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Bone tissue features a complex microarchitecture and biomolecular composition, which determine biomechanical properties. In addition to state-of-the-art technologies, innovative optical approaches allowing the characterization of the bone in native, label-free conditions can provide new, multi-level insight into this inherently challenging tissue. Here, we exploited multimodal nonlinear optical (NLO) microscopy, including co-registered stimulated Raman scattering, two-photon excited fluorescence, and second-harmonic generation, to image entire vertebrae of murine spine sections. The quantitative nature of these nonlinear interactions allowed us to extract accurate biochemical, morphological, and topological information on the bone tissue and to highlight differences between normal and pathologic samples. Indeed, in a murine model showing bone loss, we observed increased collagen and lipid content as compared to the wild type, along with a decreased craniocaudal alignment of bone collagen fibres. We propose that NLO microscopy can be implemented in standard histopathological analysis of bone in preclinical studies, with the ambitious future perspective to introduce this technique in the clinical practice for the analysis of larger tissue sections.
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We examined effects of exposing female and male mice for 33 weeks to 45% or 60% high fat diet (HFD). Males fed with either diet were more vulnerable than females, displaying higher and faster increase in body weight and more elevated cholesterol and liver enzymes levels. Higher glucose metabolism was revealed by PET in the olfactory bulbs of both sexes. However, males also displayed altered anterior cortex and cerebellum metabolism, accompanied by a more prominent brain inflammation relative to females. Although both sexes displayed reduced transcripts of neuronal and synaptic genes in anterior cortex, only males had decreased protein levels of AMPA and NMDA receptors. Oppositely, to anterior cortex, cerebellum of HFD-exposed mice displayed hypometabolism and transcriptional up-regulation of neuronal and synaptic genes. These results indicate that male brain is more susceptible to metabolic changes induced by HFD and that the anterior cortex versus cerebellum display inverse susceptibility to HFD.
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Dieta Alta en Grasa , Obesidad , Animales , Ratones , Masculino , Femenino , Dieta Alta en Grasa/efectos adversos , Obesidad/metabolismo , Encéfalo/metabolismo , Peso Corporal , Neuronas/metabolismoRESUMEN
We introduce a broadband coherent anti-Stokes Raman scattering (CARS) microscope based on a 2-MHz repetition rate ytterbium laser generating 1035-nm high-energy (≈µJ level) femtosecond pulses. These features of the driving laser allow producing broadband red-shifted Stokes pulses, covering the whole fingerprint region (400-1800 cm-1), employing supercontinuum generation in a bulk crystal. Our system reaches state-of-the-art acquisition speed (<1 ms/pixel) and unprecedented sensitivity of ≈14.1â mmol/L when detecting dimethyl sulfoxide in water. To further improve the performance of the system and to enhance the signal-to-noise ratio of the CARS spectra, we designed a convolutional neural network for spectral denoising, coupled with a post-processing pipeline to distinguish different chemical species of biological tissues.
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Aprendizaje Profundo , Espectrometría Raman , Dimetilsulfóxido , Agua , IterbioRESUMEN
Many human genetic diseases are associated with gross mutations such as aneuploidies, deletions, duplications, or inversions. For these "structural" disorders, conventional gene therapy, based on viral vectors and/or on programmable nuclease-mediated homologous recombination, is still unsatisfactory. To correct such disorders, chromosome transplantation (CT), defined as the perfect substitution of an endogenous defective chromosome with an exogenous normal one, could be applied. CT re-establishes a normal diploid cell, leaving no marker of the procedure, as we have recently shown in mouse pluripotent stem cells. To prove the feasibility of the CT approach in human cells, we used human induced pluripotent stem cells (hiPSCs) reprogrammed from Lesch-Nyhan (LN) disease patients, taking advantage of their mutation in the X-linked HPRT gene, making the LN cells selectable and distinguishable from the resistant corrected normal cells. In this study, we demonstrate, for the first time, that CT is feasible in hiPSCs: the normal exogenous X chromosome was first transferred using an improved chromosome transfer system, and the extra sex chromosome was spontaneously lost. These CT cells were functionally corrected and maintained their pluripotency and differentiation capability. By inactivation of the autologous HPRT gene, CT paves the way to the correction of hiPSCs from several X-linked disorders.
