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1.
Am J Hum Genet ; 111(10): 2265-2282, 2024 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-39293448

RESUMEN

Congenital microcoria (MCOR) is a rare hereditary developmental defect of the iris dilator muscle frequently associated with high axial myopia and high intraocular pressure (IOP) glaucoma. The condition is caused by submicroscopic rearrangements of chromosome 13q32.1. However, the mechanisms underlying the failure of iris development and the origin of associated features remain elusive. Here, we present a 3D architecture model of the 13q32.1 region, demonstrating that MCOR-related deletions consistently disrupt the boundary between two topologically associating domains (TADs). Deleting the critical MCOR-causing region in mice reveals ectopic Sox21 expression precisely aligning with Dct, each located in one of the two neighbor TADs. This observation is consistent with the TADs' boundary alteration and adoption of Dct regulatory elements by the Sox21 promoter. Additionally, we identify Tgfb2 as a target gene of SOX21 and show TGFΒ2 accumulation in the aqueous humor of an MCOR-affected subject. Accumulation of TGFB2 is recognized for its role in glaucoma and potential impact on axial myopia. Our results highlight the importance of SOX21-TGFB2 signaling in iris development and control of eye growth and IOP. Insights from MCOR studies may provide therapeutic avenues for this condition but also for glaucoma and high myopia conditions, affecting millions of people.


Asunto(s)
Glaucoma , Miopía , Factor de Crecimiento Transformador beta2 , Animales , Glaucoma/genética , Glaucoma/metabolismo , Glaucoma/patología , Ratones , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Miopía/genética , Miopía/metabolismo , Humanos , Iris/metabolismo , Iris/patología , Iris/anomalías , Presión Intraocular
2.
Am J Pathol ; 194(1): 30-51, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37827216

RESUMEN

Benign prostate hyperplasia (BPH) is caused by the nonmalignant enlargement of the transition zone of the prostate gland, leading to lower urinary tract symptoms. Although current medical treatments are unsatisfactory in many patients, the limited understanding of the mechanisms driving disease progression prevents the development of alternative therapeutic strategies. The probasin-prolactin (Pb-PRL) transgenic mouse recapitulates many histopathological features of human BPH. Herein, these alterations parallel urodynamic disturbance reminiscent of lower urinary tract symptoms. Single-cell RNA-sequencing analysis of Pb-PRL mouse prostates revealed that their epithelium mainly includes low-androgen signaling cell populations analogous to Club/Hillock cells enriched in the aged human prostate. These intermediate cells are predicted to result from the reprogramming of androgen-dependent luminal cells. Pb-PRL mouse prostates exhibited increased vulnerability to oxidative stress due to reduction of antioxidant enzyme expression. One-month treatment of Pb-PRL mice with anethole trithione (ATT), a specific inhibitor of mitochondrial ROS production, reduced prostate weight and voiding frequency. In human BPH-1 epithelial cells, ATT decreased mitochondrial metabolism, cell proliferation, and stemness features. ATT prevented the growth of organoids generated by sorted Pb-PRL basal and LSCmed cells, the two major BPH-associated, androgen-independent epithelial cell compartments. Taken together, these results support cell plasticity as a driver of BPH progression and therapeutic resistance to androgen signaling inhibition, and identify antioxidant therapy as a promising treatment of BPH.


Asunto(s)
Síntomas del Sistema Urinario Inferior , Hiperplasia Prostática , Masculino , Humanos , Ratones , Animales , Anciano , Andrógenos/farmacología , Andrógenos/metabolismo , Próstata/patología , Hiperplasia Prostática/metabolismo , Antioxidantes/farmacología , Plasticidad de la Célula , Hiperplasia/patología , Plomo/metabolismo , Plomo/uso terapéutico , Ratones Transgénicos , Prolactina/metabolismo , Prolactina/uso terapéutico , Células Epiteliales/metabolismo , Síntomas del Sistema Urinario Inferior/metabolismo , Síntomas del Sistema Urinario Inferior/patología
3.
Immunity ; 45(3): 610-625, 2016 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-27612641

