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1.
Proc Natl Acad Sci U S A ; 120(40): e2215421120, 2023 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-37756334

RESUMO

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFß and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly (P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls (n = 10). The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFß1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFß1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFß1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.


Assuntos
Fibrose Pulmonar Idiopática , Interleucina-27 , Humanos , Camundongos , Animais , Camundongos Endogâmicos C57BL , Histonas , Plaquetas , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética
2.
Blood ; 134(14): 1119-1131, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31434703

RESUMO

Antiphospholipid antibodies (aPLs) with complex lipid and/or protein reactivities cause complement-dependent thrombosis and pregnancy complications. Although cross-reactivities with coagulation regulatory proteins contribute to the risk for developing thrombosis in patients with antiphospholipid syndrome, the majority of pathogenic aPLs retain reactivity with membrane lipid components and rapidly induce reactive oxygen species-dependent proinflammatory signaling and tissue factor (TF) procoagulant activation. Here, we show that lipid-reactive aPLs activate a common species-conserved TF signaling pathway. aPLs dissociate an inhibited TF coagulation initiation complex on the cell surface of monocytes, thereby liberating factor Xa for thrombin generation and protease activated receptor 1/2 heterodimer signaling. In addition to proteolytic signaling, aPLs promote complement- and protein disulfide isomerase-dependent TF-integrin ß1 trafficking that translocates aPLs and NADPH oxidase to the endosome. Cell surface TF pathway inhibitor (TFPI) synthesized by monocytes is required for TF inhibition, and disabling TFPI prevents aPL signaling, indicating a paradoxical prothrombotic role for TFPI. Myeloid cell-specific TFPI inactivation has no effect on models of arterial or venous thrombus development, but remarkably prevents experimental aPL-induced thrombosis in mice. Thus, the physiological control of TF primes monocytes for rapid aPL pathogenic signaling and thrombosis amplification in an unexpected crosstalk between complement activation and coagulation signaling.


Assuntos
Anticorpos Antifosfolipídeos/imunologia , Monócitos/imunologia , Tromboplastina/imunologia , Trombose/imunologia , Animais , Coagulação Sanguínea , Células Cultivadas , Feminino , Humanos , Lipoproteínas/imunologia , Masculino , Camundongos Endogâmicos C57BL , Monócitos/patologia , Transdução de Sinais , Trombose/sangue , Trombose/patologia
3.
Cytogenet Genome Res ; 156(2): 95-105, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30372695

RESUMO

Although an essential component of assisted reproductive technologies, ovarian stimulation, or superovulation, may interfere with the epigenetic reprogramming machinery during early embryogenesis and gametogenesis. To investigate the possible impact of superovulation particularly on the methylation reprogramming process directly after fertilization, we performed immunofluorescence staining of pronuclear (PN) stage embryos with antibodies against 5mC and 5hmC. PN stage embryos obtained by superovulation displayed an increased incidence of abnormal methylation and hydroxymethylation patterns in both maternal and paternal pronuclear DNA. Subsequent single-cell RT-qPCR analyses of the Tet1, Tet2, and Tet3 genes revealed no significant expression differences between PN stage embryos from spontaneously and superovulated matings that could be causative for the abnormal methylation and hydroxymethylation patterns. To analyze the possible contribution of TET-independent replication-associated demethylation mechanisms, we then determined the 5mC and 5hmC levels of PN stage mouse embryos using immunofluorescence analyses after inhibition of DNA replication with aphidicolin. Inhibition of DNA replication had no effect on abnormal methylation and hydroxymethylation patterns that still persisted in the superovulated group. Interestingly, the onset of DNA replication, which was also analyzed in these experiments, was remarkably delayed in the superovulated group. Our findings imply an impact of superovulation on both replication-dependent and -independent or yet unknown demethylation mechanisms in PN stage mouse embryos. In addition, they reveal for the first time a negative effect of superovulation on the initiation of DNA replication in PN stage mouse embryos.