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BACKGROUND: Autosomal recessive osteopetrosis is a rare skeletal disorder with increased bone density due to a failure in osteoclast bone resorption. In most cases, the defect is cell-autonomous, and >50% of patients bear mutations in the TCIRG1 gene, encoding for a subunit of the vacuolar proton pump essential for osteoclast resorptive activity. The only cure is hematopoietic stem cell transplantation, which corrects the bone pathology by allowing the formation of donor-derived functional osteoclasts. Therapeutic approaches using patient-derived cells corrected ex vivo through viral transduction or gene editing can be considered, but to date functional rescue cannot be demonstrated in vivo because a relevant animal model for xenotransplant is missing. METHODS: We generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the Tcirg1 oc mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34+ cells. RESULTS: We demonstrated that neonatal murine bone marrow transplantation rescued NSG oc/oc mice, in line with previous findings in the oc/oc parental strain and with evidence from clinical practice in humans. Importantly, we also demonstrated human cell chimerism in the bone marrow of NSG oc/oc mice transplanted with human CD34+ cells. The severity and rapid progression of the disease in the mouse model prevented amelioration of the bone pathology; nevertheless, we cannot completely exclude that minor early modifications of the bone tissue might have occurred. CONCLUSION: Our work paves the way to generating an improved xenograft model for in vivo evaluation of functional rescue of patient-derived corrected cells. Further refinement of the newly generated mouse model will allow capitalizing on it for an optimized exploitation in the path to novel cell therapies.
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Controlling oxidative stress through the activation of antioxidant pathways is crucial in bone homeostasis, and impairments of the cellular defense systems involved contribute to the pathogenesis of common skeletal diseases. In this work we focused on the dipeptidyl peptidase 3 (DPP3), a poorly investigated ubiquitous zinc-dependent exopeptidase activating the Keap1-Nrf2 antioxidant pathway. We showed Dpp3 expression in bone and, to understand its role in this compartment, we generated a Dpp3 knockout (KO) mouse model and specifically investigated the skeletal phenotype. Adult Dpp3 KO mice showed a mild growth defect, a significant increase in bone marrow cellularity, and bone loss mainly caused by increased osteoclast activity. Overall, in the mouse model, lack of DPP3 resulted in sustained oxidative stress and in alterations of bone microenvironment favoring the osteoclast compared to the osteoblast lineage. Accordingly, in vitro studies revealed that Dpp3 KO osteoclasts had an inherent increased resorptive activity and ROS production, which on the other hand made them prone to apoptosis. Moreover, absence of DPP3 augmented bone loss after estrogen withdrawal in female mice, further supporting its relevance in the framework of bone pathophysiology. Overall, we show a nonredundant role for DPP3 in the maintenance of bone homeostasis and propose that DPP3 might represent a possible new osteoimmunological player and a marker of human bone loss pathology. © 2019 American Society for Bone and Mineral Research.
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Resorción Ósea , Microambiente Celular , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/deficiencia , Osteoclastos , Estrés Oxidativo , Transducción de Señal , Animales , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologíaRESUMEN
Biomimetic scaffolds are extremely versatile in terms of chemical composition and physical properties, which can be defined to accomplish specific applications. One property that can be added is the production/release of bioactive soluble factors, either directly from the biomaterial, or from cells embedded within the biomaterial. We reasoned that pursuing this strategy would be appropriate to setup a cell-based therapy for RANKL-deficient autosomal recessive osteopetrosis, a very rare skeletal genetic disease in which lack of the essential osteoclastogenic factor RANKL impedes osteoclast formation. The exogenously administered RANKL cytokine is effective in achieving osteoclast formation and function in vitro and in vivo, thus, we produced murine Rankl-/- mesenchymal stromal cells (MSCs) overexpressing human soluble RANKL (hsRL) following lentiviral transduction (LVhsRL). Here, we described a three-dimensional (3D) culture system based on a magnesium-doped hydroxyapatite/collagen I (MgHA/Col) biocompatible scaffold closely reproducing bone physicochemical properties. MgHA/Col-seeded murine MSCs showed improved properties, as compared to two-dimensional (2D) culture, in terms of proliferation and hsRL production, with respect to LVhsRL-transduced cells. When implanted subcutaneously in Rankl-/- mice, these cell constructs were well tolerated, colonized by host cells, and intensely vascularized. Of note, in the bone of Rankl-/- mice that carried scaffolds with either WT or LVhsRL-transduced Rankl-/- MSCs, we specifically observed formation of TRAP+ cells, likely due to sRL released from the scaffolds into circulation. Thus, our strategy proved to have the potential to elicit an effect on the bone; further work is required to maximize these benefits and achieve improvements of the skeletal pathology in the treated Rankl-/- mice. Stem Cells Translational Medicine 2019;8:22-34.