RESUMEN

The nature of gut intraepithelial lymphocytes (IELs) lacking antigen receptors remains controversial. Herein we showed that, in humans and in mice, innate intestinal IELs expressing intracellular CD3 (iCD3(+)) differentiate along an Id2 transcription factor (TF)-independent pathway in response to TF NOTCH1, interleukin-15 (IL-15), and Granzyme B signals. In NOTCH1-activated human hematopoietic precursors, IL-15 induced Granzyme B, which cleaved NOTCH1 into a peptide lacking transcriptional activity. As a result, NOTCH1 target genes indispensable for T cell differentiation were silenced and precursors were reprogrammed into innate cells with T cell marks including intracellular CD3 and T cell rearrangements. In the intraepithelial lymphoma complicating celiac disease, iCD3(+) innate IELs acquired gain-of-function mutations in Janus kinase 1 or Signal transducer and activator of transcription 3, which enhanced their response to IL-15. Overall we characterized gut T cell-like innate IELs, deciphered their pathway of differentiation and showed their malignant transformation in celiac disease.


Asunto(s)
Enfermedad Celíaca/inmunología , Interleucina-15/inmunología , Intestinos/inmunología , Linfoma/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Complejo CD3/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Granzimas/inmunología , Humanos , Proteína 2 Inhibidora de la Diferenciación/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Receptor Notch1/inmunología , Factor de Transcripción STAT3/inmunología , Transducción de Señal/inmunología , Transcripción Genética/inmunología
4.
Proc Natl Acad Sci U S A ; 119(18): e2115960119, 2022 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-35482924

RESUMEN

Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using a medium-throughput drug-screen in NPHP1 knockdown cells, we identified 51 Food and Drug Administration-approved compounds by their ability to alleviate the cellular phenotypes associated with the loss of NPHP1; 11 compounds were further selected for their physicochemical properties. Among those compounds, prostaglandin E1 (PGE1) rescued ciliogenesis defects in immortalized patient NPHP1 urine-derived renal tubular cells, and improved ciliary and kidney phenotypes in our NPH zebrafish and Nphp1 knockout mouse models. Furthermore, Taprenepag, a nonprostanoid prostaglandin E2 receptor agonist, alleviated the severe retinopathy observed in Nphp1−/− mice. Finally, comparative transcriptomics allowed identification of key signaling pathways downstream PGE1, including cell cycle progression, extracellular matrix, adhesion, or actin cytoskeleton organization. In conclusion, using in vitro and in vivo models, we showed that prostaglandin E2 receptor agonists can ameliorate several of the pleotropic phenotypes caused by the absence of NPHP1; this opens their potential as a first therapeutic option for juvenile NPH-associated ciliopathies.


Asunto(s)
Ciliopatías , Enfermedades Renales Poliquísticas , Animales , Cilios/metabolismo , Ciliopatías/tratamiento farmacológico , Ciliopatías/genética , Ciliopatías/metabolismo , Femenino , Humanos , Enfermedades Renales Quísticas/congénito , Masculino , Ratones , Enfermedades Renales Poliquísticas/metabolismo , Prostaglandinas/metabolismo , Receptores de Prostaglandina E/metabolismo , Pez Cebra
5.
Am J Hum Genet ; 108(6): 1126-1137, 2021 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-34010604

RESUMEN

Dysregulated transforming growth factor TGF-ß signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-ß-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-ß signaling, ipo8-/- zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8-/- zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-ß signaling during development and reinforces the existing link between TGF-ß signaling and connective tissue defects.


Asunto(s)
Enfermedades Óseas/etiología , Enfermedades Cardiovasculares/etiología , Enfermedades del Tejido Conjuntivo/etiología , Inmunidad Celular/inmunología , Mutación con Pérdida de Función , Pérdida de Heterocigocidad , beta Carioferinas/genética , Adolescente , Adulto , Animales , Enfermedades Óseas/patología , Enfermedades Cardiovasculares/patología , Niño , Enfermedades del Tejido Conjuntivo/patología , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Transducción de Señal , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Adulto Joven , Pez Cebra , beta Carioferinas/metabolismo
6.
Blood ; 139(3): 384-398, 2022 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-34232979