4.
Hepatology ; 65(6): 2074-2089, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28108987

RESUMO

Incidence and prevalence of inflammatory liver diseases has increased over the last years, but therapeutic options are limited. Pregnancy induces a state of immune tolerance, which can result in spontaneous improvement of clinical symptoms of certain autoimmune diseases including autoimmune hepatitis (AIH). We investigated the immune-suppressive mechanisms of the human pregnancy hormone, chorionic gonadotropin (hCG), in the liver. hCG signaling activates silent mating type information regulation 2 homolog 1 (SIRT1), which deacetylates forkhead box o3 (FOXO3a), leading to repression of proapoptotic gene expression, because the immunosuppressive consequence attributed to the absence of caspase-3 activity of hepatocellular interleukin 16 (IL-16) is no longer processed and released. Thus, serum levels of IL-16, a key chemotactic factor for CD4+ lymphocytes, were reduced and migration to injured hepatocytes prevented. Furthermore, elevated IL-16 levels are found in the sera from patients with AIH, hepatitis B virus, hepatitis C virus, and nonalcoholic steatohepatitis. CONCLUSION: Here, we report that hCG regulates the SIRT1/FOXO3a axis in hepatocytes, resulting in immune suppression by attenuating caspase-3-dependent IL-16 processing and release, which concomitantly prevents autoaggressive T-cell infiltration of the liver. Considering the low toxicity profile of hCG in humans, interrupting the inflammatory cycle by hCG opens new perspectives for therapeutic intervention of inflammatory liver diseases. (Hepatology 2017;65:2074-2089).


Assuntos
Gonadotropina Coriônica/farmacologia , Proteína Forkhead Box O3/efeitos dos fármacos , Hepatite Autoimune/patologia , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box O3/metabolismo , Hepatite Autoimune/imunologia , Hepatócitos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Sensibilidade e Especificidade , Sirtuína 1/metabolismo
5.
Ann Rheum Dis ; 76(5): 891-897, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27903507

RESUMO

OBJECTIVES: Hydroxychloroquine (HCQ) has been used for decades to treat patients with rheumatic diseases, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis or the antiphospholipid syndrome (APS). We hypothesise that HCQ might target endosomal NADPH oxidase (NOX), which is involved in the signal transduction of cytokines as well as antiphospholipid antibodies (aPL). METHODS: For in vitro experiments, monocytic cells were stimulated with tumour necrosis factor α (TNFα), interleukin-1ß (IL-1ß) or a human monoclonal aPL and the activity of NOX was determined by flow cytometry. The expression of genes known to be induced by these stimuli was quantified by quantitative reverse transcription PCR. Live cell imaging was performed by confocal laser scanning microscopy. Finally, the effects of HCQ on NOX-induced signal transduction were analysed in an in vivo model of venous thrombosis. RESULTS: HCQ strongly reduces or completely prevents the induction of endosomal NOX by TNFα, IL-1ß and aPL in human monocytes and MonoMac1 cells. As a consequence, induction of downstream genes by these stimuli is reduced or abrogated. This effect of HCQ is not mediated by direct interference with the agonists but by inhibiting the translocation of the catalytic subunit of NOX2 (gp91phox) into the endosome. In vivo, HCQ protects mice from aPL-induced and NOX2-mediated thrombus formation. CONCLUSIONS: We describe here a novel mechanism of action of HCQ, that is, interference with the assembly of endosomal NOX2. Since endosomal NOX2 is involved in many inflammatory and prothrombotic signalling pathways, this activity of HCQ might explain many of its beneficial effects in rheumatic diseases including the APS.


