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1.
Clin Transl Sci ; 17(2): e13724, 2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-38407540

RESUMEN

LYS006 is a novel, highly potent and selective, new-generation leukotriene A4 hydrolase (LTA4H) inhibitor in clinical development for the treatment of neutrophil-driven inflammatory diseases. We describe the complex pharmacokinetic to pharmacodynamic (PD) relationship in blood, plasma, and skin of LYS006-treated nonclinical species and healthy human participants. In a randomized first in human study, participants were exposed to single ascending doses up to 100 mg and multiple ascending doses up to 80 mg b.i.d.. LYS006 showed rapid absorption, overall dose proportional plasma exposure and nonlinear blood to plasma distribution caused by saturable target binding. The compound efficiently inhibited LTB4 production in human blood and skin blister cells, leading to greater than 90% predose target inhibition from day 1 after treatment initiation at doses of 20 mg b.i.d. and above. Slow re-distribution from target expressing cells resulted in a long terminal half-life and a long-lasting PD effect in ex vivo stimulated blood and skin cells despite low plasma exposures. LYS006 was well-tolerated and demonstrated a favorable safety profile up to highest doses tested, without any dose-limiting toxicity. This supported further clinical development in phase II studies in predominantly neutrophil-driven inflammatory conditions, such as hidradenitis suppurativa, inflammatory acne, and ulcerative colitis.


Asunto(s)
Epóxido Hidrolasas , Plasma , Humanos , Neutrófilos , Piel
2.
Front Immunol ; 14: 1180833, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37457736

RESUMEN

Introduction: Several diseases caused by the dysregulation of complement activation can be treated with inhibitors of the complement components C5 and/or C3. However, complement is required for serum bactericidal activity (SBA) against encapsulated Gram-negative bacteria. Therefore, C3 and C5 inhibition increases the risk of invasive disease, in particular by Neisseria meningitidis. As inhibitors against complement components other than C3 and C5 may carry a reduced risk of infection, we compared the effect of inhibitors targeting the terminal pathway (C5), the central complement component C3, the alternative pathway (FB and FD), and the lectin pathway (MASP-2) on SBA against serogroup B meningococci. Methods: Serum from adults was collected before and after vaccination with the meningococcal serogroup B vaccine 4CMenB and tested for meningococcal killing. Since the B capsular polysaccharide is structurally similar to certain human polysaccharides, 4CMenB was designed to elicit antibodies against meningococcal outer membrane proteins. Results: While only a few pre-vaccination sera showed SBA against the tested B meningococcal isolates, 4CMenB vaccination induced potent complement-activating IgG titers against isolates expressing a matching allele of the bacterial cell surface-exposed factor H-binding protein (fHbp). SBA triggered by these cell surface protein-specific antibodies was blocked by C5 and reduced by C3 inhibition, whereas alternative (factor B and D) and lectin (MASP-2) pathway inhibitors had no effect on the SBA of post-4CMenB vaccination sera. Discussion: Compared to the SBA triggered by A,C,W,Y capsule polysaccharide conjugate vaccination, SBA against B meningococci expressing a matching fHbp allele was remarkably resilient against the alternative pathway inhibition.


Asunto(s)
Infecciones Meningocócicas , Vacunas Meningococicas , Neisseria meningitidis , Adulto , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Infecciones Meningocócicas/prevención & control , Vía Alternativa del Complemento , Anticuerpos Antibacterianos , Vacunas Bacterianas , Proteínas del Sistema Complemento , Proteínas de la Membrana
3.
Front Immunol ; 13: 1020580, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36578495

