RESUMEN
Cholesterol seems to play a central role in the augmentation of saporin-based immunotoxin (IT) cytotoxicity by triterpenoid saponins. Endolysosomal escape has been proposed as one mechanism for the saponin-mediated enhancement of targeted toxins. We investigated the effects of lipid depletion followed by repletion on Saponinum album (SA)-induced endolysosomal escape of Alexa Fluor labelled saporin and the saporin-based immunotoxin OKT10-SAP, directed against CD38, in Daudi lymphoma cells. Lipid deprived cells showed reduced SA-induced endolysosomal escape at two concentrations of SA, as determined by a flow cytometric method. The repletion of membrane cholesterol by low density lipoprotein (LDL) restored SA-induced endolysosomal escape at a concentration of 5 µg/mL SA but not at 1 µg/mL SA. When LDL was used to restore the cholesterol levels in lipid deprived cells, the SA augmentation of OKT10-SAP cytotoxicity was partially restored at 1 µg/mL SA and fully restored at 5 µg/mL SA. These results suggest that different mechanisms of action might be involved for the two different concentrations of SA and that endosomal escape may not be the main mechanism for the augmentation of saporin IT cytotoxicity by SA at the sub-lytic concentration of 1 µg/mL SA.
Asunto(s)
Colesterol/química , Endosomas/efectos de los fármacos , Inmunotoxinas/metabolismo , Lisosomas/efectos de los fármacos , Saponinas/farmacología , Saporinas/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colesterol/metabolismo , LDL-Colesterol/farmacología , Relación Dosis-Respuesta a Droga , Endosomas/química , Endosomas/metabolismo , Fluoresceínas/química , Colorantes Fluorescentes/química , Humanos , Inmunotoxinas/química , Linfocitos/química , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Lisosomas/química , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Saporinas/química , Ácidos Sulfónicos/química , Triterpenos/farmacologíaRESUMEN
Impaired cargo trafficking and the aggregation of intracellular macromolecules are key features of neurodegeneration, and a hallmark of aged as well as diseased retinal pigment epithelial (RPE) cells in the eye. Here, photoreceptor outer segments (POS), which are internalized daily by RPE cells, were modified by UV-irradiation to create oxidatively modified POS (OxPOS). Oxidative modification was quantified by a protein carbonyl content assay. Human ARPE-19 cells were synchronously pulsed with POS or OxPOS to study whether oxidatively modified cargos can recapitulate features of RPE pathology associated with blinding diseases. Confocal immunofluorescence microscopy analysis showed that OxPOS was trafficked to LAMP1, LAMP2 lysosomes and to LC3b autophagy vacuoles. Whilst POS were eventually degraded, OxPOS cargos were sequestered in late compartments. Co-localization of OxPOS was also associated with swollen autolysosomes. Ultrastructural analysis revealed the presence of electron-dense OxPOS aggregates in RPE cells, which appeared to be largely resistant to degradation. Measurement of cellular autofluorescence, using parameters used to assess fundus autofluorescence (FAF) in age-related macular disease (AMD) patients, revealed that OxPOS contributed significantly to a key feature of aged and diseased RPE. This in vitro cell model therefore represents a versatile tool to study disease pathways linked with RPE damage and sight-loss.