RESUMEN

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy affecting adults. The NF-κB transcription factor family is activated by 2 main pathways, the canonical and the alternative NF-κB activation pathway, with different functions. The alternative NF-κB pathway leads to activation of the transcriptionally active RelB NF-κB subunit. Alternative NF-κB activation status and its role in DLBCL pathogenesis remain undefined. Here, we reveal a frequent activation of RelB in a large cohort of DLBCL patients and cell lines, independently of their activated B-cell-like or germinal center B-cell-like subtype. RelB activity defines a new subset of patients with DLBCL and a peculiar gene expression profile and mutational pattern. Importantly, RelB activation does not correlate with the MCD genetic subtype, enriched for activated B-cell-like tumors carrying MYD88L265P and CD79B mutations that cooperatively activate canonical NF-κB, thus indicating that current genetic tools to evaluate NF-κB activity in DLBCL do not provide information on the alternative NF-κB activation. Furthermore, the newly defined RelB-positive subgroup of patients with DLBCL exhibits a dismal outcome after immunochemotherapy. Functional studies revealed that RelB confers DLBCL cell resistance to DNA damage-induced apoptosis in response to doxorubicin, a genotoxic agent used in the front-line treatment of DLBCL. We also show that RelB positivity is associated with high expression of cellular inhibitor of apoptosis protein 2 (cIAP2). Altogether, RelB activation can be used to refine the prognostic stratification of DLBCL and may contribute to subvert the therapeutic DNA damage response in a segment of patients with DLBCL.


Asunto(s)
Linfoma de Células B Grandes Difuso/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción ReIB/metabolismo , Apoptosis , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma de Células B Grandes Difuso/genética , FN-kappa B/genética , Factor de Transcripción ReIB/genética , Activación Transcripcional
7.
Kidney Int ; 104(2): 367-377, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37230224

RESUMEN

X-linked Alport syndrome (XLAS) is an inherited kidney disease caused exclusively by pathogenic variants in the COL4A5 gene. In 10-20% of cases, DNA sequencing of COL4A5 exons or flanking regions cannot identify molecular causes. Here, our objective was to use a transcriptomic approach to identify causative events in a group of 19 patients with XLAS without identified mutation by Alport gene panel sequencing. Bulk RNAseq and/or targeted RNAseq using a capture panel of kidney genes was performed. Alternative splicing events were compared to those of 15 controls by a developed bioinformatic score. When using targeted RNAseq, COL4A5 coverage was found to be 23-fold higher than with bulk RNASeq and revealed 30 significant alternative splicing events in 17 of the 19 patients. After computational scoring, a pathogenic transcript was found in all patients. A causative variant affecting COL4A5 splicing and absent in the general population was identified in all cases. Altogether, we developed a simple and robust method for identification of aberrant transcripts due to pathogenic deep-intronic COL4A5 variants. Thus, these variants, potentially targetable by specific antisense oligonucleotide therapies, were found in a high percentage of patients with XLAS in whom pathogenic variants were missed by conventional DNA sequencing.


Asunto(s)
Nefritis Hereditaria , Humanos , Nefritis Hereditaria/diagnóstico , Nefritis Hereditaria/genética , Nefritis Hereditaria/patología , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Mutación , Exones , Empalme del ARN
8.
Hum Reprod ; 38(5): 992-1002, 2023 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-36952633

RESUMEN

STUDY QUESTION: Does mitochondrial deficiency affect human embryonic preimplantation development? SUMMARY ANSWER: The presence of a pathogenic mitochondrial variant triggers changes in the gene expression of preimplantation human embryos, compromising their development, cell differentiation, and survival. WHAT IS KNOWN ALREADY: Quantitative and qualitative anomalies of mitochondrial DNA (mtDNA) are reportedly associated with impaired human embryonic development, but the underlying mechanisms remain unexplained. STUDY DESIGN, SIZE, DURATION: Taking advantage of the preimplantation genetic testing for mitochondrial disorders in at-risk couples, we have compared gene expression of 9 human embryos carrying pathogenic variants in either mtDNA genes or nuclear genes encoding mitochondrial protein to 33 age-matched control embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single-embryo transcriptomic analysis was performed on whole human blastocyst embryos donated to research. MAIN RESULTS AND THE ROLE OF CHANCE: Specific pathogenic mitochondrial variants downregulate gene expression in preimplantation human embryos [566 genes in oxidative phosphorylation (OXPHOS)-deficient embryos], impacting transcriptional regulators, differentiation factors, and nuclear genes encoding mitochondrial proteins. These changes in gene expression primarily alter OXPHOS and cell survival pathways. LIMITATIONS, REASONS FOR CAUTION: The number of OXPHOS-deficient embryos available for the study was limited owing to the rarity of this material. However, the molecular signature shared by all these embryos supports the relevance of the findings. WIDER IMPLICATIONS OF THE FINDINGS: While identification of reliable markers of normal embryonic development is urgently needed in ART, our study prompts us to consider under-expression of the targeted genes reported here, as predictive biomarkers of mitochondrial dysfunction during preimplantation development. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the 'Association Française contre les Myopathies (AFM-Téléthon)' and the 'La Fondation Maladies Rares'. No competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.


