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1.
Stroke ; 53(2): 381-390, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34983246

RESUMEN

BACKGROUND AND PURPOSE: Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of death and disability in newborns. Clinical and experimental studies suggest a sexual dimorphism in HI-induced brain injury and therapy responses. A major hallmark of HI pathophysiology is the infiltration of peripheral immune cells into the injured brain. However, the specific role of regulatory T cells (Tregs) in neonatal HI is still unknown. METHODS: Nine-day-old mice were exposed to HI by ligation of the right common carotid artery followed by 1 hour hypoxia (10% oxygen). Using immunohistochemistry, flow cytometry, and microarray analyses, Tregs were investigated in the brain, spleen, and blood 24 hours post HI. The functional role of Tregs was evaluated by acute Treg depletion in depletion of regulatory T cells transgenic mice. Brain injury, neuroinflammatory responses, and vascular injury were analyzed via immunohistochemistry and Western blot 48 hours and 7 days after HI. Functional outcome was assessed 3 days and 5 weeks after HI. RESULTS: Female mice revealed an increased cerebral Treg infiltration, coinciding with elevated chemokine receptor expression. Treg depletion in females aggravated HI-induced brain tissue injury, short-term motor deficits, and long-term deficits in exploratory activity, paralleled by an increased microglia and endothelial activation and leukocyte infiltration. Treg depletion in male mice reduced HI-induced brain injury, short-term motor, and long-term cognitive deficits, associated with reduced vascular injury. Ex vivo isolated female Tregs displayed an increased immunosuppressive activity on effector T cell proliferation and an increased gene enrichment in pathways related to enhanced Treg activity. CONCLUSIONS: Tregs from neonatal female mice provide endogenous neuroprotection, whereas Tregs from male mice increase secondary neurodegeneration. As potential mechanisms, we identified intrinsic transcriptional differences associated with enhanced anti-inflammatory activity of female Tregs. Our study emphasizes the urgent need for sex-stratified clinical and preclinical analyses.


Asunto(s)
Hipoxia-Isquemia Encefálica/patología , Linfocitos T Reguladores/patología , Animales , Animales Recién Nacidos , Conducta Animal , Encéfalo/patología , Trastornos Cerebrovasculares/etiología , Trastornos Cerebrovasculares/patología , Trastornos del Conocimiento/etiología , Femenino , Hipoxia-Isquemia Encefálica/psicología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trastornos del Movimiento/etiología , Enfermedades Neuroinflamatorias/etiología , Enfermedades Neuroinflamatorias/patología , Neuronas/patología , Embarazo , Caracteres Sexuales , Linfocitos T/inmunología
2.
Immunology ; 159(3): 344-353, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31755554

RESUMEN

A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild-type littermates at different time-points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time-points after Treg cell depletion in DEREG mice from samples of early time-points before Treg cell depletion in these mice and from gut microbiota samples of wild-type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies.


Asunto(s)
Colon/microbiología , Firmicutes/crecimiento & desarrollo , Factores de Transcripción Forkhead/inmunología , Microbioma Gastrointestinal , Depleción Linfocítica , Linfocitos T Reguladores/inmunología , Animales , Cruzamiento , Colitis/inmunología , Colitis/metabolismo , Colitis/microbiología , Colon/inmunología , Colon/metabolismo , Disbiosis , Heces/microbiología , Femenino , Firmicutes/inmunología , Factores de Transcripción Forkhead/metabolismo , Interacciones Huésped-Patógeno , Vivienda para Animales , Masculino , Ratones , Factores Sexuales , Linfocitos T Reguladores/metabolismo , Factores de Tiempo
3.
Blood ; 131(5): 546-557, 2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29242186

RESUMEN

Patients chronically infected with hepatitis C virus (HCV) frequently develop mixed cryoglobulinemia (MC), a monoclonal expansion of immunoglobulin M (IgM)+ autoreactive B cells, and also have an increased B-cell lymphoma risk. Whether HCV infection also impacts the B-cell compartment and the B-cell receptor repertoire in patients not affected by MC or lymphomas is poorly understood. Flow cytometric analysis of peripheral blood B cells of 30 MC-negative HCV-infected patients and 15 healthy controls revealed that frequencies of class-switched memory B cells were increased in the patients, whereas frequencies of transitional and naive B cells were decreased. For 22 HCV+ patients and 7 healthy controls, we performed high-throughput sequencing of immunoglobulin heavy chain VDJ rearrangements of naive, mature CD5+, IgM+ memory, and class-switched memory B cells. An increased usage of several IGHV genes, including IGHV1-69 and IGHV4-59, which are closely linked to MC and HCV-associated lymphomas, was specifically seen among IgM+ memory B cells of the patients. Moreover, many, and partly very large, expanded clones were seen predominantly among IgM+ memory B cells of all HCV-infected patients analyzed. Thus, chronic HCV infection massively disturbs the B-cell compartment even in patients without clinically detectable B-cell lymphoproliferation and generates many large B-cell clones, especially among non-class-switched memory B cells. Because B-cell clones in MC and lymphomas derive from this B-cell subset, this establishes IgM+ memory B cells as a general target of lymphoproliferation in HCV+ patients, affecting apparently all patients.


