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The utilization of neurotrophins in medicine shows significant potential for addressing neurodegenerative conditions, such as age-related macular degeneration (AMD). However, the therapeutic use of neurotrophins has been restricted due to their short half-life. Here, we aimed to synthesize PEGylated nanoparticles based on electrostatic-driven interactions between human serum albumin (HSA), a carrier for adsorption; neurotrophin-3 (NT3); and brain-derived neurotrophic factor (BDNF). Electrophoretic (ELS) and multi-angle dynamic light scattering (MADLS) revealed that the PEGylated HSA-NT3-BDNF nanoparticles ranged from 10 to 430 nm in diameter and exhibited a low polydispersity index (<0.4) and a zeta potential of -8 mV. Based on microscale thermophoresis (MST), the estimated dissociation constant (Kd) from the HSA molecule of BDNF was 1.6 µM, and the Kd of NT3 was 732 µM. The nanoparticles were nontoxic toward ARPE-19 and L-929 cells in vitro and efficiently delivered BDNF and NT3. Based on the biodistribution of neurotrophins after intravitreal injection into BALB/c mice, both nanoparticles were gradually released in the mouse vitreous body within 28 days. PEGylated HSA-NT3-BDNF nanoparticles stabilize neurotrophins and maintain this characteristic in vivo. Thus, given the simplicity of the system, the nanoparticles may enhance the treatment of a variety of neurological disorders in the future.
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Factor Neurotrófico Derivado del Encéfalo , Polietilenglicoles , Ratones , Humanos , Animales , Distribución Tisular , Potenciales de la MembranaRESUMEN
Molecular dynamic modeling and various experimental techniques, including multi-angle dynamic light scattering (MADLS), streaming potential, optical waveguide light spectroscopy (OWLS), quartz crystal microbalance with dissipation (QCM), and atomic force microscopy (AFM), were applied to determine the basic physicochemical parameters of fibroblast growth factor 21 in electrolyte solutions. The protein size and shape, cross-section area, dependence of the nominal charge on pH, and isoelectric point of 5.3 were acquired. These data enabled the interpretation of the adsorption kinetics of FGF 21 on bare and macrocation-covered silica investigated by OWLS and QCM. It was confirmed that the protein molecules irreversibly adsorbed on the latter substrate, forming layers with controlled coverage up to 0.8 mg m-2, while their adsorption on bare silica was much smaller. The viability of two cell lines, CHO-K1 and L-929, on both bare and macrocation/FGF 21-covered substrates was also determined. It is postulated that the acquired results can serve as useful reference systems for designing complexes that can extend the half-life of FGF 21 in its active state.
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Factores de Crecimiento de Fibroblastos , Simulación de Dinámica Molecular , Adsorción , Dióxido de Silicio/química , Propiedades de SuperficieRESUMEN
Biomaterial science has contributed tremendously to developing nanoscale materials for delivering biologically active compounds, enhancing protein stability, and enabling its therapeutic use. This paper presents a process of formation of polyelectrolyte multilayer (PEM) prepared by sequential adsorption of positively charged polydiallyldimethylammonium chloride (PDADMAC) and negatively charged heparin sodium salt (HP), from low polyelectrolyte concentration, on a solid substrate. PEM was further applied as a platform for the adsorption of a brain-derived growth factor (BDNF), which is a protein capable of regulating neuronal cell development. The multilayers containing BDNF were thoroughly characterized by electrokinetic (streaming potential measurements, SPM) and optical (optical waveguide lightmode spectroscopy, OWLS) techniques. It was found that BDNF was significantly adsorbed onto polyelectrolyte multilayers terminated by HP under physiological conditions. We further explore the effect of established PEMs in vitro on the neuroblastoma SH-SY5Y cell line. An enzyme-linked immunosorbent assay (ELISA) confirmed that BDNF was released from multilayers, and the use of the PEMs intensified its cellular uptake. Compared to the control, PEMs with adsorbed BDNF significantly reduced cell viability and mitochondrial membrane polarization to as low as 72% and 58%, respectively. HPLC analysis showed that both PDADMAC-terminated and HP-terminated multilayers have antioxidative properties as they almost by half decreased lipid peroxidation in SH-SY5Y cells. Finally, enhanced formation of spheroid-like, 3D structures was observed by light microscopy. We offer a well-characterized PEM with antioxidant properties acting as a BDNF carrier, stabilizing BDNF and making it more accessible to cells in an inhomogeneous, dynamic, and transient in vitro environment. Described multilayers can be utilized in future biomedical applications, such as boosting the effect of treatment by selective anticancer as adjuvant therapy, and in biomedical research for future development of more precise neurodegenerative disease models, as they enhance cellular 3D structure formation.