Asunto(s)
Embrión de Mamíferos , Enfermedades Mitocondriales , Embarazo , Femenino , Humanos , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , ADN Mitocondrial/genética , Blastocisto/metabolismo , Expresión Génica
9.
Blood ; 135(15): 1219-1231, 2020 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-32040546

RESUMEN

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or ß hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.


Asunto(s)
Células Clonales/citología , Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Hemoglobinopatías/terapia , Síndrome de Wiskott-Aldrich/terapia , Diferenciación Celular , Rastreo Celular , Células Clonales/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/genética , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Hemoglobinopatías/genética , Humanos , Síndrome de Wiskott-Aldrich/genética
10.
J Cell Mol Med ; 24(24): 14453-14466, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33159500

RESUMEN

In haemophilia, the recurrence of hemarthrosis leads to irreversible arthropathy termed haemophilic arthropathy (HA). However, HA is a unique form of arthropathy in which resident cells, such as fibroblast-like synoviocytes (FLS), come into direct contact with blood. Therefore, we hypothesized that FLS in HA could have a unique inflammatory signature as a consequence of their contact with blood. We demonstrated with ELISA and ELISPOT analyses that HA-FLS expressed a unique profile of cytokine secretion, which differed from that of non-HA-FLS, mainly consisting of cytokines involved in innate immunity. We showed that unstable cytokine mRNAs were involved in this process, especially through miRNA complexes as confirmed by DICER silencing. A miRNOME analysis revealed that 30 miRNAs were expressed differently between HA and non-HA-FLS, with most miRNAs involved in inflammatory control pathways or described in certain inflammatory diseases, such as rheumatoid arthritis or lupus. Analysis of transcriptomic networks, impacted by these miRNAs, revealed that protein processes and inflammatory pathways were particularly targeted in LPS-induced FLS, and in particular vascularization and osteoarticular modulation pathways in steady-state FLS. Our study demonstrates that the presence of blood in contact with FLS may induce durable miRNA changes that likely participate in HA pathophysiology.


Asunto(s)
Biomarcadores , Hemartrosis/etiología , Hemartrosis/metabolismo , MicroARNs/genética , Sinoviocitos/metabolismo , Comunicación Celular , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Epigénesis Genética , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hemartrosis/patología , Hemofilia A/complicaciones , Humanos , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Proteómica/métodos , Transducción de Señal
11.
Blood ; 131(14): 1545-1555, 2018 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-29378696

RESUMEN

Previous data have suggested that B-cell-depletion therapy may induce the settlement of autoreactive long-lived plasma cells (LLPCs) in the spleen of patients with autoimmune cytopenia. To investigate this process, we used the AID-CreERT2-EYFP mouse model to follow plasma cells (PCs) engaged in an immune response. Multiplex polymerase chain reaction at the single-cell level revealed that only a small fraction of splenic PCs had a long-lived signature, whereas PCs present after anti-CD20 antibody treatment appeared more mature, similar to bone marrow PCs. This observation suggested that, in addition to a process of selection, a maturation induced on B-cell depletion drove PCs toward a long-lived program. We showed that B-cell activating factor (BAFF) and CD4+ T cells play a major role in the PC survival niche, because combining anti-CD20 with anti-BAFF or anti-CD4 antibody greatly reduce the number of splenic PCs. Similar results were obtained in the lupus-prone NZB/W model. These different contributions of soluble and cellular components of the PC niche in the spleen demonstrate that the LLPC expression profile is not cell intrinsic but largely depends on signals provided by the splenic microenvironment, implying that interfering with these components at the time of B-cell depletion might improve the response rate in autoimmune cytopenia.