Assuntos
Antirreumáticos/farmacologia , Hidroxicloroquina/farmacologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/biossíntese , Veia Cava Inferior , Trombose Venosa/prevenção & controle , Adulto , Idoso , Animais , Anticorpos Antifosfolipídeos/efeitos adversos , Anticorpos Antifosfolipídeos/farmacologia , Antirreumáticos/uso terapêutico , Células Cultivadas , Modelos Animais de Doenças , Endossomos/enzimologia , Indução Enzimática/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxicloroquina/uso terapêutico , Imunoglobulina G/farmacologia , Interleucina-1beta/farmacologia , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Monócitos , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , NF-kappa B/genética , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tromboplastina/genética , Fator de Necrose Tumoral alfa/farmacologia , Trombose Venosa/induzido quimicamente , Trombose Venosa/diagnóstico por imagem , Adulto Jovem
6.
Blood ; 124(1): 121-33, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24855208

RESUMO

SIRT1 is an important regulator of cellular stress response and genomic integrity. Its role in tumorigenesis is controversial. Whereas sirtuin 1 (SIRT1) can act as a tumor suppressor in some solid tumors, increased expression has been demonstrated in many cancers, including hematologic malignancies. In chronic myeloid leukemia, SIRT1 promoted leukemia development, and targeting SIRT1 sensitized chronic myeloid leukemia progenitors to tyrosine kinase inhibitor treatment. In this study, we investigated the role of SIRT1 in acute myeloid leukemia (AML). We show that SIRT1 protein, but not RNA levels, is overexpressed in AML samples harboring activating mutations in signaling pathways. In FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)(+)-cells protein, expression of SIRT1 is regulated by FLT3 kinase activity. In addition, SIRT1 function is modulated via the ATM-DBC1-SIRT1 axis in a FLT3-ITD-dependent manner. In murine leukemia models driven by MLL-AF9 or AML1-ETO coexpressing FLT3-ITD, SIRT1 acts as a safeguard to counteract oncogene-induced stress, and leukemic blasts become dependent on SIRT1 activity. Pharmacologic targeting or RNAi-mediated knockdown of SIRT1 inhibited cell growth and sensitized AML cells to tyrosine kinase inhibitor treatment and chemotherapy. This effect was a result of the restoration of p53 activity. Our data suggest that targeting SIRT1 represents an attractive therapeutic strategy to overcome primary resistance in defined subsets of patients with AML.


Assuntos
Dano ao DNA/fisiologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Ativação Enzimática/fisiologia , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Sirtuína 1/genética
7.
J Am Chem Soc ; 136(6): 2473-83, 2014 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-24460244

RESUMO

Monodisperse multifunctional and nontoxic Au@MnO Janus particles with different sizes and morphologies were prepared by a seed-mediated nucleation and growth technique with precise control over domain sizes, surface functionalization, and dye labeling. The metal oxide domain could be coated selectively with a thin silica layer, leaving the metal domain untouched. In particular, size and morphology of the individual (metal and metal oxide) domains could be controlled by adjustment of the synthetic parameters. The SiO2 coating of the oxide domain allows biomolecule conjugation (e.g., antibodies, proteins) in a single step for converting the photoluminescent and superparamagnetic Janus nanoparticles into multifunctional efficient vehicles for theranostics. The Au@MnO@SiO2 Janus particles were characterized using high-resolution transmission electron microscopy (HR-)TEM, powder X-ray diffraction (PXRD), optical (UV-vis) spectroscopy, confocal laser fluorescence scanning microscopy (CLSM), and dynamic light scattering (DLS). The functionalized nanoparticles were stable in buffer solution or serum, showing no indication of aggregation. Biocompatibility and potential biomedical applications of the Au@MnO@SiO2 Janus particles were assayed by a cell viability analysis by coincubating the Au@MnO@SiO2 Janus particles with Caki 1 and HeLa cells. Time-resolved fluorescence spectroscopy in combination with CLSM revealed the silica-coated Au@MnO@SiO2 Janus particles to be highly two-photon active; no indication for an electronic interaction between the dye molecules incorporated in the silica shell surrounding the MnO domains and the attached Au domains was found; fluorescence quenching was observed when dye molecules were bound directly to the Au domains.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Nanopartículas/química , Fótons , Dióxido de Silício/química , Transporte Biológico , Linhagem Celular Tumoral , Sobrevivência Celular , Diagnóstico por Imagem , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
8.
Hepatology ; 58(3): 1054-64, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23526469