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Neuroblastoma , Enfermedades Neurodegenerativas , Humanos , Heparina/farmacología , Heparina/química , Polielectrolitos/química , Factor Neurotrófico Derivado del Encéfalo , Neuroblastoma/tratamiento farmacológicoRESUMEN
The adsorption of biomolecules on nanoparticles' surface ultimately depends on the intermolecular forces, which dictate the mutual interaction transforming their physical, chemical, and biological characteristics. Therefore, a better understanding of the adsorption of serum proteins and their impact on nanoparticle physicochemical properties is of utmost importance for developing nanoparticle-based therapies. We investigated the interactions between potentially therapeutic proteins, neurotrophin 3 (NT3), brain-derived neurotrophic factor (BDNF), and polyethylene glycol (PEG), in a cell-free system and a retinal pigmented epithelium cell line (ARPE-19). The variance in the physicochemical properties of PEGylated NT3-BDNF nanoparticles (NPs) in serum-abundant and serum-free systems was studied using transmission electron microscopy, atomic force microscopy, multi-angle dynamic, and electrophoretic light scattering. Next, we compared the cellular response of ARPE-19 cells after exposure to PEGylated NT3-BDNF NPs in either a serum-free or complex serum environment by investigating protein release and cell cytotoxicity using ultracentrifuge, fluorescence spectroscopy, and confocal microscopy. After serum exposure, the decrease in the aggregation of PEGylated NT3-BDNF NPs was accompanied by increased cell viability and BDNF/NT3 in vitro release. In contrast, in a serum-free environment, the appearance of positively charged NPs with hydrodynamic diameters up to 900 nm correlated with higher cytotoxicity and limited BDNF/NT3 release into the cell culture media. This work provides new insights into the role of protein corona when considering the PEGylated nano-bio interface with implications for cytotoxicity, NPs' distribution, and BDNF and NT3 release profiles in the in vitro setting.
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BACKGROUND: The rational chemical design of nanoparticles can be readily controlled and optimized by quantitatively studying protein adsorption at variously charged polymer carriers, determining their fate in biological fluids. We manufactured brain-derived neurotrophic factor (BDNF) -based electrostatic nanocomplexes with a different type of dendrimer core (anionic or cationic), encapsulated or not in polyethylene glycol (PEG), and studied their physicochemical properties and behavior in a biological setting. We investigated whether the electrokinetic charge of dendrimer core influences BDNF loading and desorption from the nanoparticle and serves as a determinant of nanoparticles' behavior in in vitro setting, influencing mitochondrial dysfunction, lipid peroxidation, and general nanoparticles' cellular toxicity. RESULTS: We found that the electrokinetic charge of the dendrimer core influences nanoparticles in terms of BDNF release profile from their surfaces and their effect on cell viability, mitochondrial membrane potential, cell phenotype, and induction of oxidative stress. The electrostatic interaction of positively charged core of nanoparticles with cell membranes increases their cytotoxicity, as well as serious phenotype alterations compared to negatively charged nanoparticles core in neuron-like differentiated human neuroblastoma cells. Moreover, PEG adsorption at nanoparticles with negatively charged core presents a distinct decrease in metabolic cell activity. On the contrary, charge neutralization due to PEG adsorption on the surface of nanoparticles with positively charged core does not reduce their cytotoxicity, makes them less biocompatible with differentiated cells, and presumably shows non-specific toxicity. CONCLUSIONS: The surface charge transformation after adsorption of protein or polyelectrolyte during nanocarriers formulation has an important role not only in designing nanomaterials with potent neuroprotective and neuroregenerative properties but also in applying them in a cellular environment.
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Nanopartículas/química , Nanopartículas/toxicidad , Estrés Oxidativo , Proteínas/química , Adsorción , Humanos , Iones , Oxidopamina , Polietilenglicoles/química , Polímeros , Electricidad Estática , Propiedades de SuperficieRESUMEN
Brain-derived neurotrophic factor (BDNF) is essential for the development and function of human neurons, therefore it is a promising target for neurodegenerative disorders treatment. Here, we studied BDNF-based electrostatic complex with dendrimer nanoparticles encapsulated in polyethylene glycol (PEG) in neurotoxin-treated, differentiated neuroblastoma SH-SY5Y cells, a model of neurodegenerative mechanisms. PEG layer was adsorbed at dendrimer-protein core nanoparticles to decrease their cellular uptake and to reduce BDNF-other proteins interactions for a prolonged time. Cytotoxicity and confocal microscopy analysis revealed PEG-ylated BDNF-dendrimer nanoparticles can be used for continuous neurotrophic factor delivery to the neurotoxin-treated cells over 24 h without toxic effect. We offer a reliable electrostatic route for efficient encapsulation and controlled transport of fragile therapeutic proteins without any covalent cross-linker; this could be considered as a safe drug delivery system. Understanding the polyvalent BDNF interactions with dendrimer core nanoparticles offers new possibilities for design of well-ordered protein drug delivery systems.