Asunto(s)
Factor Activador de Células B/inmunología , Linfocitos T CD4-Positivos/inmunología , Lupus Eritematoso Sistémico/inmunología , Depleción Linfocítica , Células Plasmáticas/inmunología , Bazo/inmunología , Animales , Linfocitos T CD4-Positivos/patología , Supervivencia Celular , Modelos Animales de Enfermedad , Lupus Eritematoso Sistémico/patología , Ratones , Células Plasmáticas/patología , Bazo/patología
12.
Haematologica ; 105(1): 59-70, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31004027

RESUMEN

The molecular machinery that regulates the balance between self-renewal and differentiation properties of hematopoietic stem cells (HSC) has yet to be characterized in detail. Here we found that the tetratricopeptide repeat domain 7 A (Ttc7a) protein, a putative scaffold protein expressed by HSC, acts as an intrinsic regulator of the proliferative response and the self-renewal potential of murine HSC in vivo Loss of Ttc7a consistently enhanced the competitive repopulating ability of HSC and their intrinsic capacity to replenish the hematopoietic system after serial cell transplantations, relative to wildtype cells. Ttc7a-deficient HSC exhibit a different transcriptomic profile for a set of genes controlling the cellular response to stress, which was associated with increased proliferation in response to chemically induced stress in vitro and myeloablative stress in vivo Our results therefore revealed a previously unrecognized role of Ttc7a as a critical regulator of HSC stemness. This role is related, at least in part, to regulation of the endoplasmic reticulum stress response.


Asunto(s)
Células Madre Hematopoyéticas , Proteínas , Animales , Diferenciación Celular , Proliferación Celular , Ratones
13.
Respir Res ; 21(1): 43, 2020 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-32019538

RESUMEN

BACKGROUND: The pathophysiology of congenital cystic adenomatoid malformations (CCAM) of the lung remains poorly understood. AIM: This study aimed to identify more precisely the molecular mechanisms limited to a compartment of lung tissue, through a transcriptomic analysis of the epithelium of macrocystic forms. METHODS: Tissue fragments displaying CCAM were obtained during planned surgical resections. Epithelial mRNA was obtained from cystic and normal areas after laser capture microdissection (LCM). Transcriptomic analyses were performed and the results were confirmed by RT-PCR and immunohistochemistry in independent samples. RESULTS: After controlling for RNA quality, we analysed the transcriptomes of six cystic areas and five control areas. In total, 393 transcripts were differentially expressed in the epithelium, between CCAM and control areas. The most highly redundant genes involved in biological functions and signalling pathways differentially expressed between CCAM and control epithelium included TGFB2, TGFBR1, and MAP 2 K1. These genes were considered particularly relevant as they have been implicated in branching morphogenesis. RT-qPCR analysis confirmed in independent samples that TGFBR1 was more strongly expressed in CCAM than in control tissues (p < 0.03). Immunohistochemistry analysis showed TGFBR1 (p = 0.0007) and TGFB2 (p < 0.02) levels to be significantly higher in the epithelium of CCAM than in that of control tissues. CONCLUSIONS: This compartmentalised transcriptomic analysis of the epithelium of macrocystic lung malformations identified a dysregulation of TGFB signalling at the mRNA and protein levels, suggesting a possible role of this pathway in CCAM pathogenesis. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01732185.