RESUMO

UNLABELLED: Sirtuin 6 (SIRT6) is a member of the sirtuin family of NAD+-dependent deacetylases. Genetic deletion of Sirt6 in mice results in a severe degenerative phenotype with impaired liver function and premature death. The role of SIRT6 in development and progression of hepatocellular carcinoma is currently unknown. We first investigated SIRT6 expression in 153 primary human liver cancers and in normal and cirrhotic livers using microarray analysis. SIRT6 was significantly down-regulated in both cirrhotic livers and cancer. A Sirt6 knockout (KO) gene expression signature was generated from primary hepatoctyes isolated from 3-week-old Sirt6-deficient animals. Sirt6-deficient hepatocytes showed up-regulation of established hepatocellular carcinoma (HCC) biomarkers alpha-fetoprotein (Afp), insulin-like growth factor 2 (Igf2), H19, and glypican-3. Furthermore, decreased SIRT6 expression was observed in hepatoma cell lines that are known to be apoptosis-insensitive. Re-expression of SIRT6 in HepG2 cells increased apoptosis sensitivity to CD95-stimulation or chemotherapy treatment. Loss of Sirt6 was characterized by oncogenic changes, such as global hypomethylation, as well as metabolic changes, such as hypoglycemia and increased fat deposition. The hepatocyte-specific Sirt6-KO signature had a prognostic impact and was enriched in patients with poorly differentiated tumors with high AFP levels as well as recurrent disease. Finally, we demonstrated that the Sirt6-KO signature possessed a predictive value for tumors other than HCC (e.g., breast and lung cancer). CONCLUSION: Loss of SIRT6 induces epigenetic changes that may be relevant to chronic liver disease and HCC development. Down-regulation of SIRT6 and genes dysregulated by loss of SIRT6 possess oncogenic effects in hepatocarcinogenesis. Our data demonstrate that deficiency in one epigenetic regulator predisposes a tumorigenic phenotype that ultimately has relevance for outcome of HCC and other cancer patients.


Assuntos
Carcinoma Hepatocelular/fisiopatologia , Epigênese Genética/fisiologia , Neoplasias Hepáticas/fisiopatologia , Sirtuínas/genética , Sirtuínas/fisiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Células Cultivadas , Progressão da Doença , Regulação para Baixo/fisiologia , Feminino , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Prognóstico , Transdução de Sinais/fisiologia , Taxa de Sobrevida
9.
Gastroenterology ; 142(5): 1183-1194.e4, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22333948

RESUMO

BACKGROUND & AIMS: Transforming growth factor (TGF)-ß signaling, which is down-regulated by the E3 ubiquitin ligase Smad ubiquitin regulating factor 2 (Smurf2), promotes development of cancer. We identified a splice variant of Smurf2 (ΔE2Smurf2) and investigated its role in colon carcinogenesis in mice. METHODS: Colitis-associated colon cancer was induced in mice by administration of azoxymethane, followed by 3 cycles of oral administration of dextran sodium sulfate. Messenger RNA levels of Smurf2 in colon tumors and control tissue were measured by quantitative polymerase chain reaction; lymphocyte and cytokine levels were measured in tumor and tissue samples. RESULTS: Tumor-infiltrating CD4(+) cells expressed higher levels of ΔE2Smurf2 than CD4(+) cells from nontumor tissues of wild-type mice. T cell-specific overexpression of ΔE2Smurf2 increased TGF-ß signaling by suppressing protein levels of Smurf2, accompanied by an increase in levels of TGF-ß receptor type II. Transgenic mice that overexpress ΔE2Smurf2 were protected against development of colitis-associated tumors and down-regulated proinflammatory cytokines such as interleukin-6. Patients with chronic inflammatory bowel disease had a significantly lower ratio of Smurf2/ΔE2Smurf2 than control individuals. CONCLUSIONS: T cell-specific ΔE2Smurf2 degrades wild-type Smurf2 and controls intestinal tumor growth in mice by up-regulating TGF-ß receptor type II, reducing proliferation and production of proinflammatory cytokines.


