RESUMEN
Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Despite progress in understanding immunogenetic aspects of this disease, the mechanisms involved in lesion formation are unknown. To gain new insights into the neuropathology of MS, we used an innovative integration of Fourier transform infrared (FT-IR) microspectroscopy, bioinformatics, and a synchrotron light source to analyze macromolecular changes in the CNS during the course and prevention of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. We report that subtle chemical and structural changes not observed by conventional histology were detected before the onset of clinical signs of EAE. Moreover, trained artificial neural networks (ANNs) could discriminate, with excellent sensitivity and specificity, pathology from surrounding tissues and the early stage of the disease progression. Notably, we show that this novel measurement platform can detect characteristic differences in biochemical composition of lesion pathology in animals partially protected against EAE by vaccination with Nogo-A, an inhibitor of neural outgrowth, demonstrating the potential for automated screening and evaluation of new therapeutic agents.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/terapia , Animales , Automatización , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Femenino , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple , Proteínas de la Mielina/inmunología , Proteínas de la Mielina/uso terapéutico , Redes Neurales de la Computación , Proteínas Nogo , Péptidos/inmunología , Péptidos/uso terapéutico , Sensibilidad y Especificidad , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Factores de Tiempo , VacunaciónRESUMEN
The potential role of Nogo-66 Receptor 1 (NgR1) on immune cell phenotypes and their activation during neuroinflammatory diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is unclear. To further understand the function of this receptor on haematopoietically-derived cells, phenotypic and functional analyses were performed using NgR1-deficient (ngr1-/-) animals. Flow cytometry-based phenotypic analyses performed on blood, spleen, thymus, lymph nodes, bone marrow and central nervous-system (CNS)-infiltrating blood cells revealed no immunological defects in naïve ngr1-/- animals versus wild-type littermate (WTLM) controls. EAE was induced by either recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or by MOG35-55 peptide, a B cell-independent model. We have demonstrated that in ngr1-/- mice injected with MOG35-55, a significant reduction in the severity of EAE correlated with reduced axonal damage present in the spinal cord when compared to their WTLM controls. However, despite a reduction in axonal damage observed in the CNS of ngr1-/- mice at the chronic stage of disease, no clinical differences could be attributed to a specific genotype when rMOG was used as the encephalitogen. Following MOG35-55-induction of EAE, we could not derive any major changes to the immune cell populations analyzed between ngr1-/- and WTLM mice. Collectively, these data demonstrate that NgR1 has little if any effects on the repertoire of immune cells, their activation and trafficking to the CNS.
Asunto(s)
Linfocitos B/inmunología , Movimiento Celular/inmunología , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Proteínas de la Mielina/inmunología , Receptores de Superficie Celular/inmunología , Animales , Linfocitos B/patología , Movimiento Celular/genética , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Activación de Linfocitos/genética , Ratones , Ratones Noqueados , Esclerosis Múltiple/genética , Esclerosis Múltiple/patología , Proteínas de la Mielina/genética , Receptor Nogo 1 , Receptores de Superficie Celular/genéticaRESUMEN
The recent introduction of technologies capable of reprogramming human somatic cells into induced pluripotent stem (iPS) cells offers a unique opportunity to study many aspects of neurodegenerative diseases in vitro that could ultimately lead to novel drug development and testing. Here, we report for the first time that human dermal fibroblasts from a patient with relapsing-remitting Multiple Sclerosis (MS) were reprogrammed to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4, and c-MYC). The MSiPS cell lines resembled human embryonic stem (hES) cell-like colonies in morphology and gene expression and exhibited silencing of the retroviral transgenes after four passages. MSiPS cells formed embryoid bodies that expressed markers of all three germ layers by immunostaining and Reverse Transcriptase (RT)-PCR. The injection of undifferentiated iPS cell colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. The MSiPS cells were successfully differentiated into mature astrocytes, oligodendrocytes and neurons with normal karyotypes. Although MSiPS-derived neurons displayed some differences in their electrophysiological characteristics as compared to the control cell line, they exhibit properties of functional neurons, with robust resting membrane potentials, large fast tetrodotoxin-sensitive action potentials and voltage-gated sodium currents. This study provides for the first time proof of concept that disease cell lines derived from skin cells obtained from an MS patient can be generated and successfully differentiated into mature neural lineages. This represents an important step in a novel approach for the study of MS pathophysiology and potential drug discovery.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/patología , Esclerosis Múltiple/patología , Esclerosis Múltiple/fisiopatología , Neuronas/patología , Animales , Linaje de la Célula , Fenómenos Electrofisiológicos , Fibroblastos/patología , Humanos , Factor 4 Similar a Kruppel , Ratones , Ratones SCID , Repeticiones de Microsatélite/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Oligodendroglía/patología , Células Madre Pluripotentes/patología , Regiones Promotoras Genéticas/genética , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/patología , Transducción GenéticaRESUMEN
Bone marrow has been proposed as a possible source of cells capable of replacing injured neural cells in diseases such as Multiple Sclerosis (MS). Previous studies have reported conflicting results regarding the transformation of bone marrow cells into neural cells in vivo. This study is a detailed analysis of the fate of bone marrow derived cells (BMDC) in the CNS of C57Bl/6 mice with and without experimental autoimmune encephalomyelitis using flow cytometry to identify GFP-labeled BMDC that lacked the pan-hematopoietic marker, CD45 and co-expressed neural markers polysialic acid-neural cell adhesion molecule or A2B5. A small number of BMDC displaying neural markers and lacking CD45 expression was identified within both the non-inflamed and inflamed CNS. However, the majority of BMDC exhibited a hematopoietic phenotype.