Asunto(s)
Linfocitos B/fisiología , Evolución Clonal , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Hepacivirus/inmunología , Hepatitis C Crónica/inmunología , Exones VDJ/genética , Adulto , Estudios de Casos y Controles , Proliferación Celular/genética , Evolución Clonal/genética , Evolución Clonal/inmunología , Células Clonales/metabolismo , Células Clonales/patología , Femenino , Hepatitis C Crónica/sangre , Humanos , Recuento de Linfocitos , Masculino
4.
Eur J Haematol ; 105(6): 786-796, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32875608

RESUMEN

OBJECTIVE: Infusional alemtuzumab followed by consolidating allogeneic hematopoietic stem cell transplantation in eligible patients is considered a standard of care in T-cell prolymphocytic leukemia (T-PLL). Antibody selection against CD52 has been associated with the development of CD52-negative leukemic T cells at time of relapse. Clinical implications and molecular mechanisms underlying this phenotypic switch are unknown. METHODS: We performed flow cytometry and real-time-PCR for CD52-expression and next generation sequencing for PIGA mutational analyses. RESULTS: We identified loss of CD52 expression after alemtuzumab treatment in two of 21 T-PLL patients resulting from loss of GPI-anchor expression caused by inactivating mutations of the PIGA gene. One patient with relapsed T-PLL exhibited a single PIGA mutation, causing a CD52-negative escape variant of the initial leukemic cell clone, preventing alemtuzumab-retreatment. The second patient with continued complete remission after alemtuzumab treatment harbored three different PIGA mutations that affected either the non-neoplastic T cell or the mononuclear cell compartment and resulted in symptomatic paroxysmal nocturnal hemoglobinuria. Next generation sequencing of T-PLL cells collected before the initiation of treatment revealed PIGA wild-type sequence reads in all 16 patients with samples available for testing. CONCLUSION: These data indicate that PIGA mutations were acquired during or after completion of alemtuzumab treatment.


Asunto(s)
Alemtuzumab/farmacología , Antígeno CD52/genética , Leucemia Prolinfocítica de Células T/genética , Proteínas de la Membrana/genética , Mutación , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Alemtuzumab/uso terapéutico , Antígeno CD52/metabolismo , Análisis Mutacional de ADN , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunofenotipificación , Leucemia Prolinfocítica de Células T/tratamiento farmacológico , Leucemia Prolinfocítica de Células T/metabolismo , Proteínas de la Membrana/metabolismo , Fenotipo , Linfocitos T/patología
5.
Proc Natl Acad Sci U S A ; 114(52): E11323-E11332, 2017 12 26.
Artículo en Inglés | MEDLINE | ID: mdl-29229863

RESUMEN

Thyroid hormone (TH) and TH receptors (TRs) α and ß act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type 1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger signaling pathways independently of gene expression (noncanonical or type 3 TR signaling). To test the physiological relevance of noncanonical TR signaling, we generated knockin mice with a mutation in the TR DNA-binding domain that abrogates binding to DNA and leads to complete loss of canonical TH action. We show that several important physiological TH effects are preserved despite the disruption of DNA binding of TRα and TRß, most notably heart rate, body temperature, blood glucose, and triglyceride concentration, all of which were regulated by noncanonical TR signaling. Additionally, we confirm that TRE-binding-defective TRß leads to disruption of the hypothalamic-pituitary-thyroid axis with resistance to TH, while mutation of TRα causes a severe delay in skeletal development, thus demonstrating tissue- and TR isoform-specific canonical signaling. These findings provide in vivo evidence that noncanonical TR signaling exerts physiologically important cardiometabolic effects that are distinct from canonical actions. These data challenge the current paradigm that in vivo physiological TH action is mediated exclusively via regulation of gene transcription at the nuclear level.