Assuntos
Colite/complicações , Neoplasias do Colo/prevenção & controle , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Receptores de Hialuronatos/análise , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-kit/análise , Receptores Acoplados a Proteínas G/análise
10.
Blood ; 118(8): 2322-32, 2011 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-21734241

RESUMO

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by thromboembolic events and/or fetal loss in the presence of antiphospholipid antibodies (aPLs). The mechanisms underlying the pathogenicity of aPLs are still poorly understood. Here we show that 3 human monoclonal aPLs as well as IgG fractions from patients with the APS increase mRNA expression of the intracellular toll-like receptor (TLR) 7 in plasmacytoid dendritic cells and TLR8 in monocytes. Simultaneously they induce the translocation of TLR7 or TLR8 from the endoplasmic reticulum to the endosome. These effects depend on the uptake of aPLs into the endosome, subsequent activation of endosomal NADPH oxidase, and generation of superoxide. As a consequence cells are dramatically sensitized to ligands for TLR7 and TLR8. This observation delineates a novel signal transduction pathway in innate immunity originating from the endosome. Because the overexpression of TLR7 can also be detected in plasmacytoid dendritic cells from patients with the APS ex vivo, our results provide an explanation for proinflammatory and procoagulant effects of aPLs. Because inappropriate expression of TLR7 has been implicated in the development of systemic autoimmunity, these findings may also be relevant for the understanding of autoimmunity.


Assuntos
Anticorpos Antifosfolipídeos/administração & dosagem , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/imunologia , Receptor 8 Toll-Like/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Síndrome Antifosfolipídica/etiologia , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Feminino , Humanos , Imunidade Inata , Técnicas In Vitro , Interferon-alfa/genética , Ligantes , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Superóxidos/metabolismo , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/genética , Fator de Necrose Tumoral alfa/genética
11.
ScientificWorldJournal ; 2013: 616535, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24228010

RESUMO

In recent years, the synthetic polymer polyetheretherketone (PEEK) has increasingly been used in a number of orthopedic implementations, due to its excellent mechanical properties, bioinertness, and chemical resistance. For in vivo applications, the surface of PEEK, which does not naturally support cell adhesion, has to be modified to improve tissue integration. In the present work we demonstrate a novel wet-chemical modification of PEEK to modify the surface, enabling the covalent grafting of the cell-adhesive RGD-peptide. Modification of the polymer surface was achieved via Schiff base formation using an aliphatic diamine and subsequent crosslinker-mediated immobilization of the peptide. In cell culture experiments with primary osteoblasts it was shown that the RGD-modified PEEK not only significantly promoted cellular adhesion but also strongly enhanced the proliferation of osteoblasts on the modified polymer surface.


Assuntos
Materiais Biocompatíveis/síntese química , Cetonas/química , Oligopeptídeos/química , Osteoblastos/fisiologia , Polietilenoglicóis/química , Bases de Schiff/química , Benzofenonas , Sítios de Ligação , Adesão Celular/fisiologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Humanos , Osteoblastos/citologia , Polímeros , Ligação Proteica , Propriedades de Superfície
12.
JCI Insight ; 8(22)2023 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-37796616

RESUMO

MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.


Assuntos
Encefalopatias , Tumor de Células da Granulosa , Neoplasias Ovarianas , Feminino , Humanos , Animais , Camundongos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Tumor de Células da Granulosa/genética , Mutação , Aneuploidia
13.
Hum Mutat ; 33(4): 750-62, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22290738