Asunto(s)
Agregado de Proteínas/fisiología , Enfermedades de la Retina/patología , Epitelio Pigmentado de la Retina/patología , Autofagia/fisiología , Células Cultivadas , Humanos , Lisosomas/patología , Degeneración Macular/patología , Oxidación-Reducción , Estrés Oxidativo/fisiología , Fagocitosis/fisiología , Segmento Externo de las Células Fotorreceptoras Retinianas/patologíaRESUMEN
PURPOSE: The aim of this work was to develop a quantitative, flow cytometric method for tracking the endolysosomal escape of a fluorescently labelled saporin toxin. METHODS: Flow cytometric measurements of fluorescent pulse width and height were used to track the endocytic uptake into Daudi cells of a fluorescently labelled saporin toxin and the saporin based immunotoxin, OKT10-SAP. Subsequently, measurement of changes in pulse width were used to investigate the effect of a triterpenoid saponin on the endolysosomal escape of internalised toxin into the cytosol. Live cell confocal microscopy was used to validate the flow cytometry data. RESULTS: Increased endolysosomal escape of saporin and OKT10-SAP was observed by confocal microscopy in cells treated with saponin. Fluorescent pulse width measurements were also able to detect and quantify escape more sensitively than confocal microscopy. Saponin induced endolysosomal escape could be abrogated by treatment with chloroquine, an inhibitor of endolysosomal acidification. Chloroquine abrogation of escape was also mirrored by a concomitant abrogation of cytotoxicity. CONCLUSIONS: Poor endolysosomal escape is often a rate limiting step for the cytosolic delivery of protein toxins and other macromolecules. Pulse width analysis offers a simple method to semi-quantify the endolysosomal escape of this and similar molecules into the cytosol.
Asunto(s)
Antineoplásicos/farmacología , Citosol/metabolismo , Endosomas/metabolismo , Citometría de Flujo/métodos , Inmunoglobulina G/farmacología , Toxinas Biológicas/farmacología , Antineoplásicos/química , Transporte Biológico , Línea Celular Tumoral , Supervivencia Celular , Endocitosis , Humanos , Inmunoglobulina G/química , Inmunotoxinas/metabolismo , Lisosomas/metabolismo , Saponinas/química , Saporinas/metabolismo , Transducción de Señal , Toxinas Biológicas/química , Triterpenos/químicaRESUMEN
Extracellular matrix stiffness has a profound effect on the behavior of many cell types. Adherent cells apply contractile forces to the material on which they adhere and sense the resistance of the material to deformation-its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 µm) thicknesses. Here, we hypothesized that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic extracellular-matrix-functionalized polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker-labeled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single-cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 µm. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater half-maximal response at 54 µm. Depth-sensing was dependent on Rho-associated protein kinase-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances.
Asunto(s)
Mecanotransducción Celular , Línea Celular Tumoral , Elasticidad , Matriz Extracelular/metabolismo , Humanos , Modelos Biológicos , Seudópodos/metabolismo , Análisis de la Célula Individual , Quinasas Asociadas a rho/metabolismoRESUMEN
Tracers injected into CSF pass into the brain alongside arteries and out again. This has been recently termed the "glymphatic system" that proposes tracers enter the brain along periarterial "spaces" and leave the brain along the walls of veins. The object of the present study is to test the hypothesis that: (1) tracers from the CSF enter the cerebral cortex along pial-glial basement membranes as there are no perivascular "spaces" around cortical arteries, (2) tracers leave the brain along smooth muscle cell basement membranes that form the Intramural Peri-Arterial Drainage (IPAD) pathways for the elimination of interstitial fluid and solutes from the brain. 2 µL of 100 µM soluble, fluorescent fixable amyloid ß (Aß) were injected into the CSF of the cisterna magna of 6-10 and 24-30 month-old male mice and their brains were examined 5 and 30 min later. At 5 min, immunocytochemistry and confocal microscopy revealed Aß on the outer aspects of cortical arteries colocalized with α-2 laminin in the pial-glial basement membranes. At 30 min, Aß was colocalised with collagen IV in smooth muscle cell basement membranes in the walls of cortical arteries corresponding to the IPAD pathways. No evidence for drainage along the walls of veins was found. Measurements of the depth of penetration of tracer were taken from 11 regions of the brain. Maximum depths of penetration of tracer into the brain were achieved in the pons and caudoputamen. Conclusions drawn from the present study are that tracers injected into the CSF enter and leave the brain along separate periarterial basement membrane pathways. The exit route is along IPAD pathways in which Aß accumulates in cerebral amyloid angiopathy (CAA) in Alzheimer's disease. Results from this study suggest that CSF may be a suitable route for delivery of therapies for neurological diseases, including CAA.