RESUMEN
The inflammatory burden of the complex rheumatoid arthritis (RA) disease affects several organ-systems, including rheological properties of blood and its formed elements. Red blood cells (RBCs) are constantly exposed to circulating dysregulated inflammatory molecules that are co-transported within the vasculature; and their membranes may be particularly vulnerable to the accompanying oxidative stress. In the current study, we investigate biophysical and ultrastructural characteristics of RBCs obtained from a cohort of patients using atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy (CM). Statistical analyses of AFM data showed that RA RBCs possessed significantly reduced membrane elasticity relative to that of RBCs from healthy individuals (P-value <0.0001). SEM imaging of RA RBCs revealed increased anisocytes and poikilocytes. Poikilocytes included knizocytes, stomatocytes, dacryocytes, irregularly contracted cells, and knot cells. CM imaging of several RA RBCs, spectrin, and band 3 protein networks portrayed the similar morphological profiles. Analyses of CM images confirmed changes to distribution of band-3 skeletal protein, a protein critical for gaseous exchange functions of the RBC and preventing membrane surface loss. Decreased membrane deformability impairs the RBC's capacity to adequately adapt its shape to navigate blood vessels, especially microvasculature, and this decrease is also reflected in the cell's morphology. Changes to morphology and deformability may also indicate loss of functional domains and/or pathological protein and lipid associations. These findings suggest that RA disease and/or its concomitant factors impact on the RBC and its membrane integrity with potential for exacerbating pathological cellular function, hemorheology, and cardiovascular function.
Asunto(s)
Artritis Reumatoide/sangre , Membrana Eritrocítica/ultraestructura , Eritrocitos Anormales/ultraestructura , Artritis Reumatoide/patología , Estudios de Casos y Controles , Módulo de Elasticidad , Deformación Eritrocítica , Membrana Eritrocítica/patología , Eritrocitos Anormales/patología , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de RastreoRESUMEN
The suitability of 4 in vitro assays, commonly used for mutagenicity and genotoxicity assessment, was investigated in relation to treatment with 14 nm citrate-stabilized gold nanoparticles (AuNPs). Specifically, the Ames test was conducted without metabolic activation, where no mutagenic effects were observed. High resolution transmission electron microscopy and Cytoviva dark-field image analysis showed that AuNPs did not enter the bacterial cells, thus confirming the unreliability of the Ames test for nanoparticle mutagenicity studies. In addition, the Chinese hamster ovary (CHO) cell line was used for Comet, Chromosome aberration and Micronucleus assays. CHO cells were treated with AuNPs for 20 h at 37 °C. Cytotoxicity was not detected by cell impedance studies even though AuNP uptake was confirmed using Cytoviva image analysis. The DNA damage was statistically significant in treated cells when assessed by the Comet assay. However, minimal and nonstatistically significant chromosomal DNA damage was observed using the chromosome aberration and micronucleus assays. In this study, we showed that false positive results obtained with Comet assay may have been due to the possibility of direct contact between the residual, intracellular AuNPs and DNA during the assay procedure. Therefore, the chromosome aberration and micronucleus assays are better suited to assess the genotoxic effects of nanoparticles due to low probability of such direct contact occurring. Genotoxic effect of 14 and 20 nm citrate-stabilized, as well as, 14 nm PCOOH AuNPs were also investigated using chromosome aberration and micronucleus assays. Based on our acceptance criteria for a positive genotoxic response, none of the AuNPs were found to be genotoxic in either of these assays.