Asunto(s)
Sistema Hipotálamo-Hipofisario/metabolismo , Miocardio/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Transducción de Señal , Hormonas Tiroideas/metabolismo , Animales , Técnicas de Sustitución del Gen , Ratones , Ratones Noqueados , Receptores de Hormona Tiroidea/genética , Hormonas Tiroideas/genética
6.
J Infect Dis ; 219(3): 497-508, 2019 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-30202890

RESUMEN

Background: Human immunodeficiency virus (HIV) infection is an independent risk factor for coronary heart disease (CHD) and is associated with perturbation of the gut microbiota. Methods: We analyzed gut microbiota in 30 HIV-infected individuals with CHD (CHD+) and 30 without CHD (CHD-) of the HIV-HEART study group. Results: Gut microbiota linked to CHD was associated with lower α-diversity. Despite insignificant differences in ß-diversity, co-occurrence networks of bacterial genera clearly diverged between CHD+ and CHD- individuals. Multidimensional scaling separated HIV-infected individuals into 2 microbiome clusters, dominated by the genus Prevotella or Bacteroides. The relative abundance of 49 other genera was significantly different between both clusters. The Prevotella-rich cluster was largely composed of men who have sex with men (MSM) (97%), whereas the Bacteroides-rich cluster comprised both MSM (45%) and heterosexual individuals (55%). MSM of the Bacteroides-rich cluster were characterized by reduced α-diversity, advanced immunological HIV stage, longer antiretroviral therapy with more ART regimens, and longer use of protease inhibitors, compared with Prevotella-rich MSM. Conclusions: Community structures of gut microbiota rather than individual species might facilitate risk assessment of CHD in HIV-infected individuals. Sexual behavior appears to be an important factor affecting gut microbiota ß-diversity and should be considered in future studies.


Asunto(s)
Biodiversidad , Enfermedad Coronaria/complicaciones , Microbioma Gastrointestinal , Infecciones por VIH/complicaciones , Adulto , Anciano , Bacteroides/genética , Bacteroides/aislamiento & purificación , Bacteroides/patogenicidad , Femenino , Homosexualidad Masculina , Humanos , Masculino , Metilaminas/farmacología , Metilaminas/uso terapéutico , Persona de Mediana Edad , Prevotella/genética , Prevotella/aislamiento & purificación , Prevotella/patogenicidad , Factores de Riesgo , Conducta Sexual , Minorías Sexuales y de Género
7.
Cytogenet Genome Res ; 159(1): 1-11, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31658463

RESUMEN

The switch/sucrose non-fermenting (SWI/SNF) complex is an ATP-dependent chromatin remodeller that regulates the spacing of nucleosomes and thereby controls gene expression. Heterozygous mutations in genes encoding subunits of the SWI/SNF complex have been reported in individuals with Coffin-Siris syndrome (CSS), with the majority of the mutations in ARID1B. CSS is a rare congenital disorder characterized by facial dysmorphisms, digital anomalies, and variable intellectual disability. We hypothesized that mutations in genes encoding subunits of the ubiquitously expressed SWI/SNF complex may lead to alterations of the nucleosome profiles in different cell types. We performed the first study on CSS-patient samples and investigated the nucleosome landscapes of cell-free DNA (cfDNA) isolated from blood plasma by whole-genome sequencing. In addition, we studied the nucleosome landscapes of CD14+ monocytes from CSS-affected individuals by nucleosome occupancy and methylome-sequencing (NOMe-seq) as well as their expression profiles. In cfDNA of CSS-affected individuals with heterozygous ARID1B mutations, we did not observe major changes in the nucleosome profile around transcription start sites. In CD14+ monocytes, we found few genomic regions with different nucleosome occupancy when compared to controls. RNA-seq analysis of CD14+ monocytes of these individuals detected only few differentially expressed genes, which were not in proximity to any of the identified differential nucleosome-depleted regions. In conclusion, we show that heterozygous mutations in the human SWI/SNF subunit ARID1B do not have a major impact on the nucleosome landscape or gene expression in blood cells. This might be due to functional redundancy, cell-type specificity, or alternative functions of ARID1B.