Asunto(s)
Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/patología , Gangliósidos/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Animales , Biomarcadores/metabolismo , Médula Ósea/metabolismo , Trasplante de Médula Ósea , Sistema Nervioso Central/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , FenotipoRESUMEN
Recent evidence suggests that bone marrow (BM)-derived cells may integrate into the kidney, giving rise to functional renal cell types, including endothelial and epithelial cells and myofibroblasts. BM-derived cells can contribute to repair of the renal peritubular capillary (PTC) network following acute ischemic injury. However, the cell fate and regulation of BM-derived cells during the progression of chronic renal disease remains unclear. Using chimeric mice transplanted with enhanced green fluorescent protein (EGFP)-expressing BM, we demonstrate that the number of BM-derived myofibroblasts coincided with the development of fibrosis in a mouse adriamycin (ADR)-induced nephrosis model of chronic, progressive renal fibrosis. Four weeks after ADR injection, increased numbers of BM-derived myofibroblasts were observed in the interstitium of ADR-injected mice. Six weeks after ADR injection, more than 30% of renal alpha-smooth muscle actin (+) (alpha-SMA+) interstitial myofibroblasts were derived from the BM. In addition, BM-derived cells were observed to express the endothelial cell marker CD31 and the myofibroblast marker alpha-SMA. Blockade of p38 mitogen-activated protein kinase (MAPK) and transforming growth factor (TGF)-beta1/Smad2 signaling was found to protect BM-derived PTC endothelial cells and inhibit the number of BM-derived von Willebrand factor (vWF)(+)/EGFP(+)/alpha-SMA(+) cells, EGFP(+)/alpha-SMA(+) cells, and total alpha-SMA(+) cells in ADR-injected mice. Inhibition of the p38 MAPK and TGF-beta1/Smad signaling pathways enhanced PTC repair by decreasing endothelial-myofibroblast transformation, leading to structural and functional renal recovery and the attenuation of renal interstitial fibrosis. Investigation of the signaling pathways that regulate the differentiation and survival of BM-derived cells in a progressive disease setting is vital for the successful development of cell-based therapies for renal repair.
Asunto(s)
Células de la Médula Ósea/fisiología , Trasplante de Células/métodos , Nefritis Intersticial/fisiopatología , Animales , Trasplante de Médula Ósea , Capilares/fisiopatología , Creatinina/metabolismo , Marcadores Genéticos , Túbulos Renales/irrigación sanguínea , Ratones , Proteinuria , Urotelio/patologíaRESUMEN
All solid organs contain resident monocyte-derived cells that appear early in organogenesis and persist throughout life. These cells are critical for normal development in some organs. Here we report the use of a previously described transgenic line, with EGFP driven by the macrophage-restricted Csf1r (c-fms) promoter, to image macrophage production and infiltration accompanying organogenesis in many tissues. Using microarray analysis of FACS-isolated EGFP-positive cells, we show that fetal kidney, lung and brain macrophages show similar gene expression profiles irrespective of their tissue of origin. EGFP-positive cells appeared in the renal interstitium from 12 days post coitum, prior to nephrogenesis, and maintain a close apposition to renal tubules postnatally. CSF-1 added to embryonic kidney explants increased overall renal growth and ureteric bud branching. Expression profiling of tissue macrophages and of CSF-1-treated explants showed evidence of the alternate, pro-proliferative (M2) activation profile, including expression of macrophage mannose receptor (CD206), macrophage scavenger receptor 2 (Msr2), C1q, CD163, selenoprotein P, CCL24 and TREM2. This response has been associated with the trophic role of tumour-associated macrophages. These findings suggest a trophic role of macrophages in embryonic kidney development, which may continue to play a similar role in postnatal repair.