Asunto(s)
Oro/toxicidad , Nanopartículas del Metal/toxicidad , Mutágenos/toxicidad , Salmonella typhimurium/efectos de los fármacos , Animales , Transporte Biológico , Células CHO , Supervivencia Celular/efectos de los fármacos , Fenómenos Químicos , Aberraciones Cromosómicas/inducido químicamente , Ácido Cítrico/química , Ensayo Cometa , Cricetulus , Reacciones Falso Positivas , Oro/química , Oro/metabolismo , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Pruebas de Micronúcleos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Pruebas de Mutagenicidad , Mutágenos/química , Mutágenos/metabolismo , Tamaño de la Partícula , Conservadores Farmacéuticos/química , Reproducibilidad de los Resultados , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/ultraestructura , Propiedades de SuperficieRESUMEN
BACKGROUND: Strokes are commonly preceded by transient ischemic attacks (TIAs). TIA is often associated with metabolic syndrome (causing chronic inflammation), resulting in a proinflammatory- and procoagulant-environment. The aim of this study was to determine whether platelet- and fibrin network-morphology or coagulation profiles of individuals that suffered a TIA in the presence of metabolic syndrome was altered when compared to healthy individuals. MATERIALS AND METHODS: The study consisted of 40 voluntary participants. Twenty individuals that suffered a TIA in the previous 48 h with at least two metabolic syndrome risk factors present and twenty healthy age-matched controls. Scanning electron- and atomic force microscopy was used to study platelet- and fibrin-morphology, atomic force microscopy was used to study platelet- and fibrin fiber-elasticity and thromboelastography for the study of coagulation profiles. Statistical analysis was performed to compare the two groups. In all cases a p-value of less than 0.05 was considered statistically significant. RESULTS: Platelets of the control group appeared spherical with few pseudopodia present while the platelets of the TIA individuals presented with numerous pseudopodia and spreading, indicating activation. Platelet aggregation was also present. The fibrin networks of the healthy individuals consist of thick and thin fibers that form an organized network of fibers. The fibrin networks of the TIA individuals appeared less organized with less taut fibers. Fibrin fiber thickness was found to be significantly increased in the TIA group (p-value <0.001) when compared to healthy controls. The thicker fibers formed irregular networks with thick masses of fibrin fibers. Platelet and fibrin fiber elasticity was found to be significantly lower in the experimental group (p-value 0.0042 and p-value 0.0007 respectively). The hemostatic profiles of the diseased individuals did not differ significantly (p-value > 0.05) from the healthy controls, indicating a normal functioning coagulation cascade. CONCLUSION: The findings indicate that pathological clot formation is not caused by alterations in the coagulation cascade but rather by the premature activation of platelets (as a result of chronic inflammation) that in turn causes altered fibrin formation.
Asunto(s)
Plaquetas/metabolismo , Fibrina/metabolismo , Ataque Isquémico Transitorio/sangre , Síndrome Metabólico/sangre , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Activación Plaquetaria , Tromboelastografía , Adulto , Anciano , Coagulación Sanguínea , Plaquetas/ultraestructura , Estudios de Casos y Controles , Elasticidad , Femenino , Fibrina/ultraestructura , Fibrinólisis , Humanos , Ataque Isquémico Transitorio/complicaciones , Ataque Isquémico Transitorio/diagnóstico , Masculino , Síndrome Metabólico/complicaciones , Síndrome Metabólico/diagnóstico , Persona de Mediana Edad , Agregación Plaquetaria , Valor Predictivo de las Pruebas , Factores de RiesgoRESUMEN
Polymyxins have previously been described to have activity against Mycobacterium tuberculosis (MTB), but further research was abandoned due to systemic toxicity concerns to achieve the required MIC. Colistin methanesulfonate (CMS), a polymyxin, is well tolerated when inhaled directly into the lungs, resulting in high local concentrations. We report here for the first time, MIC and MBC data for CMS determined by the microtiter Alamar Blue assay (MABA). We also determined how the MIC would be affected by the presence of pulmonary surfactant (PS) and if any synergy with isoniazid (INH) and rifampicin (RIF) exists. The effect of CMS on the ultrastructure of MTB was also determined. The MIC for CMS was 16 mg/L, while the MBC was 256 mg/L. MIC for CMS in PS was antagonised by eight fold. For synergy, indifference was determined while time-kill assays revealed a greater killing effect when CMS was used together with INH. Ultrastructure analysis suggests that the disruption of the outer polysaccharide layer of MTB by CMS may lead to enhanced uptake of INH. Our findings may provide insight for further investigations of CMS against MTB.