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Membrana Basal/metabolismo , Encéfalo/metabolismo , Líquido Cefalorraquídeo/metabolismo , Líquido Extracelular/metabolismo , Sistema Glinfático/metabolismo , Actinas/metabolismo , Factores de Edad , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Encéfalo/citología , Colágeno Tipo IV/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Tejido Parenquimatoso/metabolismo , Receptores de Superficie Celular/metabolismo , Factores de TiempoRESUMEN
Deposition of amyloid ß (Aß) in the walls of cerebral arteries as cerebral amyloid angiopathy (CAA) suggests an age-related failure of perivascular drainage of soluble Aß from the brain. As CAA is associated with Alzheimer's disease and with intracerebral haemorrhage, the present study determines the unique sequence of changes that occur as Aß accumulates in artery walls. Paraffin sections of post-mortem human occipital cortex were immunostained for collagen IV, fibronectin, nidogen 2, Aß and smooth muscle actin and the immunostaining was analysed using Image J and confocal microscopy. Results showed that nidogen 2 (entactin) increases with age and decreases in CAA. Confocal microscopy revealed stages in the progression of CAA: Aß initially deposits in basement membranes in the tunica media, replaces first the smooth muscle cells and then the connective tissue elements to leave artery walls completely or focally replaced by Aß. The pattern of development of CAA in the human brain suggests expansion of Aß from the basement membranes to progressively replace all tissue elements in the artery wall. Establishing this full picture of the development of CAA is pivotal in understanding the clinical presentation of CAA and for developing therapies to prevent accumulation of Aß in artery walls. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Angiopatía Amiloide Cerebral/patología , Arterias Cerebrales/patología , Adulto , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/análisis , Membrana Basal/metabolismo , Membrana Basal/patología , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Encéfalo/patología , Angiopatía Amiloide Cerebral/metabolismo , Arterias Cerebrales/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Túnica Media/metabolismo , Túnica Media/patología , Adulto JovenRESUMEN
Triterpenoid saponins from Saponinum Album (SA) exert potent lytic effects on eukaryotic cell plasma membranes and, when used at sub-lytic concentrations, significantly augment the cytotoxicity of saporin-based immunotoxins (IT). To help elucidate the mechanism(s) behind these two phenomena we investigated the role of cholesterol to both. Human Daudi lymphoma cells were lipid deprived using a combination of three different approaches. Following treatment, the total cellular lipid content was analyzed by electrospray ionization mass spectrometry (ESI-MS) and plasma membrane (PM) cholesterol content measured using the lipophilic fluorescent probe NR12S. Maximal lipid deprivation of cells resulted in a complete loss of sensitivity to lysis by SA. Similarly augmentation of the anti-CD19 immunotoxin (IT) BU12-SAPORIN by SA was lost but without a concomitant loss of intrinsic IT cytotoxicity. The lytic activity of SA was restored following incubation of lipid deprived Daudi cells with Synthecol or LDL. The augmentative effect of SA on IT cytotoxicity for Daudi cells was restored following repletion of PM cholesterol levels with LDL. NR12S fluorescence and ESI-MS analysis of cellular lipids demonstrated that restoration of SA lytic activity by Synthecol was entirely due to increased PM cholesterol levels. Restoration of cellular and PM cholesterol levels by LDL also restored the augmentative effect of SA for IT, an effect associated with repletion of PM cholesterol with minor changes in some phospholipid species. These results indicate that the lytic and IT augmentative properties of SA are cholesterol-dependent in contrast to intrinsic IT cytotoxicity that is at least partially cholesterol independent.