RESUMEN

Defense against Haemophilus influenzae type b (Hib) is dependent on antibodies and complement, which mediate both serum bactericidal activity (SBA) and opsonophagocytosis. Here we evaluated the influence of capsule-specific antibodies and complement inhibitors targeting the central component C3, the alternative pathway (AP; fB, fD), the lectin pathway (LP; MASP-2) and the terminal pathway (C5) on both effector functions. Findings may be relevant for the treatment of certain diseases caused by dysregulation of the complement system, where inhibitors of complement factors C3 or C5 are used. Inhibitors against other complement components are being evaluated as potential alternative treatment options that may carry a reduced risk of infection by encapsulated bacteria. Serum and reconstituted blood of healthy adults were tested for bactericidal activity before and after vaccination with the Hib capsule-conjugate vaccine ActHIB. Most sera had bactericidal activity prior to vaccination, but vaccination significantly enhanced SBA titers. Independently of the vaccination status, both C3 and C5 inhibition abrogated SBA, whereas inhibition of the LP had no effect. AP inhibition had a major inhibitory effect on SBA of pre- vaccination serum, but vaccination mitigated this inhibition for all disease isolates tested. Despite this, SBA-mediated killing of some Hib isolates remained retarded. Even for the most serum-resistant isolate, SBA was the dominating defense mechanism in reconstituted whole blood, as addition of blood cells to the serum did not enhance bacterial killing. Limited Fc receptor-mediated opsonophagocytosis was unmasked when bacterial killing by the membrane attack complex was blocked. In the presence of C3 or C5 inhibitors, addition of post-vaccination, but not of pre-vaccination serum to the blood cells triggered opsonophagocytosis, leading to suppression of bacterial multiplication. Taken together, our data indicate that for host defense against Hib, killing by SBA is more efficient than by blood cell opsonophagocytosis. However, additional defense mechanisms, such as bacterial clearance by spleen and liver, may play an important role in preventing Hib-mediated sepsis, in particular for Hib isolates with increased serum-resistance. Results indicate potentially improved safety profile of AP inhibitors over C3 and C5 inhibitors as alternative therapeutic agents in patients with increased susceptibility to Hib infection.


Asunto(s)
Infecciones por Haemophilus , Haemophilus influenzae tipo b , Adulto , Humanos , Opsonización , Anticuerpos Antibacterianos , Proteínas del Sistema Complemento
4.
Front Immunol ; 12: 747594, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34691058

RESUMEN

Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the complement component C5 and prevent formation of the membrane attack complex (MAC). While MAC is involved in uncontrolled lysis of erythrocytes in these patients, it is also required for serum bactericidal activity (SBA), i.e. clearance of encapsulated bacteria. Therefore, terminal complement blockage in these patients increases the risk of invasive disease by Neisseria meningitidis more than 1000-fold compared to the general population, despite obligatory vaccination. It is assumed that alternative instead of terminal pathway inhibition reduces the risk of meningococcal disease in vaccinated individuals. To address this, we investigated the SBA with alternative pathway inhibitors. Serum was collected from adults before and after vaccination with a meningococcal serogroup A, C, W, Y capsule conjugate vaccine and tested for meningococcal killing in the presence of factor B and D, C3, C5 and MASP-2 inhibitors. B meningococci were not included in this study since the immune response against protein-based vaccines is more complex. Unsurprisingly, inhibition of C5 abrogated killing of meningococci by all sera. In contrast, both factor B and D inhibitors affected meningococcal killing in sera from individuals with low, but not with high bactericidal anti-capsular titers. While the anti-MASP-2 mAb did not impair SBA, inhibition of C3 impeded meningococcal killing in most, but not in all sera. These data provide evidence that vaccination can provide protection against invasive meningococcal disease in patients treated with alternative pathway inhibitors.