Asunto(s)
Malformación Adenomatoide Quística Congénita del Pulmón/genética , Malformación Adenomatoide Quística Congénita del Pulmón/patología , Perfilación de la Expresión Génica/métodos , Mucosa Respiratoria/patología , Malformación Adenomatoide Quística Congénita del Pulmón/metabolismo , Factores de Transcripción de la Respuesta de Crecimiento Precoz/biosíntesis , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Femenino , Estudios de Seguimiento , Humanos , Lactante , Factores de Transcripción de Tipo Kruppel/biosíntesis , Factores de Transcripción de Tipo Kruppel/genética , Captura por Microdisección con Láser/métodos , Masculino , Estudios Prospectivos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Mucosa Respiratoria/metabolismo
14.
J Am Soc Nephrol ; 30(4): 692-709, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30850439

RESUMEN

BACKGROUND: Although anti-HLA antibodies (Abs) cause most antibody-mediated rejections of renal allografts, non-anti-HLA Abs have also been postulated to contribute. A better understanding of such Abs in rejection is needed. METHODS: We conducted a nationwide study to identify kidney transplant recipients without anti-HLA donor-specific Abs who experienced acute graft dysfunction within 3 months after transplantation and showed evidence of microvascular injury, called acute microvascular rejection (AMVR). We developed a crossmatch assay to assess serum reactivity to human microvascular endothelial cells, and used a combination of transcriptomic and proteomic approaches to identify non-HLA Abs. RESULTS: We identified a highly selected cohort of 38 patients with early acute AMVR. Biopsy specimens revealed intense microvascular inflammation and the presence of vasculitis (in 60.5%), interstitial hemorrhages (31.6%), or thrombotic microangiopathy (15.8%). Serum samples collected at the time of transplant showed that previously proposed anti-endothelial cell Abs-angiotensin type 1 receptor (AT1R), endothelin-1 type A and natural polyreactive Abs-did not increase significantly among patients with AMVR compared with a control group of stable kidney transplant recipients. However, 26% of the tested AMVR samples were positive for AT1R Abs when a threshold of 10 IU/ml was used. The crossmatch assay identified a common IgG response that was specifically directed against constitutively expressed antigens of microvascular glomerular cells in patients with AMVR. Transcriptomic and proteomic analyses identified new targets of non-HLA Abs, with little redundancy among individuals. CONCLUSIONS: Our findings indicate that preformed IgG Abs targeting non-HLA antigens expressed on glomerular endothelial cells are associated with early AMVR, and that in vitro cell-based assays are needed to improve risk assessments before transplant.


Asunto(s)
Rechazo de Injerto/inmunología , Hemorragia/inmunología , Inmunoglobulina G/sangre , Receptor de Angiotensina Tipo 1/inmunología , Microangiopatías Trombóticas/inmunología , Vasculitis/inmunología , Enfermedad Aguda , Adulto , Anciano , Células Endoteliales/inmunología , Endotelina-1/inmunología , Femenino , Rechazo de Injerto/patología , Rechazo de Injerto/fisiopatología , Hemorragia/patología , Humanos , Glomérulos Renales/patología , Trasplante de Riñón/efectos adversos , Masculino , Microvasos/patología , Persona de Mediana Edad , Microangiopatías Trombóticas/patología , Factores de Tiempo , Vasculitis/patología
16.
Hum Mol Genet ; 26(1): 90-108, 2017 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-28007902

RESUMEN

Human doublecortin (DCX) mutations are associated with severe brain malformations leading to aberrant neuron positioning (heterotopia), intellectual disability and epilepsy. The Dcx protein plays a key role in neuronal migration, and hippocampal pyramidal neurons in Dcx knockout (KO) mice are disorganized. The single CA3 pyramidal cell layer observed in wild type (WT) is present as two abnormal layers in the KO, and CA3 KO pyramidal neurons are more excitable than WT. Dcx KO mice also exhibit spontaneous epileptic activity originating in the hippocampus. It is unknown, however, how hyperexcitability arises and why two CA3 layers are observed.Transcriptome analyses were performed to search for perturbed postnatal gene expression, comparing Dcx KO CA3 pyramidal cell layers with WT. Gene expression changes common to both KO layers indicated mitochondria and Golgi apparatus anomalies, as well as increased cell stress. Intriguingly, gene expression analyses also suggested that the KO layers differ significantly from each other, particularly in terms of maturity. Layer-specific molecular markers and BrdU birthdating to mark the final positions of neurons born at distinct timepoints revealed inverted layering of the CA3 region in Dcx KO animals. Notably, many early-born 'outer boundary' neurons are located in an inner position in the Dcx KO CA3, superficial to other pyramidal neurons. This abnormal positioning likely affects cell morphology and connectivity, influencing network function. Dissecting this Dcx KO phenotype sheds light on coordinated developmental mechanisms of neuronal subpopulations, as well as gene expression patterns contributing to a bi-layered malformation associated with epilepsy.