Asunto(s)
Antituberculosos/farmacología , Colistina/análogos & derivados , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/química , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Colistina/química , Colistina/farmacología , Sinergismo Farmacológico , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Microscopía Electrónica de Transmisión , Estructura Molecular , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/ultraestructura , Surfactantes Pulmonares/farmacología , Rifampin/farmacología , Relación Estructura-Actividad , Factores de TiempoRESUMEN
We have noted in previous work, in a variety of inflammatory diseases, where iron dysregulation occurs, a strong tendency for erythrocytes to lose their normal discoid shape and to adopt a skewed morphology (as judged by their axial ratios in the light microscope and by their ultrastructure in the SEM). Similarly, the polymerization of fibrinogen, as induced in vitro by added thrombin, leads not to the common 'spaghetti-like' structures but to dense matted deposits. Type 2 diabetes is a known inflammatory disease. In the present work, we found that the axial ratio of the erythrocytes of poorly controlled (as suggested by increased HbA1c levels) type 2 diabetics was significantly increased, and that their fibrin morphologies were again highly aberrant. As judged by scanning electron microscopy and in the atomic force microscope, these could be reversed, to some degree, by the addition of the iron chelators deferoxamine (DFO) or deferasirox (DFX). As well as their demonstrated diagnostic significance, these morphological indicators may have prognostic value.
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/diagnóstico , Eritrocitos/patología , Eritrocitos/ultraestructura , Fibrinógeno/ultraestructura , Trombina/ultraestructura , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Distribución AleatoriaRESUMEN
A major trend in recent Parkinson's disease (PD) research is the investigation of biological markers that could help in identifying at-risk individuals or to track disease progression and response to therapies. Central to this is the knowledge that inflammation is a known hallmark of PD and of many other degenerative diseases. In the current work, we focus on inflammatory signalling in PD, using a systems approach that allows us to look at the disease in a more holistic way. We discuss cyclooxygenases, prostaglandins, thromboxanes and also iron in PD. These particular signalling molecules are involved in PD pathophysiology, but are also very important in an aberrant coagulation/hematology system. We present and discuss a hypothesis regarding the possible interaction of these aberrant signalling molecules implicated in PD, and suggest that these molecules may affect the erythrocytes of PD patients. This would be observable as changes in the morphology of the RBCs and of PD patients relative to healthy controls. We then show that the RBCs of PD patients are indeed rather dramatically deranged in their morphology, exhibiting eryptosis (a kind of programmed cell death). This morphological indicator may have useful diagnostic and prognostic significance.
Asunto(s)
Eritrocitos/patología , Ferritinas/sangre , Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/patología , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Femenino , Humanos , Masculino , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Persona de Mediana EdadRESUMEN
INTRODUCTION: Unliganded iron both contributes to the pathology of Alzheimer's disease (AD) and also changes the morphology of erythrocytes (RBCs). We tested the hypothesis that these two facts might be linked, i.e., that the RBCs of AD individuals have a variant morphology, that might have diagnostic or prognostic value. METHODS: We included a literature survey of AD and its relationships to the vascular system, followed by a laboratory study. Four different microscopy techniques were used and results statistically compared to analyze trends between high and normal serum ferritin (SF) AD individuals. RESULTS: Light and scanning electron microscopies showed little difference between the morphologies of RBCs taken from healthy individuals and from normal SF AD individuals. By contrast, there were substantial changes in the morphology of RBCs taken from high SF AD individuals. These differences were also observed using confocal microscopy and as a significantly greater membrane stiffness (measured using force-distance curves). CONCLUSION: We argue that high ferritin levels may contribute to an accelerated pathology in AD. Our findings reinforce the importance of (unliganded) iron in AD, and suggest the possibility both of an early diagnosis and some means of treating or slowing down the progress of this disease.
RESUMEN
Smoking affects the general health of an individual, however, the red blood cells (RBCs) and their architecture are particularly vulnerable to inhaled toxins related to smoking. Smoking is one of the lifestyle diseases that are responsible for the most deaths worldwide and an individual who smokes is exposed to excessive amounts of oxidants and toxins which generate up to 10(18) free radicals in the human body. Recently, it was reported that smoking decreases RBC membrane fluidity. Here we confirm this and we show changes visible in the topography of RBC membranes, using scanning electron microscopy (SEM). RBC membranes show bubble formation of the phospholipid layer, as well as balloon-like smooth areas; while their general discoid shapes are changed to form pointed extensions. We also investigate membrane roughness using atomic force microscopy (AFM) and these results confirm SEM results. Due to the vast capability of RBCs to be adaptable, their state of well-being is a major indication for the general health status of an individual. We conclude that these changes, using an old technique in a novel application, may provide new insights and new avenues for future improvements in clinical medicine pertaining to conditions like COPD.