RESUMO

The multidrug resistance-associated protein 2 (MRP2/ABCC2) is involved in the efflux of endogenous and xenobiotic substrates, including several anticancer and antiviral drugs. The functional consequences of ABCC2 protein variants remain inconsistent, which may be due to shortcomings of the in vitro assays used. To study systematically the functional consequences of nonsynonymous ABCC2 variants, we used a novel "Screen and Insert" (ScIn) technology to achieve stable and highly reproducible expression of 13 ABCC2 variants in HT1080 cells. Western blotting revealed lower (30-65%) ABCC2 expression for D333G, R1174H, and R1181L as compared with wild type (WT; 100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum-like distribution. Variants N1244K and R1174H decreased transport of glutathione-methylfluorescein (GS-MF) and glutathione-monochlorobimane (GS-MCB) by 80% and 50%, respectively, whereas R1181L and P1291L reduced only GS-MCB transport by 50% as compared with WT. Contrary to protein data, the double variant V1188E/C1515Y decreased specific transport activity for GS-MF and GS-MCB by 40%. The ScIn approach is a feasible and reliable method to functionally characterize systematically ABCC2 variants. D333G, R1174H, R1181L, N1244K, P1291L, and double variant V1188E/C1515Y have been identified as most promising for further clinical evaluation.


Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Negro ou Afro-Americano/genética , Asiático/genética , Linhagem Celular Tumoral , Cloraminas/metabolismo , Fibrossarcoma/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Variação Genética , Células HEK293 , Haplótipos , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Mutação de Sentido Incorreto , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tetraciclina/farmacologia
14.
Immunology ; 136(2): 208-17, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22348538

RESUMO

Although allergen-specific immunotherapy is a clinically effective therapy for IgE-mediated allergic diseases, the risk of IgE-mediated adverse effects still exists. For this reason, chemically modified allergoids have been introduced, which may destroy IgE-binding sites while T-cell activation should be retained. The aim of the study was to analyse the differences between intact allergens and differently modified/aggregated allergoids concerning their internalization as well as T-cell and basophil activation. For this purpose human monocyte-derived immature dendritic cells (DC) were incubated with Phleum pratense or Betula verrucosa pollen extract or with the corresponding allergoids, modified with formaldehyde or glutaraldehyde. After an additional maturation process, the antigen-loaded mature DC were co-cultured with autologous CD4(+) T cells. Allergenicity was tested by leukotriene release from basophils. In addition, the uptake of intact allergens and allergoids by immature DC was analysed. The proliferation of, as well as the interleukin-4 (IL-4), IL-10, IL-13 and interferon-γ production by, CD4(+) T cells which had been stimulated with glutaraldehyde allergoid-treated DC was reduced compared with CD4(+) T cells stimulated with intact allergen-treated or formaldehyde allergoid-treated DC. In line with this, glutaraldehyde-modified allergoids were more aggregated and were internalized more slowly. Furthermore, only the allergoids modified with glutaraldehyde induced a decreased leukotriene release by activated basophils. These findings suggest that IgE-reactive epitopes were destroyed more efficiently by modification with glutaraldehyde than with formaldehyde under the conditions chosen for these investigations. Glutaraldehyde-modified allergoids also displayed lower T-cell stimulatory capacity, which is mainly the result of greater modification/aggregation and diminished uptake by DC.


Assuntos
Alérgenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Glutaral/imunologia , Extratos Vegetais/imunologia , Alérgenos/química , Basófilos/efeitos dos fármacos , Basófilos/imunologia , Betula/química , Betula/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Dessensibilização Imunológica/métodos , Epitopos/química , Epitopos/imunologia , Formaldeído/química , Formaldeído/imunologia , Glutaral/química , Humanos , Leucotrienos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Phleum/química , Phleum/imunologia , Extratos Vegetais/química
15.
Biol Chem ; 393(1-2): 23-35, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22628296

RESUMO

Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.