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Células Madre Mesenquimatosas/citología , Osteopetrosis/metabolismo , Osteopetrosis/terapia , Ligando RANK/metabolismo , Biomimética/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Humanos , Células Madre Mesenquimatosas/fisiología , Osteopetrosis/genética , Ligando RANK/genética , Ingeniería de Tejidos/métodosRESUMEN
Acrofrontofacionasal Dysostosis type 1 (AFFND1) is an extremely rare, autosomal recessive syndrome, comprising facial and skeletal abnormalities, short stature and intellectual disability. We analyzed an Indian family with two affected siblings by exome sequencing and identified a novel homozygous truncating mutation in the Neuroblastoma-Amplified Sequence (NBAS) gene in the patients' genome. Mutations in the NBAS gene have recently been associated with different phenotypes mainly involving skeletal formation, liver and cognitive development. The NBAS protein has been implicated in two key cellular processes, namely the non-sense mediated decay and the Golgi-to-Endoplasmic Reticulum retrograde traffic. Both functions were impaired in HEK293T cells overexpressing the truncated NBAS protein, as assessed by Real-Time PCR, Western blot analysis, co-immunoprecipitation, and immunofluorescence analysis. We examined the expression of NBAS protein in mouse embryos at various developmental stages by immunohistochemistry, and detected expression in developing chondrogenic and osteogenic structures of the skeleton as well as in the cortex, hippocampus and cerebellum, which is compatible with a role in bone and brain development. Functional genetics in the zebrafish model showed that depletion of endogenous z-nbas in fish embryos results in defective morphogenesis of chondrogenic cranial skeletal elements. Overall, our data point to a conserved function of NBAS in skeletal morphogenesis during development, support the hypothesis of a causative role of the mutated NBAS gene in the pathogenesis of AFFND1 and extend the spectrum of phenotypes associated with defects in this gene.
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Disostosis Mandibulofacial/diagnóstico por imagen , Disostosis Mandibulofacial/genética , Mutación/genética , Proteínas de Neoplasias/genética , Hermanos , Animales , Animales Modificados Genéticamente , Femenino , Células HEK293 , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos C57BL , Embarazo , Pez CebraRESUMEN
Adult vascular smooth muscle cells (VSMCs) dedifferentiate in response to extracellular cues such as vascular damage and inflammation. Dedifferentiated VSMCs are proliferative, migratory, less contractile, and can contribute to vascular repair as well as to cardiovascular pathologies such as intimal hyperplasia/restenosis in coronary artery and arterial aneurysm. We here demonstrate the role of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) as an epigenetic master regulator of VSMC plasticity. UHRF1 expression correlated with the development of vascular pathologies associated with modulation of noncoding RNAs, such as microRNAs. miR-145 - pivotal in regulating VSMC plasticity, which is reduced in vascular diseases - was found to control Uhrf1 mRNA translation. In turn, UHRF1 triggered VSMC proliferation, directly repressing promoters of cell-cycle inhibitor genes (including p21 and p27) and key prodifferentiation genes via the methylation of DNA and histones. Local vascular viral delivery of Uhrf1 shRNAs or Uhrf1 VSMC-specific deletion prevented intimal hyperplasia in mouse carotid artery and decreased vessel damage in a mouse model of aortic aneurysm. Our study demonstrates the fundamental role of Uhrf1 in regulating VSMC phenotype by promoting proliferation and dedifferentiation. UHRF1 targeting may hold therapeutic potential in vascular pathologies.