Asunto(s)
Anticuerpos Antibacterianos/inmunología , Inactivadores del Complemento/farmacología , Infecciones Meningocócicas/inmunología , Vacunas Meningococicas/inmunología , Adulto , Anciano , Vía Alternativa del Complemento/efectos de los fármacos , Femenino , Humanos , Masculino
5.
Front Immunol ; 12: 732146, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34707606

RESUMEN

To assess the relative contribution of opsonisation by antibodies, classical and alternative complement pathways to pneumococcal phagocytosis, we analyzed killing of pneumococci by human blood leukocytes collected from vaccine-naïve and PCV13-vaccinated subjects. With serotype 4 pneumococci as model, two different physiologic opsonophagocytosis assays based on either hirudin-anticoagulated whole blood or on washed cells from EDTA-anticoagulated blood reconstituted with active serum, were compared. Pneumococcal killing was measured in the presence of inhibitors targeting the complement components C3, C5, MASP-2, factor B or factor D. The two assay formats yielded highly consistent and comparable results. They highlighted the importance of alternative complement pathway activation for efficient opsonophagocytic killing in blood of vaccine-naïve subjects. In contrast, alternative complement pathway inhibition did not affect pneumococcal killing in PCV13-vaccinated individuals. Independent of amplification by the alternative pathway, even low capsule-specific antibody concentrations were sufficient to efficiently trigger classical pathway mediated opsonophagocytosis. In heat-inactivated or C3-inhibited serum, high concentrations of capsule-specific antibodies were required to trigger complement-independent opsonophagocytosis. Our findings suggest that treatment with alternative complement pathway inhibitors will increase susceptibility for invasive pneumococcal infection in non-immune subjects, but it will not impede pneumococcal clearance in vaccinated individuals.


Asunto(s)
Vía Alternativa del Complemento , Proteínas del Sistema Complemento/inmunología , Opsonización , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/administración & dosificación , Streptococcus pneumoniae/inmunología , Vacunación , Adulto , Anciano , Proteínas Inactivadoras de Complemento/inmunología , Proteínas Inactivadoras de Complemento/metabolismo , Proteínas del Sistema Complemento/metabolismo , Femenino , Interacciones Huésped-Patógeno , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Infecciones Neumocócicas/sangre , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/microbiología , Vacunas Neumococicas/inmunología , Streptococcus pneumoniae/patogenicidad
6.
J Immunol ; 203(11): 2791-2806, 2019 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-31659015

RESUMEN

The paracaspase Malt1 is a key regulator of canonical NF-κB activation downstream of multiple receptors in both immune and nonimmune cells. Genetic disruption of Malt1 protease function in mice and MALT1 mutations in humans results in reduced regulatory T cells and a progressive multiorgan inflammatory pathology. In this study, we evaluated the altered immune homeostasis and autoimmune disease in Malt1 protease-deficient (Malt1PD) mice and the Ags driving disease manifestations. Our data indicate that B cell activation and IgG1/IgE production is triggered by microbial and dietary Ags preferentially in lymphoid organs draining mucosal barriers, likely as a result of dysregulated mucosal immune homeostasis. Conversely, the disease was driven by a polyclonal T cell population directed against self-antigens. Characterization of the Malt1PD T cell compartment revealed expansion of T effector memory cells and concomitant loss of a CD4+ T cell population that phenotypically resembles anergic T cells. Therefore, we propose that the compromised regulatory T cell compartment in Malt1PD animals prevents the efficient maintenance of anergy and supports the progressive expansion of pathogenic, IFN-γ-producing T cells. Overall, our data revealed a crucial role of the Malt1 protease for the maintenance of intestinal and systemic immune homeostasis, which might provide insights into the mechanisms underlying IPEX-related diseases associated with mutations in MALT1.


Asunto(s)
Autoinmunidad/inmunología , Homeostasis/inmunología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/inmunología , Linfocitos T Reguladores/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/deficiencia , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética
7.
Sci Rep ; 6: 21917, 2016 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-26912421

