic stem (ES) cells. We provide genetic and molecular evidence that the ability and sensitivity of ES cells to differentiate into the three germ layers is inhibited by increased doses of beta-catenin by specific Apc mutations. These range from a severe differentiation blockade in Apc alleles completely deficient in beta-catenin regulation to more specific neuroectodermal, dorsal mesodermal and endodermal defects in more hypomorphic alleles. Accordingly, a targeted oncogenic mutation in Catnb also affects the differentiation potential of ES cells. Expression profiling of wildtype and Apc-mutated teratomas supports the differentiation defects at the molecular level and pinpoints a large number of downstream structural and regulating genes. Chimeric experiments showed that this effect is cell-autonomous. Our results imply that constitutive activation of the Apc/beta-catenin signaling pathway results in differentiation defects in tissue homeostasis, and possibly underlies tumorigenesis in the colon and other self-renewing tissues.
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http://dx.doi.org/10.1038/ng1045DOI Listing
December 2002

A 10-Mb paracentric inversion of chromosome arm 2p inactivates MSH2 and is responsible for hereditary nonpolyposis colorectal cancer in a North-American kindred.

Genes Chromosomes Cancer 2002 Sep;35(1):49-57

MGC-Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Genomic deletions of the MSH2 gene are a frequent cause of hereditary nonpolyposis colorectal cancer (HNPCC), a common hereditary predisposition to the development of tumors in several organs including the gastrointestinal and urinary tracts and endometrium. The mutation spectrum at the MSH2 gene is extremely heterogeneous because it includes nonsense and missense point mutations, small insertions and deletions leading to frameshifts, and larger genomic deletions, the latter representing approximately 25% of the total mutation burden. Here, we report the identification and molecular characterization of the first paracentric inversion of the MSH2 locus known to cause HNPCC. Southern blot analysis and inverse PCR showed that the centromeric and telomeric breakpoints of the paracentric inversion map within intron 7 and to a contig 10 Mb 3' of MSH2, respectively. Pathogenicity of the paracentric inversion was demonstrated by conversion analysis. The patient's lymphocytes were employed to generate somatic cell hybrids to analyze the expression of the inverted MSH2 allele in an Msh2-deficient rodent cellular background. The inversion was shown to abolish MSH2 expression by both northern and western analysis. This study confirms that Southern blot analysis still represents a useful and informative tool to screen for and identify complex genomic rearrangements in HNPCC. Moreover, monoallelic expression analysis represents an attractive approach to demonstrate pathogenicity of unusual mutations in autosomal dominant hereditary conditions.
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http://dx.doi.org/10.1002/gcc.10094DOI Listing
September 2002

The 'just-right' signaling model: APC somatic mutations are selected based on a specific level of activation of the beta-catenin signaling cascade.

Hum Mol Genet 2002 Jun;11(13):1549-60

Centro de Investigação de Patobiologia Molecular-CIPM, Instituto Português de Oncologia Francisco Gentil, 1093 Lisbon, Portugal.

According to the classical interpretation of Knudson's 'two-hit' hypothesis for tumorigenesis, the two 'hits' are independent mutation events, the end result of which is loss of a tumor suppressing function. Recently, it has been shown that the APC (adenomatous polyposis coli) gene does not entirely follow this model. Both the position and type of the second hit in familial adenomatous polyposis (FAP) polyps depend on the localization of the germline mutation. This non-random distribution of somatic hits has been interpreted as the result of selection for more advantageous mutations during tumor formation. However, the APC gene encodes for a multifunctional protein, and the exact cellular function upon which this selection is based is yet unknown. In this study, we have analyzed somatic APC point mutations and loss of heterozygosity (LOH) in 133 colorectal adenomas from six FAP patients. We observed that when germline mutations result in truncated proteins without any of the seven beta-catenin downregulating 20-amino-acid repeats distributed in the central domain of APC, the majority of the corresponding somatic point mutations retain one or, less frequently, two of the same 20-amino-acid repeats. Conversely, when the germline mutation results in a truncated protein retaining one 20-amino-acid repeat, most second hits remove all 20-amino-acid repeats. The latter is frequently accomplished by allelic loss. Notably, and in contrast to previous observations, in a patient where the germline APC mutation retains two such repeats, the majority of the somatic hits are point mutations (and not LOH) located upstream and removing all of the 20-amino-acid repeats. These results indicate selection for APC genotypes that are likely to retain some activity in downregulating beta-catenin signaling. We propose that this selection process is aimed at a specific degree of beta-catenin signaling optimal for tumor formation, rather than at its constitutive activation by deletion of all of the beta-catenin downregulating motifs in APC.
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http://dx.doi.org/10.1093/hmg/11.13.1549DOI Listing
June 2002
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