Asunto(s)
Antígenos CD19/inmunología , LDL-Colesterol/fisiología , Inmunotoxinas/farmacología , Linfoma/tratamiento farmacológico , Lípidos de la Membrana/fisiología , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Saponinas/farmacología , Triterpenos/farmacología , Línea Celular Tumoral , Humanos , Linfoma/química , SaporinasRESUMEN
Mammalian extra-embryonic lineages perform the crucial role of nutrient provision during gestation to support embryonic and fetal growth. These lineages derive from outer trophectoderm (TE) and internal primitive endoderm (PE) in the blastocyst and subsequently give rise to chorio-allantoic and visceral yolk sac placentae, respectively. We have shown maternal low protein diet exclusively during mouse preimplantation development (Emb-LPD) is sufficient to cause a compensatory increase in fetal and perinatal growth that correlates positively with increased adult-onset cardiovascular, metabolic and behavioural disease. Here, to investigate early mechanisms of compensatory nutrient provision, we assessed the influence of maternal Emb-LPD on endocytosis within extra-embryonic lineages using quantitative imaging and expression of markers and proteins involved. Blastocysts collected from Emb-LPD mothers within standard culture medium displayed enhanced TE endocytosis compared with embryos from control mothers with respect to the number and collective volume per cell of vesicles with endocytosed ligand and fluid and lysosomes, plus protein expression of megalin (Lrp2) LDL-family receptor. Endocytosis was also stimulated using similar criteria in the outer PE-like lineage of embryoid bodies formed from embryonic stem cell lines generated from Emb-LPD blastocysts. Using an in vitro model replicating the depleted amino acid (AA) composition found within the Emb-LPD uterine luminal fluid, we show TE endocytosis response is activated through reduced branched-chain AAs (leucine, isoleucine, valine). Moreover, activation appears mediated through RhoA GTPase signalling. Our data indicate early embryos regulate and stabilise endocytosis as a mechanism to compensate for poor maternal nutrient provision.
Asunto(s)
Endocitosis/fisiología , Animales , Blastocisto/citología , Células Cultivadas , Dieta con Restricción de Proteínas , Endodermo/citología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Masculino , Ratones , Embarazo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Bacterial infections of central venous catheters (CVCs) cause much morbidity and mortality, and are usually diagnosed by concordant culture of blood and catheter tip. However, studies suggest that culture often fails to detect biofilm bacteria. This study optimizes X-ray micro-focus computed tomography (X-ray µCT) for the quantification and determination of distribution and heterogeneity of biofilms in in vitro CVC model systems.Bacterial culture and scanning electron microscopy (SEM) were used to detect Staphylococcus epidermidis ATCC 35984 biofilms grown on catheters in vitro in both flow and static biofilm models. Alongside this, X-ray µCT techniques were developed in order to detect biofilms inside CVCs. Various contrast agent stains were evaluated using energy-dispersive X-ray spectroscopy (EDS) to further optimize these methods. Catheter material and biofilm were segmented using a semi-automated matlab script and quantified using the Avizo Fire software package. X-ray µCT was capable of distinguishing between the degree of biofilm formation across different segments of a CVC flow model. EDS screening of single- and dual-compound contrast stains identified 10 nm gold and silver nitrate as the optimum contrast agent for X-ray µCT. This optimized method was then demonstrated to be capable of quantifying biofilms in an in vitro static biofilm formation model, with a strong correlation between biofilm detection via SEM and culture. X-ray µCT has good potential as a direct, non-invasive, non-destructive technology to image biofilms in CVCs, as well as other in vivo medical components in which biofilms accumulate in concealed areas.