RESUMEN

Melanocytes of the hair follicle produce melanin and are essential in determining the differences in hair color. Pigment cell-specific MELanocyte Protein (PMEL17) plays a crucial role in melanogenesis. One of the critical steps is the amyloid-like functional oligomerization of PMEL17. Beta Site APP Cleaving Enzyme-2 (BACE2) and γ-secretase have been shown to be key players in generating the proteolytic fragments of PMEL17. The ß-secretase (BACE1) is responsible for the generation of amyloid-ß (Aß) fragments in the brain and is therefore proposed as a therapeutic target for Alzheimer's disease (AD). Currently BACE1 inhibitors, most of which lack selectivity over BACE2, have demonstrated efficacious reduction of amyloid-ß peptides in animals and the CSF of humans. BACE2 knock-out mice have a deficiency in PMEL17 proteolytic processing leading to impaired melanin storage and hair depigmentation. Here, we confirm BACE2-mediated inhibition of PMEL17 proteolytic processing in vitro in mouse and human melanocytes. Furthermore, we show that wildtype as well as bace2(+/-) and bace2(-/-) mice treated with a potent dual BACE1/BACE2 inhibitor NB-360 display dose-dependent appearance of irreversibly depigmented hair. Retinal pigmented epithelium showed no morphological changes. Our data demonstrates that BACE2 as well as additional BACE1 inhibition affects melanosome maturation and induces hair depigmentation in mice.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Cabello/metabolismo , Antígeno gp100 del Melanoma/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/genética , Western Blotting , Línea Celular Tumoral , Femenino , Cabello/efectos de los fármacos , Cabello/patología , Humanos , Masculino , Melaninas/metabolismo , Melanocitos/citología , Melanocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Fragmentos de Péptidos/metabolismo , Ácidos Picolínicos/farmacología , Pigmentación/efectos de los fármacos , Prosencéfalo/metabolismo , Prosencéfalo/patología , Inhibidores de Proteasas/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Tiazinas/farmacología , Úvea/efectos de los fármacos , Úvea/metabolismo , Úvea/patología , Antígeno gp100 del Melanoma/antagonistas & inhibidores
8.
J Immunol ; 194(8): 3723-34, 2015 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-25762782

RESUMEN

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.


Asunto(s)
Linfocitos B Reguladores/inmunología , Caspasas/inmunología , Diferenciación Celular/inmunología , Proliferación Celular , Encefalomielitis Autoinmune Experimental/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Linfocitos B Reguladores/patología , Caspasas/genética , Diferenciación Celular/genética , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Humanos , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Ratones , Ratones Noqueados , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Linfocitos T Reguladores/patología , Células TH1/inmunología , Células TH1/patología , Células Th17/inmunología , Células Th17/patología
9.
Toxicol Sci ; 131(2): 375-86, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23091169

RESUMEN

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for ß-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and ß-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.


Asunto(s)
Biomarcadores de Tumor/genética , Impresión Genómica , Péptidos y Proteínas de Señalización Intercelular/genética , Yoduro Peroxidasa/genética , Neoplasias Hepáticas Experimentales/genética , Familia de Multigenes , ARN no Traducido/genética , Animales , Proteínas de Unión al Calcio , Receptor de Androstano Constitutivo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Reacción en Cadena de la Polimerasa , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Transcriptoma , beta Catenina/metabolismo
10.
PLoS One ; 6(3): e18216, 2011 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-21455306

RESUMEN

Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.


Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Epigenómica/métodos , Fenobarbital/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Esteroide Hidroxilasas/genética , Animales , Inmunoprecipitación de Cromatina , Receptor de Androstano Constitutivo , Islas de CpG/efectos de los fármacos , Islas de CpG/genética , Familia 2 del Citocromo P450 , Metilación de ADN/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa
11.
Bone ; 46(3): 680-94, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19857617