Assuntos
Encéfalo/citologia , Células Endoteliais/citologia , Corantes Fluorescentes/análise , RNA Interferente Pequeno/análise , Animais , Encéfalo/metabolismo , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Microscopia Confocal , RNA Interferente Pequeno/química , Ratos
16.
Artif Organs ; 36(9): 839-44, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22747750

RESUMO

Polyvinyl chloride (PVC) is one of the most frequently used polymers for the manufacturing of medical devices. Limitations for its usage are based upon unfavorable surface properties of the polymer including its hydrophobicity and lack of functionalities in order to increase its versatility. To address this issue, wet chemical modification of PVC was performed through surface amination using the bifunctional compound ethylene diamine. The reaction was conducted in order to achieve maximum surface amination while leaving the bulk material unaffected. The initial activation step was characterized by means of various methods including contact angle measurements, colorimetric amine quantification, infrared spectroscopy, and gel permeation chromatography. Depth profiles were obtained by a confocal microscopic method using fluorescence labeling. Exclusive surface modification was thus confirmed. To demonstrate biological applications of the presented technique, two examples were chosen: The covalent immobilization of the cell adhesive Asp-Gly-Asp-Ser-peptide (RGD) onto PVC samples yielded a surface that strongly supported cellular adhesion and proliferation of fibroblasts. In contrast, the decoration of PVC with the hydrophilic polymer polyethylene glycol prevented cellular adhesion to a large extent. The impact of these modifications was demonstrated by cell culture experiments.


Assuntos
Fibroblastos/citologia , Oligopeptídeos/química , Cloreto de Polivinila/química , Alicerces Teciduais/química , Sequência de Aminoácidos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Adesão Celular , Células Cultivadas , Humanos , Oligopeptídeos/metabolismo , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Cloreto de Polivinila/metabolismo , Molhabilidade
17.
Int J Cancer ; 128(6): 1259-68, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20506153

RESUMO

A genetic basis of hepatocellular carcinoma (HCC) has been well-established and major signaling pathways, such as p53, Wnt-signaling, transforming growth factor-ß (TGF-ß) and Ras pathways, have been identified to be essential to HCC development. Lately, the family of platelet-derived growth factors (PDGFs) has shifted to the center of interest. We have reported on spontaneously developing liver fibrosis in PDGF-B transgenic mice. Since HCC rarely occurs in healthy liver, but dramatically increases at the cirrhosis stage of which liver fibrosis is a preliminary stage, we investigated liver cancer development in chemically induced liver carcinogenesis in these mice. HCC induction was performed by treatment of the mice with diethylnitrosamine and phenobarbital. At an age of 6 months, the tumor development of these animals was analyzed. Not only the development of dysplastic lesions in PDGF-B transgenic mice was significantly increased but also their malignant transformation to HCC. Furthermore, we were able to establish a key role of PDGF-B signaling at diverse stages of liver cancer development. Here, we show that development of liver fibrosis is likely through upregulation of TGF-ß receptors by PDGF-B. Additionally, overexpression of PDGF-B also leads to an increased expression of ß-catenin as well as vascular endothelial growth factor and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), all factors with established roles in carcinogenesis. We were able to extend the understanding of key genetic regulators in HCC development by PDGF-B and decode essential downstream signals.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Alquilantes/toxicidade , Animais , Anticonvulsivantes/toxicidade , Western Blotting , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Dietilnitrosamina/toxicidade , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Transgênicos , Fenobarbital/toxicidade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
18.
Mod Pathol ; 24(8): 1101-10, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21499232

RESUMO

Podocin is a critical component of the glomerular filtration barrier, its mutations causing recessive steroid-resistant nephrotic syndrome. A GenBank analysis of the human podocin (NPHS2) gene resulted in the possible existence of a new splice variant of podocin in the kidney, missing the in-frame of exon 5, encoding the prohibitin homology domain. Using RT-polymerase chain reaction and immunoblotting followed by sequence analysis, we are for the first time able to prove the expression of a novel podocin isoform (isoform 2), exclusively and constitutively expressed in human podocytes. Furthermore, we reveal singular extrarenal podocin expression in human and murine testis. Our data show the Sertoli cells of the seminiferous tubules to be the origin of testicular podocin. Confocal laser microscopy illustrates the co-localization of podocin with filamentous actin within Sertoli cells, suggesting a role of podocin in the blood/testis barrier. These results led to the rationale to examine podocin expression in testes of men with Sertoli cell-only syndrome, a disorder characterized by azoospermia. Interestingly, we observed a complete down-regulation of podocin mRNA in Sertoli cell-only syndrome, indicating a possible role of podocin in the pathogenesis of this germinal aplasia. Men with Sertoli cell-only syndrome show normal renal podocin expression, suggesting an alternate regulation of the testicular promoter. Our findings may change the perception of podocin and give new insights into the ultrastructure of glomerular slit diaphragm and the blood/testis barrier.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Podócitos/metabolismo , Síndrome de Células de Sertoli/metabolismo , Células de Sertoli/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Perfilação da Expressão Gênica , Humanos , Immunoblotting , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Microscopia Confocal , Pessoa de Meia-Idade , Dados de Sequência Molecular , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
19.
Blood ; 113(23): 5891-5, 2009 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-19342479