Asunto(s)
Biopelículas , Infecciones Relacionadas con Catéteres/microbiología , Catéteres Venosos Centrales/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus epidermidis/fisiología , Humanos , Infecciones Estafilocócicas/diagnóstico por imagen , Staphylococcus epidermidis/ultraestructura , TomografíaRESUMEN
Age-related Macular Degeneration (AMD) is a common, irreversible blinding condition that leads to the loss of central vision. AMD has a complex aetiology with both genetic as well as environmental risks factors, and share many similarities with Alzheimer's disease. Recent findings have contributed significantly to unravelling its genetic architecture that is yet to be matched by molecular insights. Studies are made more challenging by observations that aged and AMD retinas accumulate the highly pathogenic Alzheimer's-related Amyloid beta (Aß) group of peptides, for which there appears to be no clear genetic basis. Analyses of human donor and animal eyes have identified retinal Aß aggregates in retinal ganglion cells (RGC), the inner nuclear layer, photoreceptors as well as the retinal pigment epithelium. Aß is also a major drusen constituent; found correlated with elevated drusen-load and age, with a propensity to aggregate in retinas of advanced AMD. Despite this evidence, how such a potent driver of neurodegeneration might impair the neuroretina remains incompletely understood, and studies into this important aspect of retinopathy remains limited. In order to address this we exploited R28 rat retinal cells which due to its heterogeneous nature, offers diverse neuroretinal cell-types in which to study the molecular pathology of Aß. R28 cells are also unaffected by problems associated with the commonly used RGC-5 immortalised cell-line, thus providing a well-established model in which to study dynamic Aß effects at single-cell resolution. Our findings show that R28 cells express key neuronal markers calbindin, protein kinase C and the microtubule associated protein-2 (MAP-2) by confocal immunofluorescence which has not been shown before, but also calretinin which has not been reported previously. For the first time, we reveal that retinal neurons rapidly internalised Aß1-42, the most cytotoxic and aggregate-prone amongst the Aß family. Furthermore, exposure to physiological amounts of Aß1-42 for 24 h correlated with impairment to neuronal MAP-2, a cytoskeletal protein which regulates microtubule dynamics in axons and dendrites. Disruption to MAP-2 was transient, and had recovered by 48 h, although internalised Aß persisted as discrete puncta for as long as 72 h. To assess whether Aß could realistically localise to living retinas to mediate such effects, we subretinally injected nanomolar levels of oligomeric Aß1-42 into wildtype mice. Confocal microscopy revealed the presence of focal Aß deposits in RGC, the inner nuclear and the outer plexiform layers 8 days later, recapitulating naturally-occurring patterns of Aß aggregation in aged retinas. Our novel findings describe how retinal neurons internalise Aß to transiently impair MAP-2 in a hitherto unreported manner. MAP-2 dysfunction is reported in AMD retinas, and is thought to be involved in remodelling and plasticity of post-mitotic neurons. Our insights suggest a molecular pathway by which this could occur in the senescent eye leading to complex diseases such as AMD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Degeneración Macular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Degeneración Macular/diagnóstico , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Electrónica de Transmisión , Epitelio Pigmentado de la Retina/ultraestructuraRESUMEN
Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.
Asunto(s)
Genoma de los Helmintos/genética , Schistosoma mansoni/genética , Animales , Evolución Biológica , Exones/genética , Genes de Helminto/genética , Interacciones Huésped-Parásitos/genética , Intrones/genética , Datos de Secuencia Molecular , Mapeo Físico de Cromosoma , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/embriología , Schistosoma mansoni/fisiología , Esquistosomiasis mansoni/tratamiento farmacológico , Esquistosomiasis mansoni/parasitologíaRESUMEN