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Aneurisma de la Aorta/metabolismo , Arterias Carótidas/metabolismo , Epigénesis Genética , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Animales , Aneurisma de la Aorta/genética , Aneurisma de la Aorta/patología , Proteínas Potenciadoras de Unión a CCAAT , Arterias Carótidas/patología , Diferenciación Celular/genética , Línea Celular , Proliferación Celular/genética , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Proteínas Nucleares/genética , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by eczema, infections, and susceptibility to autoimmunity and malignancies. Thrombocytopenia is a constant finding, but its pathogenesis remains elusive. OBJECTIVE: To dissect the basis of the WAS platelet defect, we used a novel conditional mouse model (CoWas) lacking Wiskott-Aldrich syndrome protein (WASp) only in the megakaryocytic lineage in the presence of a normal immunologic environment, and in parallel we analyzed samples obtained from patients with WAS. METHODS: Phenotypic and functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic interaction network. Platelet immunogenicity was tested by using ELISAs and B-cell and platelet cocultures. RESULTS: CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis in vitro, but WASp-deficient platelets had short lifespan and high expression of activation markers. Proteomic analysis identified signatures compatible with defects in cytoskeletal reorganization and metabolism yet surprisingly increased antigen-processing capabilities. In addition, WASp-deficient platelets expressed high levels of surface and soluble CD40 ligand and were capable of inducing B-cell activation in vitro. WASp-deficient platelets were highly immunostimulatory in mice and triggered the generation of antibodies specific for WASp-deficient platelets, even in the context of a normal immune system. Patients with WAS also showed platelet hyperactivation and increased plasma soluble CD40 ligand levels correlating with the presence of autoantibodies. CONCLUSION: Overall, these findings suggest that intrinsic defects in WASp-deficient platelets decrease their lifespan and dysregulate immune responses, corroborating the role of platelets as modulators of inflammation and immunity.
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Plaquetas/inmunología , Síndrome de Wiskott-Aldrich/inmunología , Adolescente , Adulto , Animales , Autoinmunidad , Ligando de CD40/inmunología , Niño , Preescolar , Femenino , Humanos , Lactante , Inflamación/sangre , Inflamación/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Recuento de Plaquetas , Síndrome de Wiskott-Aldrich/sangre , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/inmunología , Adulto JovenRESUMEN
Hepatocellular carcinoma (HCC) is a frequent neoplasia and a leading cause of inflammation-related cancer mortality. Despite that most HCCs arise from persistent inflammatory conditions, pathways linking chronic inflammation to cancer development are still incompletely elucidated. We dissected the role of adaptive immunity in the Mdr2 knockout (Mdr2-/- ) mouse, a model of inflammation-associated cancer, in which ablation of adaptive immunity has been induced genetically (Rag2-/- Mdr2-/- and µMt-Mdr2-/- mice) or with in vivo treatments using lymphocyte-specific depleting antibodies (anti-CD20 or anti-CD4/CD8). We found that activated B and T lymphocytes, secreting fibrogenic tumor necrosis factor alpha (TNFα) and other proinflammatory cytokines, infiltrated liver of the Mdr2-/- mice during chronic fibrosing cholangitis. Lymphocyte ablation, in the Rag2-/- Mdr2-/- and µMt-Mdr2-/- mice, strongly suppressed hepatic stellate cell (HSC) activation and extracellular matrix deposition, enhancing HSC transition to cellular senescence. Moreover, lack of lymphocytes changed the intrahepatic metabolic/oxidative state, resulting in skewed macrophage polarization toward an anti-inflammatory M2 phenotype. Remarkably, hepatocarcinogenesis was significantly suppressed in the Rag2-/- Mdr2-/- mice, correlating with reduced TNFα/NF-κB (nuclear factor kappa B) pathway activation. Ablation of CD20+ B cells, but not of CD4+ /CD8+ T cells, in Mdr2-/- mice, promoted senescence-mediated fibrosis resolution and inhibited the protumorigenic TNFα/NF-κB pathway. Interestingly, presence of infiltrating B cells correlated with increased tumor aggressiveness and reduced disease-free survival in human HCC. CONCLUSION: Adaptive immunity sustains liver fibrosis (LF) and favors HCC growth in chronic injury, by modulating innate components of inflammation and limiting the extent of HSC senescence. Therapies designed for B-cell targeting may be an effective strategy in LF. (Hepatology 2018;67:1970-1985).