Asunto(s)
Anomalías Múltiples/genética , Proteínas de Unión al ADN/genética , Cara/anomalías , Deformidades Congénitas de la Mano/genética , Discapacidad Intelectual/genética , Micrognatismo/genética , Cuello/anomalías , Proteínas Nucleares/genética , Nucleosomas/genética , Factores de Transcripción/genética , Adolescente , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , Niño , Preescolar , Femenino , Genoma Humano/genética , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Monocitos/citología , Adulto Joven
8.
Gut ; 66(12): 2110-2120, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28615301

RESUMEN

OBJECTIVE: Postoperative ileus (POI), the most frequent complication after intestinal surgery, depends on dendritic cells (DCs) and macrophages. Here, we have investigated the mechanism that activates these cells and the contribution of the intestinal microbiota for POI induction. DESIGN: POI was induced by manipulating the intestine of mice, which selectively lack DCs, monocytes or macrophages. The disease severity in the small and large intestine was analysed by determining the distribution of orally applied fluorescein isothiocyanate-dextran and by measuring the excretion time of a retrogradely inserted glass ball. The impact of the microbiota on intestinal peristalsis was evaluated after oral antibiotic treatment. RESULTS: We found that Cd11c-Cre+ Irf4flox/flox mice lack CD103+CD11b+ DCs, a DC subset unique to the intestine whose function is poorly understood. Their absence in the intestinal muscularis reduced pathogenic inducible nitric oxide synthase (iNOS) production by monocytes and macrophages and ameliorated POI. Pathogenic iNOS was produced in the jejunum by resident Ly6C- macrophages and infiltrating chemokine receptor 2-dependent Ly6C+ monocytes, but in the colon only by the latter demonstrating differential tolerance mechanisms along the intestinal tract. Consistently, depletion of both cell subsets reduced small intestinal POI, whereas the depletion of Ly6C+ monocytes alone was sufficient to prevent large intestinal POI. The differential role of monocytes and macrophages in small and large intestinal POI suggested a potential role of the intestinal microbiota. Indeed, antibiotic treatment reduced iNOS levels and ameliorated POI. CONCLUSIONS: Our findings reveal that CD103+CD11b+ DCs and the intestinal microbiome are a prerequisite for the activation of intestinal monocytes and macrophages and for dysregulating intestinal motility in POI.


Asunto(s)
Células Dendríticas/citología , Microbioma Gastrointestinal , Ileus/inmunología , Ileus/microbiología , Activación de Macrófagos , Monocitos/inmunología , Peristaltismo/inmunología , Complicaciones Posoperatorias/inmunología , Complicaciones Posoperatorias/microbiología , Animales , Antígenos CD/inmunología , Antígeno CD11b/inmunología , Modelos Animales de Enfermedad , Tránsito Gastrointestinal , Ileus/fisiopatología , Cadenas alfa de Integrinas/inmunología , Ratones , Ratones Transgénicos , Complicaciones Posoperatorias/fisiopatología
9.
Blood ; 125(8): 1207-16, 2015 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-25480661

RESUMEN

Hairy cell leukemia (HCL) shows unique clinicopathological and biological features. HCL responds well to purine analogs but relapses are frequent and novel therapies are required. BRAF-V600E is the key driver mutation in HCL and distinguishes it from other B-cell lymphomas, including HCL-like leukemias/lymphomas (HCL-variant and splenic marginal zone lymphoma). The kinase-activating BRAF-V600E mutation also represents an ideal therapeutic target in HCL. Here, we investigated the biological and therapeutic importance of the activated BRAF-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway in HCL by exposing in vitro primary leukemic cells purified from 26 patients to clinically available BRAF (vemurafenib; dabrafenib) or MEK (trametinib) inhibitors. Results were validated in vivo in samples from vemurafenib-treated HCL patients within a phase 2 clinical trial. BRAF and MEK inhibitors caused, specifically in HCL (but not HCL-like) cells, marked MEK/ERK dephosphorylation, silencing of the BRAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene expression signature, downregulation of the HCL markers CD25, tartrate-resistant acid phosphatase, and cyclin D1, smoothening of leukemic cells' hairy surface, and, eventually, apoptosis. Apoptosis was partially blunted by coculture with bone marrow stromal cells antagonizing MEK-ERK dephosphorylation. This protective effect could be counteracted by combined BRAF and MEK inhibition. Our results strongly support and inform the clinical use of BRAF and MEK inhibitors in HCL.


Asunto(s)
Antineoplásicos , Imidazoles , Indoles , Leucemia de Células Pilosas/tratamiento farmacológico , Leucemia de Células Pilosas/genética , Oximas , Piridonas , Pirimidinonas , Sulfonamidas , Transcriptoma/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Indoles/farmacología , Indoles/uso terapéutico , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Oximas/farmacología , Oximas/uso terapéutico , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Piridonas/farmacología , Piridonas/uso terapéutico , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Células Tumorales Cultivadas , Vemurafenib
10.
Int J Cancer ; 138(1): 121-4, 2016 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-26199174

RESUMEN

The pathogenesis of T-cell large granular lymphocytic leukemia (T-LGL) is poorly understood, as STAT3 mutations are the only known frequent genetic lesions. Here, we identified non-synonymous alterations in the TNFAIP3 tumor suppressor gene in 3 of 39 T-LGL. In two cases these were somatic mutations, in one case the somatic origin was likely. A further case harbored a SNP that is a known risk allele for autoimmune diseases and B cell lymphomas. Thus, TNFAIP3 mutations represent recurrent genetic lesions in T-LGL that affect about 8% of cases, likely contributing to deregulated NF-κB activity in this leukemia.