RESUMEN

The transcription factor Hey1, a known Notch target gene of the HES family, has recently been described as a target gene of bone morphogenetic protein-2 (BMP-2) during osteoblastic differentiation in vitro. As the role of Hey1 in skeletal physiology is unknown, we analyzed bones of mice ubiquitously lacking or overexpressing Hey1. This strategy enabled us to evaluate whether Hey1 modulation in the whole organism could serve as a drug or antibody target for therapy of diseases associated with bone loss. Hey1 deficiency resulted in modest osteopenia in vivo and increased number and activity of osteoclasts generated ex vivo. Hey1 overexpression resulted in distinct progressive osteopenia and inhibition of osteoblasts ex vivo, an effect apparently dominant to a mild inhibition of osteoclasts. In both Hey1 deficient and overexpressing mice, males were less affected than females and skeleton was not affected during development. Bone histomorphometry did not reveal major changes in animals at 20 weeks, suggesting that modulation had occurred before. Adult Hey1 transgenics also displayed increased type X collagen expression and an enlarged hypertrophic zone in the growth plate. Taken together, our data suggest that ubiquitous in vivo Hey1 regulation affects osteoblasts, osteoclasts and chondrocytes. Due to the complex role of Hey1 in bone, inhibition of Hey1 does not appear to be a straightforward therapeutic strategy to increase the bone mass.


Asunto(s)
Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/patología , Proteínas de Ciclo Celular/biosíntesis , Condrocitos/patología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/biosíntesis , Animales , Animales Recién Nacidos , Enfermedades Óseas Metabólicas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Células Cultivadas , Condrocitos/metabolismo , Femenino , Genes Dominantes , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/fisiología , Hipertrofia , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Osteoblastos/patología , Osteoclastos/patología , Factores de Transcripción/genética , Factores de Transcripción/fisiología
12.
J Biol Chem ; 279(36): 37704-15, 2004 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-15178686

RESUMEN

To examine early events in osteoblast differentiation, we analyzed the expression of about 9,400 genes in the murine MC3T3 cell line, whose robust differentiation was documented cytochemically and molecularly. The cells were stimulated for 1 and 3 days with the osteogenic stimulus containing bone morphogenic protein 2. Total RNA was extracted and analyzed by Affymetrix GeneChip oligonucleotide arrays. A regulated expression of 394 known genes and 295 expressed sequence tags was detected. The sensitivity and reliability of detection by microarrays was shown by confirming the expression pattern for 20 genes by radioactive quantitative reverse transcription-PCR. Functional classification of regulated genes was performed, defining the groups of regulated growth factors, receptors, and transcription factors. The most interesting finding was concomitant activation of transforming growth factor-beta, Wnt, and Notch signaling pathways, confirmed by strong up-regulation of their target genes by PCR. The transforming growth factor-beta pathway is activated by stimulated production of the growth factor itself, while the exact mechanism of Wnt and Notch activation remains elusive. We showed that bone morphogenic protein 2 stimulated expression of Hey1, a direct Notch target gene, in mouse MC3T3 and C2C12 cells, in human mesenchymal cells, and in mouse calvaria. Small interfering RNA-mediated inhibition of Hey1 induction led to an increase in osteoblast matrix mineralization, suggesting that Hey1 is a negative regulator of osteoblast maturation. This negative regulation is apparently achieved via interaction with Runx2: Hey1 completely abrogated Runx2 transcriptional activity. These findings identify the Notch-Hey1 pathway as a negative regulator of osteoblast differentiation/maturation, which is a completely novel aspect of osteogenesis and could point to possible new targets for bone anabolic agents.


Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Calcificación Fisiológica/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/fisiología , Osteogénesis/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Factores de Transcripción/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Células 3T3 , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 2 , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Cartilla de ADN , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Notch , Transcripción Genética/fisiología , Proteínas Wnt
13.
J Transl Med ; 2(1): 6, 2004 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-15025786