Asunto(s)
Desarrollo Embrionario/fisiología , Macrófagos/citología , Macrófagos/metabolismo , Animales , Encéfalo/citología , Encéfalo/embriología , Encéfalo/metabolismo , Desarrollo Embrionario/genética , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Riñón/citología , Riñón/embriología , Riñón/metabolismo , Pulmón/citología , Pulmón/embriología , Pulmón/metabolismo , Masculino , Ratones , Ratones Transgénicos , Organogénesis/genética , Organogénesis/fisiología , Embarazo , Regiones Promotoras Genéticas , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The peritubular capillary (PTC) network is a component of the tubulointerstitium of the kidney with important roles in renal function and hemodynamics. Bone marrow (BM)-derived cells can contribute to repair of the renal PTC network after ischemic injury. However, the cell fate and the regulation of renal BM-derived cell engraftment in comparison with somatic cells during disease progression are unclear. This study characterized the time course and regulation of PTC endothelial cell injury in adriamycin (ADR)-induced nephropathy in mice, a model of chronic, irreversible, progressive renal disease. Enhanced green fluorescence protein-positive BM cells that coexpressed two endothelial cell markers, von Willebrand factor and CD31, were found to engraft into the PTC of chimeric ADR-injected mice in a time-dependent manner. The number of BM-derived PTC endothelial cells peaked 2 wk after ADR injection, then declined dramatically thereafter. In these mice, apoptosis was evident in BM-derived PTC endothelial cells, and the p38 mitogen-activated protein kinase (MAPK) and TGF-beta1/Smad signaling pathways were activated. Blocking both the p38 MAPK and TGF-beta1/Smad signaling pathways by administration of a p38 MAPK inhibitor (SB203580) and a TGF-beta receptor 1 inhibitor (ALK5I) to ADR-injected mice rescued BM-derived PTC endothelial cells from apoptosis, reduced the loss of PTC, and restored kidney function. Investigation into the signaling pathways that regulate the differentiation and survival of BM-derived cells that engraft into the kidney in the proinflammatory setting of progressive renal disease is vital for the successful development of cell-based therapies to promote renal regeneration and repair.
Asunto(s)
Células de la Médula Ósea/fisiología , Doxorrubicina/toxicidad , Endotelio Vascular/fisiología , Nefrosis/metabolismo , Transducción de Señal , Proteínas Smad/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Apoptosis , Células Endoteliales , Activación Enzimática/efectos de los fármacos , Riñón/citología , Riñón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Nefrosis/inducido químicamente , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Inflammation and fibrogenesis are the two determinants of the progression of renal fibrosis, the common pathway leading to end-stage renal disease. The p38 mitogen-activated protein kinase (MAPK) and transforming growth factor (TGF)-beta1/Smad signaling pathways play critical roles in inflammation and fibrogenesis, respectively. The present study examined the beneficial renoprotective effect of combination therapy using the p38 MAPK pathway inhibitor (SB203580) and a TGF-beta receptor I (ALK5) inhibitor (ALK5I) in a mouse model of adriamycin (ADR) nephrosis. The p38 MAPK and TGF-beta1/Smad2 signaling pathways were activated in ADR-induced nephropathy in a sequential time course manner. Two weeks after ADR injection, the combined administration of SB203580 (1 mg/kg/24 hours) and ALK5I (1 mg/kg/24 hours) markedly reduced p38 MAPK and Smad2 activities. Moreover, the co-administration of SB203580 and ALK5I to ADR-injected mice resulted in a down-regulation of total and active TGF-beta1 production, reduced myofibroblast accumulation, and decreased expression of collagen type IV and fibronectin. In these mice, retardation in the development of glomerulosclerosis and interstitial fibrosis was observed. In conclusion, although p38 MAPK and TGF-beta1/Smad signaling pathways are distinct they coordinate the progression of renal fibrosis in ADR nephrosis. The co-administration of a p38 MAPK inhibitor and an ALK5 inhibitor may have potential applications in the treatment of renal fibrosis.