Asunto(s)
Proteínas de Unión al ADN/genética , Variación Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Linfocítica Granular Grande/genética , Proteínas Nucleares/genética , Estudios de Cohortes , Variaciones en el Número de Copia de ADN , Proteínas de Unión al ADN/metabolismo , Exones , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patología , Mutación , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple , Factor de Transcripción STAT3/genética , Análisis de Secuencia de ADN , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa
11.
Hum Mol Genet ; 22(25): 5121-35, 2013 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-23906836

RESUMEN

Chromatin remodeling complexes are known to modify chemical marks on histones or to induce conformational changes in the chromatin in order to regulate transcription. De novo dominant mutations in different members of the SWI/SNF chromatin remodeling complex have recently been described in individuals with Coffin-Siris (CSS) and Nicolaides-Baraitser (NCBRS) syndromes. Using a combination of whole-exome sequencing, NGS-based sequencing of 23 SWI/SNF complex genes, and molecular karyotyping in 46 previously undescribed individuals with CSS and NCBRS, we identified a de novo 1-bp deletion (c.677delG, p.Gly226Glufs*53) and a de novo missense mutation (c.914G>T, p.Cys305Phe) in PHF6 in two individuals diagnosed with CSS. PHF6 interacts with the nucleosome remodeling and deacetylation (NuRD) complex implicating dysfunction of a second chromatin remodeling complex in the pathogenesis of CSS-like phenotypes. Altogether, we identified mutations in 60% of the studied individuals (28/46), located in the genes ARID1A, ARID1B, SMARCB1, SMARCE1, SMARCA2, and PHF6. We show that mutations in ARID1B are the main cause of CSS, accounting for 76% of identified mutations. ARID1B and SMARCB1 mutations were also found in individuals with the initial diagnosis of NCBRS. These individuals apparently belong to a small subset who display an intermediate CSS/NCBRS phenotype. Our proposed genotype-phenotype correlations are important for molecular screening strategies.


Asunto(s)
Anomalías Múltiples/genética , Ensamble y Desensamble de Cromatina/genética , Cara/anomalías , Deformidades Congénitas del Pie/genética , Deformidades Congénitas de la Mano/genética , Hipotricosis/genética , Discapacidad Intelectual/genética , Micrognatismo/genética , Cuello/anomalías , Eliminación de Secuencia/genética , Anomalías Múltiples/patología , Adolescente , Adulto , Proteínas Portadoras/genética , Niño , Preescolar , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Exoma/genética , Cara/patología , Facies , Femenino , Deformidades Congénitas del Pie/patología , Deformidades Congénitas de la Mano/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hipotricosis/patología , Lactante , Recién Nacido , Discapacidad Intelectual/patología , Cariotipificación , Masculino , Micrognatismo/patología , Mutación Missense , Cuello/patología , Proteínas Represoras , Proteína SMARCB1 , Factores de Transcripción/genética
13.
Pflugers Arch ; 466(10): 1953-61, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24390754

RESUMEN

Ischemic postconditioning (PoCo) reduces infarct size following myocardial ischemia/reperfusion. To protect, PoCo must be performed early during reperfusion, and causal cardioprotective signaling must occur then. The role of microRNA (miRNA) in PoCo is unclear. Anesthetized pigs were subjected to 60 min left anterior descending coronary artery (LAD) occlusion and 180 min reperfusion. Immediate full reperfusion (IFR, n = 5) was compared to PoCo (four cycles of 60 s/60 s reperfusion/reocclusion, n = 5). Transmural myocardial biopsies from the LAD territory were sampled at baseline, 60 min ischemia, 10 and 180 min reperfusion. RNA was isolated. The expression of 11 miRNAs, including muscle-specific (miRNA-1, -133a, -206, -208b, -214, and -499), fibrosis- (miRNA-21, -24, and -29b), neovascularization- (miRNA-92a), and inflammation-associated (miRNA-146b) candidates, was quantified using real-time PCR (RT-PCR). mRNA expression at baseline and 180 min reperfusion was quantified and validated (microarray and RT-PCR). PoCo reduced infarct size from 44.9 ± 7.7 to 34.8 ± 5.3% of the area at risk. The expression of miRNA-1, -24, -29b, -133a, -146b, -208b, and -499 was increased at 10 min reperfusion with PoCo vs. IFR; however, that of miRNA-1, -24, -208b, and -499 was already increased at 60 min ischemia and probably reflects falsely positive results. Five mRNAs were different with PoCo vs. IFR. In silico analysis identified a tentative connection between three miRNAs and five mRNAs with the biological functions "cell death", "inflammatory response" and/or "glucose metabolism". If at all, only miRNA-29b, -133a, and -146b fulfill the minimal temporal requirements for a potential causal involvement in cardioprotection by PoCo.