RESUMO

Deubiquitination of NF-kappaB members by CYLD is crucial in controlling the magnitude and nature of cell activation. The role of the naturally occurring CYLD splice variant in dendritic cell (DC) function was analyzed using CYLD(ex7/8) mice, which lack the full-length CYLD (flCYLD) transcript and overexpress the short splice variant (sCYLD). Bone marrow-derived DCs from CYLD(ex7/8) mice display a hyperactive phenotype in vitro and in vivo and have a defect in establishing tolerance with the use of DEC-205-mediated antigen targeting to resting DCs. The combination of sCYLD overexpression and lack of flCYLD in CYLD(ex7/8) DCs leads to enhanced NF-kappaB activity accompanied by an increased nuclear translocation of the IkappaB molecule Bcl-3, along with nuclear p50 and p65. This suggests that, in contrast to flCYLD, sCYLD is a positive regulator of NF-kappaB activity, and its overexpression induces a hyperactive phenotype in DCs.


Assuntos
Processamento Alternativo/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Animais , Enzima Desubiquitinante CYLD , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/metabolismo , Fenótipo , Transdução de Sinais
20.
BMC Biol ; 8: 33, 2010 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-20374622

RESUMO

BACKGROUND: Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. Precancerous cells are often removed by cell death from normal tissues in the early steps of the tumourigenic process, but the molecules responsible for such a fundamental safeguard process remain in part elusive. With the aim to investigate the molecular crosstalk occurring between precancerous and normal cells in vivo, we took advantage of the clonal analysis methods that are available in Drosophila for studying the phenotypes due to lethal giant larvae (lgl) neoplastic mutation induced in different backgrounds and tissues. RESULTS: We observed that lgl mutant cells growing in wild-type imaginal wing discs show poor viability and are eliminated by Jun N-terminal Kinase (JNK)-dependent cell death. Furthermore, they express very low levels of dMyc oncoprotein compared with those found in the surrounding normal tissue. Evidence that this is a cause of lgl mutant cells elimination was obtained by increasing dMyc levels in lgl mutant clones: their overgrowth potential was indeed re-established, with mutant cells overwhelming the neighbouring tissue and forming tumourous masses displaying several cancer hallmarks. Moreover, when lgl mutant clones were induced in backgrounds of slow-dividing cells, they upregulated dMyc, lost apical-basal cell polarity and were able to overgrow. Those phenotypes were abolished by reducing dMyc levels in the mutant clones, thereby confirming its key role in lgl-induced tumourigenesis. Furthermore, we show that the eiger-dependent Intrinsic Tumour Suppressor pathway plays only a minor role in eliminating lgl mutant cells in the wing pouch; lgl-/- clonal death in this region is instead driven mainly by dMyc-induced Cell Competition. CONCLUSIONS: Our results provide the first evidence that dMyc oncoprotein is required in lgl tumour suppressor mutant tissue to promote invasive overgrowth in larval and adult epithelial tissues. Moreover, we show that dMyc abundance inside versus outside the mutant clones plays a key role in driving neoplastic overgrowth.


Assuntos
Morte Celular/genética , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fenótipo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Células Epiteliais/metabolismo , Imunofluorescência , Técnicas de Inativação de Genes , Mutação/genética
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