Rituximab, a monoclonal antibody that targets CD20 on B cells, is now central to the treatment of a variety of malignant and autoimmune disorders. Despite this success, a substantial proportion of B-cell lymphomas are unresponsive or develop resistance, hence more potent anti-CD20 monoclonal antibodies (mAbs) are continuously being sought. Here we demonstrate that type II (tositumomab-like) anti-CD20 mAbs are 5 times more potent than type I (rituximab-like) reagents in depleting human CD20 Tg B cells, despite both operating exclusively via activatory Fcgamma receptor-expressing macrophages. Much of this disparity in performance is attributable to type I mAb-mediated internalization of CD20 by B cells, leading to reduced macrophage recruitment and the degradation of CD20/mAb complexes, shortening mAb half-life. Importantly, human B cells from healthy donors and most cases of chronic lymphatic leukemia and mantle cell lymphoma, showed rapid CD20 internalization that paralleled that seen in the Tg mouse B cells, whereas most follicular lymphoma and diffuse large B-cell lymphoma cells were far more resistant to CD20 loss. We postulate that differences in CD20 modulation may play a central role in determining the relative efficacy of rituximab in treating these diseases and strengthen the case for focusing on type II anti-CD20 mAb in the clinic.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD20/inmunología , Antineoplásicos/farmacología , Linfoma de Células B/tratamiento farmacológico , Macrófagos/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales de Origen Murino , Complejo Antígeno-Anticuerpo/inmunología , Antineoplásicos/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Depleción Linfocítica , Linfoma de Células B/inmunología , Ratones , Ratones Noqueados , RituximabRESUMEN
A defining pathological feature of human lung fibrosis is localized tissue heterogeneity, which challenges the interpretation of transcriptomic studies that typically lose spatial information. Here we investigate spatial gene expression in diagnostic tissue using digital profiling technology. We identify distinct, region-specific gene expression signatures as well as shared gene signatures. By integration with single-cell data, we spatially map the cellular composition within and distant from the fibrotic niche, demonstrating discrete changes in homeostatic and pathologic cell populations even in morphologically preserved lung, while through ligand-receptor analysis, we investigate cellular cross-talk within the fibrotic niche. We confirm findings through bioinformatic, tissue, and in vitro analyses, identifying that loss of NFKB inhibitor zeta in alveolar epithelial cells dysregulates the TGFß/IL-6 signaling axis, which may impair homeostatic responses to environmental stress. Thus, spatially resolved deconvolution advances understanding of cell composition and microenvironment in human lung fibrogenesis.
Asunto(s)
Fibrosis Pulmonar , Células Epiteliales Alveolares/metabolismo , Fibrosis , Humanos , Pulmón/patología , Fibrosis Pulmonar/metabolismo , Transducción de SeñalAsunto(s)
Infecciones por Haemophilus/microbiología , Mastocitos/microbiología , Pólipos Nasales/microbiología , Infecciones por Pseudomonas/microbiología , Rinitis/microbiología , Sinusitis/microbiología , Infecciones Estafilocócicas/microbiología , Biopelículas/crecimiento & desarrollo , Estudios de Casos y Controles , Enfermedad Crónica , Infecciones por Haemophilus/complicaciones , Infecciones por Haemophilus/inmunología , Infecciones por Haemophilus/patología , Haemophilus influenzae/fisiología , Humanos , Mastocitos/inmunología , Mastocitos/patología , Pólipos Nasales/complicaciones , Pólipos Nasales/inmunología , Pólipos Nasales/patología , Estudios Prospectivos , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/fisiología , Rinitis/complicaciones , Rinitis/inmunología , Rinitis/patología , Sinusitis/complicaciones , Sinusitis/inmunología , Sinusitis/patología , Infecciones Estafilocócicas/complicaciones , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/patología , Staphylococcus aureus/fisiologíaRESUMEN
Microglia are the brain immune cells and their function is highly dependent on cell motility. It was hypothesised that morphological variability leads to differences in motility, ultimately impacting on the microglial function. Here, we assessed microglial morphology in 32 controls, 44 Alzheimer's disease (AD) cases and 16 AD cases from patients immunised against Aß42 (iAD) using 2D and 3D approaches. Our 2D assessment showed an increased number of microglia in iAD vs. AD (P = 0.032) and controls (P = 0.018). Ramified microglia were fewer in AD vs. controls (P = 0.041) but increased in iAD compared to AD (P < 0.001) and controls (P = 0.006). 3D reconstructions highlighted larger cell bodies in AD vs. controls (P = 0.049) and increased total process length in iAD vs. AD (P = 0.032), with negative correlations detected for pan-Aß load with total process length (P < 0.001) in AD and number of primary processes (P = 0.043) in iAD. In summary, reactive/amoeboid microglia are the most represented population in the aged human brain. AD does not affect the number of microglia, but the ramified population is decreased adopting a more reactive morphology. Aß removal by immunotherapy leads to increased ramified microglia, implying that the cells retain plasticity in an aged disease brain meriting further investigation.