Asunto(s)
Poscondicionamiento Isquémico , MicroARNs/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Animales , MicroARNs/genética , Daño por Reperfusión Miocárdica/terapia , Porcinos , Porcinos Enanos
14.
BMC Genomics ; 15: 291, 2014 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-24734910

RESUMEN

BACKGROUND: It has been shown previously that aminocoumarin antibiotics such as novobiocin lead to immediate downregulation of recA expression and thereby inhibit the SOS response, mutation frequency and recombination capacity in Staphylococcus aureus. Aminocoumarins function by inhibiting the ATPase activity of DNA gyrase subunit B with a severe impact on DNA supercoiling. RESULTS: Here, we have analysed the global impact of the DNA relaxing agent novobiocin on gene expression in S. aureus. Using a novobiocin-resistant mutant, it became evident that the change in recA expression is due to gyrase inhibition. Microarray analysis and northern blot hybridisation revealed that the expression levels of a distinct set of genes were increased (e.g., recF-gyrB-gyrA, the rib operon and the ure operon) or decreased (e.g., arlRS, recA, lukA, hlgC and fnbA) by novobiocin. The two-component ArlRS system was previously found to decrease the level of supercoiling in S. aureus. Thus, downregulation of arlRS might partially compensate for the relaxing effect of novobiocin. Global analysis and gene mapping of supercoiling-sensitive genes did not provide any indication that they are clustered in the genome. Promoter fusion assays confirmed that the responsiveness of a given gene is intrinsic to the promoter region but independent of the chromosomal location. CONCLUSIONS: The results indicate that the molecular properties of a given promoter, rather than the chromosomal topology, dictate the responsiveness to changes in supercoiling in the pathogen Staphylococcus aureus.


Asunto(s)
Aminocumarinas/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Antineoplásicos/farmacología , Proteínas Bacterianas/genética , Girasa de ADN/metabolismo , ADN Superhelicoidal/efectos de los fármacos , ADN Superhelicoidal/genética , Perfilación de la Expresión Génica , Genoma Bacteriano , Familia de Multigenes , Regiones Promotoras Genéticas , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Virulencia/genética
15.
Nucleic Acids Res ; 40(1): 65-74, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21908408

RESUMEN

The MYST HAT Sas2 is part of the SAS-I complex that acetylates histone H4 lysine 16 (H4 K16Ac) and blocks the propagation of heterochromatin at the telomeres of Saccharomyces cerevisiae. In this study, we investigated Sas2-mediated H4 K16Ac on a genome-wide scale. Interestingly, H4 K16Ac loss in sas2Δ cells outside of the telomeric regions showed a distinctive pattern in that there was a pronounced decrease of H4 K16Ac within the majority of open reading frames (ORFs), but little change in intergenic regions. Furthermore, regions of low histone H3 exchange and low H3 K56 acetylation showed the most pronounced loss of H4 K16Ac in sas2Δ, indicating that Sas2 deposited this modification on chromatin independently of histone exchange. In agreement with the effect of Sas2 within ORFs, sas2Δ caused resistance to 6-azauracil, indicating a positive effect on transcription elongation in the absence of H4 K16Ac. In summary, our data suggest that Sas2-dependent H4 K16Ac is deposited into chromatin independently of transcription and histone exchange, and that it has an inhibitory effect on the ability of PolII to travel through the body of the gene.