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Linfocitos B/inmunología , Carcinogénesis/inmunología , Carcinoma Hepatocelular/inmunología , Cirrosis Hepática/inmunología , Neoplasias Hepáticas/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Inmunidad Adaptativa/inmunología , Animales , Carcinogénesis/patología , Técnicas de Cultivo de Célula , Senescencia Celular/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Hígado/inmunología , Hígado/patología , Cirrosis Hepática/patología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Cell fusion between neoplastic and normal cells has been suggested to play a role in the acquisition of a malignant phenotype. Several studies have pointed to the macrophage as the normal partner in this fusion, suggesting that the fused cells could acquire new invasive properties and become able to disseminate to distant organs. However, this conclusion is mainly based on studies with transplantable cell lines. We tested the occurrence of cell fusion in the MMTV-neu model of mouse mammary carcinoma. In the first approach, we generated aggregation chimeras between GFP/neu and RFP/neu embryos. Tumor cells would display both fluorescent proteins only if cell fusion with normal cells occurred. In addition, if cell fusion conferred a growth/dissemination advantage, cells with both markers should be detectable in lung metastases at increased frequency. We confirmed that fused cells are present at low but consistent levels in primary neoplasms and that the macrophage is the normal partner in the fusion events. Similar results were obtained using a second approach in which bone marrow from mice carrying the Cre transgene was transplanted into MMTV-neu/LoxP-tdTomato transgenic animals, in which the Tomato gene is activated only in the presence of CRE recombinase. However, no fused cells were detected in lung metastases in either model. We conclude that fusion between macrophages and tumor cells does not confer a selective advantage in our spontaneous model of breast cancer, although these data do not rule out a possible role in models in which an inflammation environment is prominent.
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Macrófagos/metabolismo , Neoplasias Mamarias Animales/metabolismo , Receptor ErbB-2/metabolismo , Animales , Apoptosis , Separación Celular , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Inflamación , Integrasas/metabolismo , Masculino , Neoplasias Mamarias Experimentales/metabolismo , Virus del Tumor Mamario del Ratón , Ratones , Metástasis de la Neoplasia , Fagocitosis , Fenotipo , TransgenesRESUMEN
Omenn syndrome (OS) is caused by hypomorphic Rag mutations and characterized by a profound immunodeficiency associated with autoimmune-like manifestations. Both in humans and mice, OS is mediated by oligoclonal activated T and B cells. The role of microbial signals in disease pathogenesis is debated. Here, we show that Rag2(R229Q) knock-in mice developed an inflammatory bowel disease affecting both the small bowel and colon. Lymphocytes were sufficient for disease induction, as intestinal CD4 T cells with a Th1/Th17 phenotype reproduced the pathological picture when transplanted into immunocompromised hosts. Moreover, oral tolerance was impaired in Rag2(R229Q) mice, and transfer of wild-type (WT) regulatory T cells ameliorated bowel inflammation. Mucosal immunoglobulin A (IgA) deficiency in the gut resulted in enhanced absorption of microbial products and altered composition of commensal communities. The Rag2(R229Q) microbiota further contributed to the immunopathology because its transplant into WT recipients promoted Th1/Th17 immune response. Consistently, long-term dosing of broad-spectrum antibiotics (ABXs) in Rag2(R229Q) mice ameliorated intestinal and systemic autoimmunity by diminishing the frequency of mucosal and circulating gut-tropic CCR9(+) Th1 and Th17 T cells. Remarkably, serum hyper-IgE, a hallmark of the disease, was also normalized by ABX treatment. These results indicate that intestinal microbes may play a critical role in the distinctive immune dysregulation of OS.
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Autoinmunidad , Proteínas de Unión al ADN/metabolismo , Microbioma Gastrointestinal , Inflamación/inmunología , Inflamación/patología , Traslado Adoptivo , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Autoinmunidad/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Carga Bacteriana/efectos de los fármacos , Traslocación Bacteriana/efectos de los fármacos , Colitis/inmunología , Colitis/patología , Proteínas de Unión al ADN/deficiencia , Microbioma Gastrointestinal/efectos de los fármacos , Tolerancia Inmunológica/efectos de los fármacos , Inmunoglobulina E/metabolismo , Inmunofenotipificación , Inflamación/microbiología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/microbiología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/metabolismo , Células TH1/inmunología , Células Th17/inmunología , Tropismo/efectos de los fármacosRESUMEN
Desmoid tumor is a fibroproliferative neoplasm with an intermediate malignancy and it can be localized in every bodily district: some locations are considered exceptional, like the urogenital localization. The Author point out a rare case of giant idiopathic scrotal fibromatosis that was found during an autopsy. A widower, that lived alone in poor hygienic conditions, was found dead in his house. The Judicial Authority ordered the autopsy, that was performed two days later at the Medico-Legal Section of Milan University. External examinations revealed only the considerable dimension of the scrotum (cm 24 × 41). The cause of death was fixed in a cardiac tamponade due to a natural heart laceration localized in correspondence of a transmural infarction. The toxicological exam resulted negative, while the histopathological and immunohistochemical analysis qualify the scrotal mass as a desmoids tumor. Due to the absence of predisposing conditions and of fibroproliferative infiltration in bladder and retroperitoneal space, the neoplasm was configured as an idiopathic desmoid tumor. The presented case gives the reason for the discussion concerning medico-legal aspects that are typical of rare neoplasms.