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Enfermedad de Alzheimer/fisiopatología , Microglía/patología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/inmunología , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Femenino , Humanos , Inmunoterapia/métodos , Masculino , Microglía/metabolismo , Ovillos Neurofibrilares/metabolismo , Bancos de TejidosRESUMEN
Monoclonal antibodies (mAb) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses. The mechanisms underlying these differential agonistic activities remain poorly understood. Here, we employ a panel of experimental and clinically-relevant molecules targeting human CD40, 4-1BB and OX40 to examine this issue. Confocal and STORM microscopy reveal that strongly agonistic reagents induce clusters characterized by small area and high receptor density. Using antibody pairs differing only in isotype we show that hIgG2 confers significantly more receptor clustering than hIgG1 across all three receptors, explaining its greater agonistic activity, with receptor clustering shielding the receptor-agonist complex from further molecular access. Nevertheless, discrete receptor clustering patterns are observed with different hIgG2 mAb, with a unique rod-shaped assembly observed with the most agonistic mAb. These findings dispel the notion that larger receptor clusters elicit greater agonism, and instead point to receptor density and subsequent super-structure as key determinants.
Asunto(s)
Receptores del Factor de Necrosis Tumoral/agonistas , Animales , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Antígenos CD40/agonistas , Antígenos CD40/química , Línea Celular , Humanos , Inmunoglobulina G/farmacología , Ratones , Microscopía Confocal , Receptores OX40/agonistas , Receptores del Factor de Necrosis Tumoral/química , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistasRESUMEN
Alzheimer's disease-associated amyloid beta (Aß) proteins accumulate in the outer retina with increasing age and in eyes of age-related macular degeneration (AMD) patients. To study Aß-induced retinopathy, wild-type mice were injected with nanomolar human oligomeric Aß1-42, which recapitulate the Aß burden reported in human donor eyes. In vitro studies investigated the cellular effects of Aß in endothelial and retinal pigment epithelial (RPE) cells. Results show subretinal Aß-induced focal AMD-like pathology within 2 weeks. Aß exposure caused endothelial cell migration, and morphological and barrier alterations to the RPE. Aß co-localized to late-endocytic compartments of RPE cells, which persisted despite attempts to clear it through upregulation of lysosomal cathepsin B, revealing a novel mechanism of lysosomal impairment in retinal degeneration. The rapid upregulation of cathepsin B was out of step with the prolonged accumulation of Aß within lysosomes, and contrasted with enzymatic responses to internalized photoreceptor outer segments (POS). Furthermore, RPE cells exposed to Aß were identified as deficient in cargo-carrying lysosomes at time points that are critical to POS degradation. These findings imply that Aß accumulation within late-endocytic compartments, as well as lysosomal deficiency, impairs RPE function over time, contributing to visual defects seen in aging and AMD eyes.