Asunto(s)
Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Acetilación , Eliminación de Gen , Genoma Fúngico , Histona Acetiltransferasas/genética , Sistemas de Lectura Abierta , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Uracilo/análogos & derivados , Uracilo/farmacología
16.
Cancer Immunol Immunother ; 62(11): 1697-1709, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24022692

RESUMEN

Altered numbers and functions of T cells have previously been demonstrated in chronic lymphocytic leukemia (CLL) patients. However, dynamics and specific T-cell subset alterations have not been studied in great detail. Therefore, we studied CLL blood lymphocyte subsets of individual patients in a longitudinal manner. Dynamic expansions of blood CD4 + and CD8 + T-cell numbers were consistently associated with a progressively increasing CLL leukemic compartment. Interestingly, the T-cell subset expansion over time was more pronounced in CD38 + CLL. Additionally, we performed gene expression profiling of CD3 + T cells of CLL patients and normal donors. Using gene set enrichment analysis, we found significant enrichment of genes with higher expression in CLL T cells within CD8+ effector memory and terminal effector T-cell gene signatures. In agreement with these data, we observed a marked expansion of phenotypic CD8 + effector memory T cells in CLL by flow cytometry. Moreover, we observed that increments of CD8 + effector memory T cells in human CLL and also mouse CLL (Eµ-TCL1 model) were due to an expansion of the inhibitory killer cell lectin-like receptor G1 (KLRG1) expressing cellular subset. Furthermore, higher plasma levels of the natural KLRG1 ligand E-cadherin were detected in CLL patients compared to normal donor controls. The predominance of KLRG1+ expression within CD8+ T cells in conjunction with increased systemic soluble E-cadherin might significantly contribute to CLL immune dysfunction and might additionally represent an important component of the CLL microenvironment.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Lectinas Tipo C/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Receptores Inmunológicos/inmunología , Transactivadores/inmunología , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Cadherinas/genética , Cadherinas/inmunología , Cadherinas/metabolismo , Membrana Celular/inmunología , Membrana Celular/metabolismo , Proliferación Celular , Femenino , Citometría de Flujo , Humanos , Memoria Inmunológica/genética , Inmunofenotipificación , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Transcriptoma/genética , Transcriptoma/inmunología
17.
J Immunol ; 187(6): 2982-92, 2011 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-21841135

RESUMEN

Miz-1 is a Broad-complex, Tramtrack and Bric-à-brac/pox virus zinc finger domain (BTB/POZ)-containing protein expressed in lymphoid precursors that can activate or repress transcription. We report in this article that mice expressing a nonfunctional Miz-1 protein lacking the BTB/POZ domain (Miz-1(ΔPOZ)) have a severe differentiation block at the pre-T cell "ß-selection" checkpoint, evident by a drastic reduction of CD4(-)CD8(-) double-negative-3 (DN3) and DN4 cell numbers. T cell-specific genes including Rag-1, Rag-2, CD3ε, pTα, and TCRß are expressed in Miz-1-deficient cells and V(D)J recombination is intact, but few DN3/DN4 cells express a surface pre-TCR. Miz-1-deficient DN3 cells are highly apoptotic and do not divide, which is consistent with enhanced expression of p53 target genes such as Cdkn1a, PUMA, and Noxa. However, neither coexpression of the antiapoptotic protein Bcl2 nor the deletion of p21(CIP1) nor the combination of both relieved Miz-1-deficient DN3/DN4 cells from their differentiation block. Only the coexpression of rearranged TCRαß and Bcl2 fully rescued Miz-1-deficient DN3/DN4 cell numbers and enabled them to differentiate into DN4TCRß(+) and double-positive cells. We propose that Miz-1 is a critical factor for the ß-selection checkpoint and is required for both the regulation of p53 target genes and proper expression of the pre-TCR to support the proliferative burst of DN3 cells during T cell development.


Asunto(s)
Diferenciación Celular/inmunología , Regulación de la Expresión Génica/inmunología , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Proteínas Nucleares/inmunología , Proteínas Inhibidoras de STAT Activados/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Ciclo Celular , Separación Celular , Citometría de Flujo , Expresión Génica , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Inhibidoras de STAT Activados/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/citología , Linfocitos T/inmunología , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas
18.
Front Immunol ; 14: 1227191, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37790937

RESUMEN

Introduction: Streptococcus pneumoniae is one of the main causes of community-acquired infections in the lung alveoli in children and the elderly. Alveolar macrophages (AM) patrol alveoli in homeostasis and under infectious conditions. However, the molecular adaptations of AM upon infections with Streptococcus pneumoniae are incompletely resolved. Methods: We used a comparative transcriptomic and proteomic approach to provide novel insights into the cellular mechanism that changes the molecular signature of AM during lung infections. Using a tandem mass spectrometry approach to murine cell-sorted AM, we revealed significant proteomic changes upon lung infection with Streptococcus pneumoniae. Results: AM showed a strong neutrophil-associated proteomic signature, such as expression of CD11b, MPO, neutrophil gelatinases, and elastases, which was associated with phagocytosis of recruited neutrophils. Transcriptomic analysis indicated intrinsic expression of CD11b by AM. Moreover, comparative transcriptomic and proteomic profiling identified CD11b as the central molecular hub in AM, which influenced neutrophil recruitment, activation, and migration. Discussion: In conclusion, our study provides novel insights into the intrinsic molecular adaptations of AM upon lung infection with Streptococcus pneumoniae and reveals profound alterations critical for effective antimicrobial immunity.