Asunto(s)
Hipocampo/metabolismo , Hipocampo/patología , Proteínas Asociadas a Microtúbulos/fisiología , Neuronas/metabolismo , Neuronas/patología , Neuropéptidos/fisiología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Región CA1 Hipocampal/ultraestructura , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/patología , Región CA3 Hipocampal/ultraestructura , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Femenino , Hipocampo/ultraestructura , Procesamiento de Imagen Asistido por Computador , Captura por Microdisección con Láser , Masculino , Ratones , Ratones Noqueados , Microscopía Confocal , Neuronas/ultraestructura
17.
Immunity ; 33(3): 375-86, 2010 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-20832340

RESUMEN

Monocytes are effectors of the inflammatory response to microbes. Human CD14(+) monocytes specialize in phagocytosis and production of reactive oxygen species and secrete inflammatory cytokines in response to a broad range of microbial cues. Here, we have characterized the functions of human monocytes that lack CD14 (CD14(dim)) and express CD16. CD14(dim) monocytes were genetically distinct from natural killer cells. Gene expression analyses indicated similarities with murine patrolling Gr1(dim) monocytes, and they patrolled the endothelium of blood vessels after adoptive transfer, in a lymphocyte function-associated antigen-1-dependent manner. CD14(dim) monocytes were weak phagocytes and did not produce ROS or cytokines in response to cell-surface Toll-like receptors. Instead, they selectively produced TNF-α, IL-1ß, and CCL3 in response to viruses and immune complexes containing nucleic acids, via a proinflammatory TLR7-TLR 8-MyD88-MEK pathway. Thus, CD14(dim) cells are bona fide monocytes involved in the innate local surveillance of tissues and the pathogenesis of autoimmune diseases.


Asunto(s)
Receptores de Lipopolisacáridos/fisiología , Monocitos/fisiología , Ácidos Nucleicos/fisiología , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 8/fisiología , Virus/inmunología , Animales , Presentación de Antígeno , Citocinas/biosíntesis , Proteínas Ligadas a GPI , Antígenos HLA-DR/análisis , Humanos , Lupus Eritematoso Sistémico/inmunología , Ratones , Factor 88 de Diferenciación Mieloide/fisiología , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/análisis
18.
J Immunol ; 199(7): 2408-2420, 2017 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-28807996

RESUMEN

Klhl6 belongs to the KLHL gene family, which is composed of an N-terminal BTB-POZ domain and four to six Kelch motifs in tandem. Several of these proteins function as adaptors of the Cullin3 E3 ubiquitin ligase complex. In this article, we report that Klhl6 deficiency induces, as previously described, a 2-fold reduction in mature B cells. However, we find that this deficit is centered on the inability of transitional type 1 B cells to survive and to progress toward the transitional type 2 B cell stage, whereas cells that have passed this step generate normal germinal centers (GCs) upon a T-dependent immune challenge. Klhl6-deficient type 1 B cells showed a 2-fold overexpression of genes linked with cell proliferation, including most targets of the anaphase-promoting complex/cyclosome complex, a set of genes whose expression is precisely downmodulated upon culture of splenic transitional B cells in the presence of BAFF. These results thus suggest a delay in the differentiation process of Klhl6-deficient B cells between the immature and transitional stage. We further show, in the BL2 Burkitt's lymphoma cell line, that KLHL6 interacts with Cullin3, but also that it binds to HBXIP/Lamtor5, a protein involved in cell-cycle regulation and cytokinesis. Finally, we report that KLHL6, which is recurrently mutated in B cell lymphomas, is an off-target of the normal somatic hypermutation process taking place in GC B cells in both mice and humans, thus leaving open whether, despite the lack of impact of Klhl6 deficiency on GC B cell expansion, mutants could contribute to the oncogenic process.