Asunto(s)
Membrana Eritrocítica/patología , Membrana Eritrocítica/fisiología , Fumar/sangre , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Fluidez de la Membrana/fisiología , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Fumar/epidemiología , Superóxidos/sangre , Adulto JovenRESUMEN
Thrombo-embolic ischemic stroke is a serious and debilitating disease, and it remains the second most common cause of death worldwide. Tobacco smoke exposure continues to be responsible for preventable deaths around the world, and is a major risk factor for stroke. Platelets play a fundamental role in clotting, and their pathophysiological functioning is present in smokers and stroke patients, resulting in a pro-thrombotic state. In the current manuscript, atomic force and scanning electron microscopy were used to compare the platelets of smokers, stroke patients and healthy individuals. Results showed that the elastic modulus of stroke platelets is decreased by up to 40%, whereas there is an elasticity decrease of up to 20% in smokers' platelets. This indicates a biophysical alteration of the platelets. Ultrastructurally, both the stroke patients and smokers' platelets are more activated than the healthy control group, with prominent cytoskeletal rearrangement involved; but to a more severe extent in the stroke group than in the smokers. Importantly, this is a confirmation of the extent of smoking as risk factor for stroke. We conclude by suggesting that the combined AFM and SEM analyses of platelets might give valuable information about the disease status of patients. Efficacy of treatment regimes on the integrity, cell shape, roughness and health status of platelets may be tracked, as this cell's health status is crucial in the over-activated coagulation system of conditions like stroke.
Asunto(s)
Plaquetas/fisiología , Plaquetas/ultraestructura , Fumar/efectos adversos , Accidente Cerebrovascular/fisiopatología , Plaquetas/efectos de los fármacos , Módulo de Elasticidad/fisiología , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de RastreoRESUMEN
Red blood cells (RBCs) are highly deformable and possess a robust membrane that can withstand shear force. Previous research showed that in diabetic patients, there is a changed RBC ultrastructure, where these cells are elongated and twist around spontaneously formed fibrin fibers. These changes may impact erythrocyte function. Ultrastructural analysis of RBCs in inflammatory and degenerative diseases can no longer be ignored and should form a fundamental research tool in clinical studies. Consequently, we investigated the membrane roughness and ultrastructural changes in type 2 diabetes. Atomic force microscopy (AFM) was used to study membrane roughness and we correlate this with scanning electron microscopy (SEM) to compare results of both the techniques with the RBCs of healthy individuals. We show that the combined AFM and SEM analyses of RBCs give valuable information about the disease status of patients with diabetes. Effectiveness of treatment regimes on the integrity, cell shape and roughness of RBCs may be tracked, as this cell's health status is crucial to the overall wellness of the diabetic patient.
Asunto(s)
Estructuras de la Membrana Celular/patología , Diabetes Mellitus Tipo 2/patología , Eritrocitos/patología , Microscopía de Fuerza Atómica , Estructuras de la Membrana Celular/ultraestructura , Bases de Datos Factuales , Eritrocitos/ultraestructura , Femenino , Humanos , Masculino , Microscopía de Fuerza Atómica/métodos , Microscopía Electrónica de Rastreo/métodos , Propiedades de SuperficieRESUMEN
For the study of cellular ultrastructure, the sample needs to be stabilized by fixation, with the ultimate aim to preserve the native tissue organization and to protect the tissue against later stages of preparation. Chemical and freezing fixation are most used, and chemical fixation employs agents that permeate tissues and cells by diffusion and covalently bind with their major biochemical constituents to fix them. Most widely used chemical fixatives are aldehydes, e.g., formaldehyde and glutaraldehyde, which are noncoagulating, crosslinking agents. Cryofixation methods for ultrastructural studies are also popular, and high-pressure freezing immobilizes all cell constituents and arrests biological activity by removing the thermal energy from the system. In the current research, we used platelet-rich plasma (PRP) to study expansive fibrin fibers and platelet ultrastructure to compare the two fixation techniques. We also used thrombin and calcium chloride as a clotting agent to determine the technique most suitable for the formation of extensive fibrin networks. Chemically fixated fibrin fibers were more compact and condensed and also showed a banding pattern on longitudinal sections. High-pressure frozen samples were more dispersed while platelets fixated showed better preserved cellular membranes and organelle structure. PRP coagulated by addition of CaCl(2) showed blood platelets that are noticeably more activated compared with PRP; however, with thrombin, a sharp ultrastructure was seen. We conclude that PRP mixed with thrombin, and freeze substituted, is the most suitable method for the study of extensive fibrin fibers as well as platelets.