Asunto(s)
Antígeno CD11b , Neumonía Neumocócica , Animales , Ratones , Integrinas , Pulmón , Macrófagos Alveolares , Proteómica , Streptococcus pneumoniae , Transcriptoma
19.
Genes Chromosomes Cancer ; 50(5): 327-37, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21305643

RESUMEN

In addition to mutations in both alleles of the retinoblastoma gene (RB1) alleles, retinoblastomas frequently show additional alterations including loss of chromosome arm 16q. In a previous study, the presence of 16q alterations was found to be associated with diffuse vitreous seeding of this tumor. This growth pattern is clinically important as it determines therapeutic decisions. The present study was designed to test this association and to narrow down the list of candidate genes in the minimal region of genomic loss on chromosome arm 16q. Our data confirm the association of 16q loss and diffuse vitreous seeding and define a minimal region of genomic loss of 6.6 Mb on 16q containing 86 known genes. As retinoblastoma is an embryonic tumor, we assumed that any gene relevant for its progression is likely to show regulated expression during retinogenesis. Microarray expression analysis of RNA from a continuous developmental series of murine retinas identified murine orthologs with regulated expression and these data helped to narrow the number of candidate genes in minimal region to 35. Analysis of gene expression in retinoblastomas with and without the loss of heterozygosity (LOH) on chromosome 16q further reduced this number to 26 candidate genes. One of these genes is cadherin 13 (CDH13) and notably, downregulation of CHD13 has previously been associated with poorer prognosis in various other cancers.


Asunto(s)
Cromosomas Humanos Par 16 , Genes de Retinoblastoma , Neoplasias de la Retina/genética , Retinoblastoma/genética , Cuerpo Vítreo/patología , Alelos , Animales , Cadherinas/genética , Deleción Cromosómica , ADN de Neoplasias/sangre , Regulación Neoplásica de la Expresión Génica , Genes Reguladores , Humanos , Pérdida de Heterocigocidad , Ratones , Ratones Endogámicos C57BL , Mutación , Polimorfismo de Nucleótido Simple , Neoplasias de la Retina/sangre , Neoplasias de la Retina/patología , Retinoblastoma/sangre , Retinoblastoma/patología , Eliminación de Secuencia
20.
Front Immunol ; 13: 945409, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36148245

RESUMEN

Sepsis is associated with profound immune dysregulation that increases the risk for life-threatening secondary infections: Dendritic cells (DCs) undergo functional reprogramming due to yet unknown changes during differentiation in the bone marrow (BM). In parallel, lymphopenia and exhaustion of T lymphocytes interfere with antigen-specific adaptive immunity. We hypothesized that there exists a link between T cells and the modulation of DC differentiation in the BM during murine polymicrobial sepsis. Sepsis was induced by cecal ligation and puncture (CLP), a model for human bacterial sepsis. At different time points after CLP, the BM and spleen were analyzed in terms of T-cell subpopulations, activation, and Interferon (IFN)-γ synthesis as well as the number of pre-DCs. BM-derived DCs were generated in vitro. We observed that naïve and virtual memory CD8+ T cells, but not CD4+ T cells, were activated in an antigen-independent manner and accumulated in the BM early after CLP, whereas lymphopenia was evident in the spleen. The number of pre-DCs strongly declined during acute sepsis in the BM and almost recovered by day 4 after CLP, which required the presence of CD8+ T cells. Adoptive transfer experiments and in vitro studies with purified T cells revealed that Toll-like receptor 2 (TLR2) signaling in CD8+ T cells suppressed their capacity to secrete IFN-γ and was sufficient to change the transcriptome of the BM during sepsis. Moreover, the diminished IFN-γ production of CD8+ T cells favored the differentiation of DCs with increased production of the immune-activating cytokine Interleukin (IL)-12. These data identify a novel role of CD8+ T cells in the BM during sepsis as they sense TLR2 ligands and control the number and function of de novo differentiating DCs.


Asunto(s)
Linfopenia , Sepsis , Animales , Antígenos , Médula Ósea , Linfocitos T CD8-positivos , Diferenciación Celular , Citocinas , Células Dendríticas , Humanos , Interferón gamma , Interleucina-12 , Ratones , Receptor Toll-Like 2
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