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Péptidos beta-Amiloides/metabolismo , Lisosomas/metabolismo , Degeneración Macular/metabolismo , Fragmentos de Péptidos/metabolismo , Fenotipo , Animales , Autofagia/fisiología , Ratones , Retina/metabolismo , Enfermedades de la Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
INTRODUCTION: Pericytes are a common feature in the placental microvasculature but their roles are not well understood. Pericytes may provide physical or endocrine support for endothelium and in some tissues mediate vasoconstriction. METHODS: This study uses serial block-face scanning electron microscopy (SBFSEM) to generate three-dimensional (3D) reconstructions of placental pericytes of the terminal villi and transmission electron microscopy (TEM) to study pericyte endothelial cell interactions. The proportion of endothelial cell junctions covered by pericytes was determined. RESULTS: The detailed 3D models of placental pericytes show pericyte structure at a new level of detail. Placental pericytes have many fingers extending from the cell body which can span multiple capillary branches. The proportion of endothelial cell-cell junctions covered by pericytes was significantly higher than pericyte coverage of capillary endothelium as a whole (endothelium: 14%, junctions: 43%, p < 0.0001). However, the proportion of endothelial cell-cell junctions covered by pericytes in regions adjacent to trophoblast was reduced compared to regions >3 µm away from trophoblast (27% vs 62% respectively, p < 0.001). No junctional complexes were observed connecting pericytes and endothelial cells but there were regions of cell membrane with features suggestive of intercellular adhesions. DISCUSSION: These data suggest that the localisation of pericytes on the villous capillary is not random but organised in relation to both endothelial junctions and the location of adjacent trophoblast. This further suggests that pericyte coverage may favour capillary permeability in regions that are most important for exchange, but limit capillary permeability in other regions.
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Capilares/metabolismo , Vellosidades Coriónicas/metabolismo , Pericitos/citología , Placenta/irrigación sanguínea , Trofoblastos/citología , Actinas/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Femenino , Humanos , Microscopía Electrónica de Rastreo , Pericitos/metabolismo , Placenta/citología , Placenta/metabolismo , Embarazo , Trofoblastos/metabolismoRESUMEN
Angiotensin-converting enzyme 2 (ACE2) is the main entry point in airway epithelial cells for SARS-CoV-2. ACE2 binding to the SARS-CoV-2 protein spike triggers viral fusion with the cell plasma membrane, resulting in viral RNA genome delivery into the host. Despite ACE2's critical role in SARS-CoV-2 infection, full understanding of ACE2 expression, including in response to viral infection, remains unclear. ACE2 was thought to encode five transcripts and one protein of 805 amino acids. In the present study, we identify a novel short isoform of ACE2 expressed in the airway epithelium, the main site of SARS-CoV-2 infection. Short ACE2 is substantially upregulated in response to interferon stimulation and rhinovirus infection, but not SARS-CoV-2 infection. This short isoform lacks SARS-CoV-2 spike high-affinity binding sites and, altogether, our data are consistent with a model where short ACE2 is unlikely to directly contribute to host susceptibility to SARS-CoV-2 infection.
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Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Células Epiteliales/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Chlorocebus aethiops , Exones , Células HEK293 , Humanos , Interferones/inmunología , Unión Proteica , Isoformas de Proteínas/genética , Sitios de Empalme de ARN , RNA-Seq , Sistema Respiratorio/citología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Transcriptoma , Regulación hacia Arriba , Células VeroRESUMEN
CA88 is the first long nuclear repetitive DNA sequence identified in the blood fluke, Schistosoma mansoni. The assembled S. mansoni sequence, which contains the CA88 repeat, has 8,887 nucleotides and at least three repeat units of approximately 360 bp. In addition, CA88 also possesses an internal CA microsatellite, identified as SmBr18. Both PCR and BLAST analysis have been used to analyse and confirm the CA88 sequence in other S. mansoni sequences in the public database. PCR-acquired nuclear repetitive DNA sequence profiles from nine Schistosoma species were used to classify this organism into four genotypes. Included among the nine species analysed were five sequences of both African and Asian lineages that are known to infect humans. Within these genotypes, three of them refer to recognised species groups. A panel of four microsatellite loci, including SmBr18 and three previously published loci, has been used to characterise the nine Schistosoma species. Each species has been identified and classified based on its CA88 DNA fingerprint profile. Furthermore, microsatellite sequences and intra-specific variation have also been observed within the nine Schistosoma species sequences. Taken together, these results support the use of these markers in studying the population dynamics of Schistosoma isolates from endemic areas and also provide new methods for investigating the relationships between different populations of parasites. In addition, these data also indicate that Schistosoma magrebowiei is not a sister taxon to Schistosoma mattheei, prompting a new designation to a basal clade.