Asunto(s)
Nefrosis/inducido químicamente , Nefrosis/patología , Transducción de Señal , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Actinas/metabolismo , Receptores de Activinas Tipo I/antagonistas & inhibidores , Animales , Doxorrubicina/farmacología , Activación Enzimática/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibrosis , Imidazoles/farmacología , Riñón/citología , Riñón/patología , Riñón/fisiopatología , Pruebas de Función Renal , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Células Musculares/efectos de los fármacos , Nefrosis/fisiopatología , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas , Piridinas/farmacología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/biosíntesis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The end point of immune and nonimmune renal injury typically involves glomerular and tubulointerstitial fibrosis. Although numerous studies have focused on the events that lead to renal fibrosis, less is known about the mechanisms that promote cellular repair and tissue remodeling. Described is a model of renal injury and repair after the reversal of unilateral ureteral obstruction (UUO) in male C57bl/6J mice. Male mice (20 to 25 g) underwent 10 d of UUO with or without 1, 2, 4, or 6 wk of reversal of UUO (R-UUO). UUO resulted in cortical tubular cell atrophy and tubular dilation in conjunction with an almost complete ablation of the outer medulla. This was associated with interstitial macrophage infiltration; increased hydroxyproline content; and upregulated type I, III, IV, and V collagen expression. The volume density of kidney occupied by renal tubules that exhibited a brush border was measured as an assessment of the degree of repair after R-UUO. After 6 wk of R-UUO, there was an increase in the area of kidney occupied by repaired tubules (83.7 +/- 5.9%), compared with 10 d UUO kidneys (32.6 +/- 7.3%). This coincided with reduced macrophage numbers, decreased hydroxyproline content, and reduced collagen accumulation and interstitial matrix expansion, compared with obstructed kidneys from UUO mice. GFR in the 6-wk R-UUO kidneys was restored to 43 to 88% of the GFR in the contralateral unobstructed kidneys. This study describes the regenerative potential of the kidney after the established interstitial matrix expansion and medullary ablation associated with UUO in the adult mouse.
Asunto(s)
Corteza Renal/fisiología , Túbulos Renales/fisiología , Obstrucción Ureteral/patología , Obstrucción Ureteral/fisiopatología , Análisis de Varianza , Animales , Biopsia con Aguja , Modelos Animales de Enfermedad , Tasa de Filtración Glomerular , Inmunohistoquímica , Pruebas de Función Renal , Masculino , Ratones , Ratones Endogámicos C57BL , Probabilidad , Valores de Referencia , Regeneración , Reoperación , Sensibilidad y Especificidad , Índice de Severidad de la EnfermedadRESUMEN
The degradation of hemoglobin by the malaria parasite, Plasmodium falciparum, produces free ferriprotoporphyrin IX (FP) as a toxic by-product. In the presence of FP-binding drugs such as chloroquine, FP detoxification is inhibited, and the build-up of free FP is thought to be a key mechanism in parasite killing. In an effort to identify parasite proteins that might interact preferentially with FP, we have used a mass spectrometry approach. Proteins that bind to FP immobilized on agarose include P. falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), P. falciparum glutathione reductase (PfGR), and P. falciparum protein disulfide isomerase. To examine the potential consequences of FP binding, we have examined the ability of FP to inhibit the activities of GAPDH and GR from P. falciparum and other sources. FP inhibits the enzymic activity of PfGAPDH with a Ki value of 0.2 microm, whereas red blood cell GAPDH is much less sensitive. By contrast, PfGR is more resistant to FP inhibition (Ki > 25 microm) than its human counterpart. We also examined the ability of FP to inhibit the activities of the additional antioxidant enzymes, P. falciparum thioredoxin reductase, which exhibits a Ki value of 1 microm, and P. falciparum glutaredoxin, which shows more moderate sensitivity to FP. The exquisite sensitivity of PfGAPDH to FP may indicate that the glycolytic pathway of the parasite is particularly susceptible to modulation by FP stress. Inhibition of this pathway may drive flux through the pentose phosphate pathway ensuring sufficient production of reducing equivalents to counteract the oxidative stress induced by FP build-up.