RESUMEN

Osteoclasts are cells of hematopoietic origin with a unique property of dissolving bone; their inhibition is a principle for treatment of diseases of bone loss. Protocols for generation of human osteoclasts in vitro have been described, but they often result in cells of low activity, raising questions on cell phenotype and suitability of such assays for screening of bone resorption inhibitors. Here we describe an optimized protocol for the production of stable amounts of highly active human osteoclasts. Mononuclear cells were isolated from human peripheral blood by density centrifugation, seeded at 600,000 cells per 96-well and cultured for 17 days in alpha-MEM medium, supplemented with 10% of selected fetal calf serum, 1 microM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFkappaB ligand (RANKL, 50 ng/ml), and transforming growth factor-beta1 (TGF-beta1, 5 ng/ml). Thus, in addition to widely recognized osteoclast-generating factors M-CSF and RANKL, other medium supplements and lengthy culture times were necessary. This assay reliably detected inhibition of osteoclast formation (multinucleated cells positive for tartrate-resistant acid phosphatase) and activity (resorbed area and collagen fragments released from bone slices) in dose response curves with several classes of bone resorption inhibitors. Therefore, this assay can be applied for monitoring bone-resorbing activity of novel drugs and as an clinical test for determining the capacity of blood cells to generate bone-resorbing osteoclasts. Isolation of large quantities of active human osteoclast mRNA and protein is also made possible by this assay.

14.
Srp Arh Celok Lek ; 130(5-6): 154-8, 2002.
Artículo en Serbio | MEDLINE | ID: mdl-12395434

RESUMEN

Duchenne's and Becker's muscular dystrophy (DMD & BMD) is a X linked disease caused by mutations in the dystrophic gene. DMD is the malign form of the disease, which significantly shortens the lifetime of the patient, while BMD has late onset with slow progression. Sixty five percent of DMD and BMD cases are caused by deletion of one or more exons in the dystrophic gene, while duplications cause these diseases in 6 to 7% of the cases. There are two hot spots for deletions and duplications. These are exons in the proximal part of the gene (3rd to 18th) and exons of a distal part of the gene (45th to 52nd). The remaining 30% of DMD and BMD cases are caused by point mutations, small deletions or inversions in the dystrophic gene. The correlation between the severity of the disease and the position of deletion shows that most of the out of frame deletions cause DMD phenotype, while in frame deletions result in BMD phenotype. We report on the results of 28 non-related DMD and BMD patients. In 57% of cases deletions were detected and all were found in the distal hot spot of the gene. These results suggest that in most of the cases, out of frame deletions produce DMD phenotype while in frame deletions result in BMD phenotype. This is in compliance with data from literature.


Asunto(s)
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Mutación , Fenotipo , Exones/genética , Humanos , Reacción en Cadena de la Polimerasa
15.
Srp Arh Celok Lek ; 130(3-4): 59-63, 2002.
Artículo en Serbio | MEDLINE | ID: mdl-12154515

RESUMEN

Charcot-Marie-Tooth type 1A disease (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are common inherited disorders of the peripheral nervous system associated with duplication and deletion, respectively, of the 17p11.2 segment including the gene of peripheral myelin protein 22. We studied 48 subjects belonging to 29 families with clinical and electrophysiological signs of definite CMT1, 20 patients with suspected CMT phenotype, and 17 patients and healthy members of their families with HNPP. Blood sampling and DNA isolation, PCR, restriction analysis, southern blotting were performed using standard procedures. Of 48 patients with diagnosis of definite CMT1 in 25 (52%) we found a 1.5 Mb tandem duplication in chromosome 17p11.2. These duplications were not found in any of 20 sporadic cases with the clinical phenotype of CMT but without reliable electrophysiological data. Only 13 (44.8%) of 29 unrelated CMT1 patients from the first group had 17p11.2 duplications. Three of 4 sporadic cases (75%) with definite CMT1 had 17p11.2 duplications. Of 17 patients from 6 families with HNPP deletion of 17p11.2 segment was found in 15 (88.2%), as well as in 5 (83.3%) of six unrelated cases. Detection of CMT1A/HNPP recombination hotspot is a simple and reliable DNA diagnostic method, which is useful only for the patients with clinically already verified CMT1, and HNPP for further genetic counselling of patients and members of their families.


Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Cromosomas Humanos Par 17 , Neuropatía Hereditaria Motora y Sensorial/genética , Mutación , Femenino , Duplicación de Gen , Humanos , Masculino , Linaje
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