Asunto(s)
Linfocitos B/fisiología , Proteínas Portadoras/fisiología , Centro Germinal/citología , Animales , Linfocitos B/inmunología , Linfoma de Burkitt/patología , Proteínas Portadoras/genética , Diferenciación Celular , Línea Celular , Proliferación Celular , Centro Germinal/inmunología , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patología , Ratones , Mutación , Células Precursoras de Linfocitos B/fisiología
19.
Eur Heart J ; 39(20): 1835-1847, 2018 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-29420830

RESUMEN

Aims: We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. Methods and results: The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left ventricular end diastolic volume: -4%, P = 0.002), and increased LVEF (+14%, P < 0.0001) relative to baseline values. Gene profiling revealed that EV-treated hearts were enriched for tissue reparative pathways. Conclusion: Extracellular vesicles secreted by iPSC-Pg are effective in the treatment of CHF, possibly, in part, through their specific miRNA signature and the associated stimulation of distinct cardioprotective pathways. The processing and regulatory advantages of EV could make them effective substitutes for cell transplantation.


Asunto(s)
Vesículas Extracelulares/trasplante , Insuficiencia Cardíaca/terapia , Animales , Proliferación Celular , Supervivencia Celular , Células Madre Embrionarias/ultraestructura , Vesículas Extracelulares/genética , Insuficiencia Cardíaca/patología , Humanos , Ratones Desnudos , MicroARNs/análisis , Infarto del Miocardio/patología , Infarto del Miocardio/terapia , Miocitos Cardíacos/ultraestructura , Células Madre Pluripotentes/ultraestructura , Resultado del Tratamiento
20.
J Pathol ; 243(1): 51-64, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28603917

RESUMEN

Castration-resistant prostate cancer is a lethal disease. The cell type(s) that survive androgen deprivation remain poorly described, despite global efforts to understand the various mechanisms of therapy resistance. We recently identified in wild-type (WT) mouse prostates a rare population of luminal progenitor cells that we called LSCmed according to their FACS profile (Lin- /Sca-1+ /CD49fmed ). Here, we investigated the prevalence and castration resistance of LSCmed in various mouse models of prostate tumourigenesis (Pb-PRL, Ptenpc-/- , and Hi-Myc mice). LSCmed prevalence is low (∼8%, similar to WT) in Hi-Myc mice, where prostatic androgen receptor signalling is unaltered, but is significantly higher in the two other models, where androgen receptor signalling is decreased, rising up to more than 80% in Ptenpc-/- prostates. LSCmed tolerate androgen deprivation and persist or are enriched 2-3 weeks after castration. The tumour-initiating properties of LSCmed from Ptenpc-/- mice were demonstrated by regeneration of tumours in vivo. Transcriptomic analysis revealed that LSCmed represent a unique cell entity as their gene expression profile is different from luminal and basal/stem cells, but shares markers of each. Their intrinsic androgen signalling is markedly decreased, explaining why LSCmed tolerate androgen deprivation. This also illuminates why Ptenpc-/- tumours are castration-resistant since LSCmed represent the most prevalent cell type in this model. We validated CK4 as a specific marker for LSCmed on sorted cells and prostate tissues by immunostaining, allowing for the detection of LSCmed in various mouse prostate specimens. In castrated Ptenpc-/- prostates, there was significant proliferation of CK4+ cells, further demonstrating their key role in castration-resistant prostate cancer progression. Taken together, this study identifies LSCmed as a probable source of prostate cancer relapse after androgen deprivation and as a new therapeutic target for the prevention of castrate-resistant prostate cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Asunto(s)
Biomarcadores de Tumor/deficiencia , Proliferación Celular , Células Madre Neoplásicas/enzimología , Fosfohidrolasa PTEN/deficiencia , Neoplasias de la Próstata Resistentes a la Castración/enzimología , Antagonistas de Andrógenos/farmacología , Animales , Antineoplásicos Hormonales/farmacología , Ataxina-1/metabolismo , Biomarcadores de Tumor/genética , Linaje de la Célula , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Integrina alfa6/metabolismo , Queratina-4/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Recurrencia Local de Neoplasia , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/trasplante , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfohidrolasa PTEN/genética , Fenotipo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/efectos de los fármacos , Receptores Androgénicos/metabolismo , Transducción de Señal
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