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Fibromatosis Agresiva/patología , Neoplasias de los Genitales Masculinos/patología , Escroto/patología , Anciano , Patologia Forense , Humanos , Hallazgos Incidentales , Masculino , Invasividad Neoplásica , Espacio Retroperitoneal/patología , Vejiga Urinaria/patologíaRESUMEN
The congenital malformation split hand/foot (SHFM) is characterized by missing central fingers and dysmorphology or fusion of the remaining ones. Type-1 SHFM is linked to deletions/rearrangements of the DLX5-DLX6 locus and point mutations in the DLX5 gene. The ectrodactyly phenotype is reproduced in mice by the double knockout (DKO) of Dlx5 and Dlx6. During limb development, the apical ectodermal ridge (AER) is a key-signaling center responsible for early proximal-distal growth and patterning. In Dlx5;6 DKO hindlimbs, the central wedge of the AER loses multilayered organization and shows down-regulation of FGF8 and Dlx2. In search for the mechanism, we examined the non-canonical Wnt signaling, considering that Dwnt-5 is a target of distalless in Drosophila and the knockout of Wnt5, Ryk, Ror2 and Vangl2 in the mouse causes severe limb malformations. We found that in Dlx5;6 DKO limbs, the AER expresses lower levels of Wnt5a, shows scattered ß-catenin responsive cells and altered basolateral and planar cell polarity (PCP). The addition of Wnt5a to cultured embryonic limbs restored the expression of AER markers and its stratification. Conversely, the inhibition of the PCP molecule c-jun N-terminal kinase caused a loss of AER marker expression. In vitro, the addition of Wnt5a on mixed primary cultures of embryonic ectoderm and mesenchyme was able to confer re-polarization. We conclude that the Dlx-related ectrodactyly defect is associated with the loss of basoapical and PCP, due to reduced Wnt5a expression and that the restoration of the Wnt5a level is sufficient to partially reverts AER misorganization and dysmorphology.
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Proteínas de Homeodominio/genética , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/patología , Proteína Wnt-5a/farmacología , Animales , Polaridad Celular/efectos de los fármacos , Polaridad Celular/fisiología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Ectodermo/metabolismo , Ectodermo/patología , Proteínas de Homeodominio/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Deformidades Congénitas de las Extremidades/tratamiento farmacológico , Deformidades Congénitas de las Extremidades/metabolismo , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Transactivadores/genética , Vía de Señalización Wnt , Proteína Wnt-5a/biosíntesis , Proteína Wnt-5a/deficiencia , Proteína Wnt-5a/genética , beta Catenina/metabolismoRESUMEN
Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41(+) cells gradually gave rise to CD45(+) cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.
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Células Madre Pluripotentes Inducidas/citología , Osteoclastos/citología , Osteopetrosis/terapia , Reparación del Gen Blanco/métodos , ATPasas de Translocación de Protón Vacuolares/genética , Animales , Diferenciación Celular , Línea Celular , Hematopoyesis , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Células Mieloides/citología , Células Mieloides/metabolismo , Osteoclastos/metabolismo , Osteopetrosis/genéticaRESUMEN
OS is a severe combined immunodeficiency characterized by erythrodermia and protracted diarrhea as a result of infiltration of oligoclonal-activated T cells, caused by hypomorphic mutations in RAGs. The RAG2(R229Q) mouse model fully recapitulates the clinical OS phenotype. We evaluated whether T and B cell defects, together with the abnormal lymphoid structure, could affect DC homeostasis and function. High density of LCs was observed in skin biopsies of Omenn patients and in the derma of RAG2(R229Q) mice, correlating with the presence of erythrodermia. In vivo models of cutaneous skin painting and CHS demonstrated a decreased migration of RAG2(R229Q) DCs-in particular, LCs-into draining LNs. Interestingly, at steady state, RAG2(R229Q) mice showed a reduction in DC number in all hematopoietic organs except LNs. Analysis of the MHCII marker revealed a diminished expression also upon the LPS-driven inflammatory condition. Despite the decreased number of peripheral DCs, BM pre-cDCs were present in normal number compared with RAG2(+/+) controls, whereas pDCs and monocytes were reduced significantly. Overall, these results point to a secondary defect in the DC compartment, which contributes to clinical manifestations and autoimmunity in OS.
Asunto(s)
Movimiento Celular/inmunología , Proteínas de Unión al ADN/inmunología , Dermis/inmunología , Células de Langerhans/inmunología , Mutación , Inmunodeficiencia Combinada Grave/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Movimiento Celular/genética , Proteínas de Unión al ADN/genética , Dermatitis Exfoliativa/genética , Dermatitis Exfoliativa/inmunología , Dermatitis Exfoliativa/patología , Dermis/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Células de Langerhans/patología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Ratones , Ratones Transgénicos , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/patología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Extremidades/crecimiento & desarrollo , Proteínas de Homeodominio/genética , Factor de Transcripción MSX1/genética , Animales , Ectodermo/crecimiento & desarrollo , Ectodermo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Mesodermo/crecimiento & desarrollo , Mesodermo/metabolismo , Ratones , Morfogénesis , MutaciónRESUMEN
In the last decades the molecular basis of monogenic diseases has been largely unraveled, although their treatment has often remained unsatisfactory. Autosomal recessive osteopetrosis (ARO) belongs to the small group of genetic diseases that are usually treated with hematopoietic stem cell transplantation (HSCT). However, this approach is not effective in the recently identified form carrying mutations in the receptor activator of NF-κB ligand (RANKL) gene. In this subset, therapy replacement approach based on RANKL delivery has a strong rationale. Here we demonstrate that the systematic administration of RANKL for 1 month to Rankl(-/-) mice, which closely resemble the human disease, significantly improves the bone phenotype and has beneficial effects on bone marrow, spleen and thymus; major adverse effects arise only when mice are clearly overtreated. Overall, we provide evidence that the pharmacological administration of RANKL represents the appropriate treatment option for RANKL-deficient ARO patients, to be validated in a pilot clinical trial.
Asunto(s)
Osteopetrosis/tratamiento farmacológico , Osteopetrosis/genética , Ligando RANK/uso terapéutico , Animales , Células de la Médula Ósea/efectos de los fármacos , Resorción Ósea/inducido químicamente , Huesos/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Osteopetrosis/patología , Fenotipo , Ligando RANK/administración & dosificación , Ligando RANK/efectos adversos , Ligando RANK/genética , Receptor Activador del Factor Nuclear kappa-B/deficiencia , Receptor Activador del Factor Nuclear kappa-B/genéticaRESUMEN
BACKGROUNDS & AIMS: Cholangiocarcinoma (CCA) is highly fatal because of early invasion, widespread metastasis, and lack of an effective therapy. Migration, invasion, and metastasis of CCA cells are modulated by signals received from stromal cells. The SDF-1-CXCR4 axis emerges as a pivotal regulator of migration and survival of different tumor cells. The aim of the present study was to characterize the interaction between CCA cells and human hepatic stellate cells (hHSC) focusing on the role of SDF-1. METHODS: The intrahepatic CCA cell line HuCCT-1 and primary hHSC were used for this study. RNA expression was examined by RTQ-PCR and protein expression by Western blotting. Immunofluorescence microscopy and immunohistochemistry were also employed. Migration of CCA cells was assessed using modified Boyden chambers. RESULTS: CXCR4 was clearly expressed in CCA cells of human CCA liver specimens. SDF-1 and hHSC conditioned medium (CM) promoted HuCCT-1 cell migration, which was abrogated by pre-incubation with AMD3100, a non-peptide antagonist of the CXCR4 receptor. In addition, HuCCT-1 cells silenced for CXCR4 did not migrate in presence of SDF-1. Both P-ERK and p-AKT were implicated in HuCCT-1 migration and showed a biphasic trend under stimulation of SDF-1. Finally, SDF-1 induced apoptotic rescue of HuCCT-1 cells by binding to CXCR4. CONCLUSIONS: Our study demonstrates that CCA cells migration and survival are modulated by the crosstalk between SDF-1, released by hHSC, and HuCCT-1 cells bearing CXCR4.