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Factor Neurotrófico Derivado del Encéfalo , Dendrímeros/química , Portadores de Fármacos , Nanopartículas/química , Neuroblastoma/metabolismo , Factor Neurotrófico Derivado del Encéfalo/química , Factor Neurotrófico Derivado del Encéfalo/farmacocinética , Línea Celular Tumoral , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Humanos , Neurotoxinas/efectos adversos , Polietilenglicoles/química , Electricidad EstáticaRESUMEN
Background: NT4 has been regarded as a promising therapeutic protein for treatment of damaged retinal pigment epithelium cells. Purpose: Here, we studied physicochemical parameters of an NT4-polyamidoamine (PAMAM) electrostatic complex, which can provide a sustained concentration of protein in intraocular space over an extended period after delivery. Adsorption/desorption of NT4 molecules to/from positively charged PAMAM dendrimers were precisely determined to control the concentration of bounded/unbounded protein molecules, diffusion coefficient, and size of a protein-laden dendrimer structure. We determined kinetics of NT4 desorption in PBS, vitreous, and damaged retina. Methods: Initially, adsorption of NT4 molecules on PAMAM dendrimers was studied in PBS using dynamic light scattering, electrophoresis, solution depletion, ELISA, and atomic force microscopy. This allowed us precisely to determine desorption of NT4 from nanoparticles under in situ conditions. The maximum coverage of irreversibly adsorbed NT4 determined by ELISA allowed us to devise a robust procedure for preparing stable and well-controlled coverage of NT4 on PAMAM nanoparticles. Thereafter, we studied diffusion of nanospheres containing NT4 molecules by injecting them into vitreous cavities of mice exposed to intravenous injections of sodium iodate and evaluated their intraocular desorption kinetics from drug carriers in vivo. Results: Our measurements revealed NT4-dendrimer nanoparticles can be used for continuous neurotrophic factor delivery, enhancing its distribution into mouse vitreous, as well as damaged retina over 28 days of postinjury observation. Conclusion: Understanding of polyvalent neurotrophin interactions with dendrimer nanoparticles might be useful to obtain well-ordered protein layers, targeting future development of drug-delivery systems, especially for neuroprotection of damaged retinal neurons.
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Dendrímeros/química , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Factores de Crecimiento Nervioso/farmacología , Electricidad Estática , Adsorción , Animales , Preparaciones de Acción Retardada/farmacología , Modelos Animales de Enfermedad , Femenino , Ratones Endogámicos BALB C , Retina/efectos de los fármacos , Retina/lesiones , Cuerpo Vítreo/efectos de los fármacos , Cuerpo Vítreo/lesionesRESUMEN
Adsorption of human serum albumin (HSA), recombinant HSA (rHSA) and the albumin dimer (dHSA) at solid/electrolyte interfaces is reviewed with the emphasis put on quantitative analysis of this process. Initially, various physicochemical data characterizing bulk properties of albumin molecules are discussed such as electrophoretic mobility, electrokinetic charge, zeta potential and diffusion coefficient. Adsorption kinetics of HSA, rHSA and dHSA at mica derived from AFM, streaming potential and XPS measurements is analyzed. Maximum coverages of irreversibly adsorbed molecules under various ionic strengths and pHs are quantitatively interpreted in terms of the random sequential adsorption model. Thorough acid-basic characteristic of albumin monolayers of well-controlled coverage are also presented. The results derived from the colloid deposition method that unveil albumin molecule orientation and charge distribution are discussed and interpreted in terms of the random site theory. Subsequently, adsorption of albumins at negatively and positively charged polymeric microparticles studied by the electrokinetic and the AFM aided concentration depletion methods is analyzed. These results are theoretically interpreted by applying the bead model of HSA and dHSA molecules. Orientation of adsorbed molecules and the stability of albumin monolayers in respect to pH cyclic changes are discussed. A universal, electrostatic interaction driven, mechanism of albumin adsorption at macroscopic surfaces and polymer microparticles is confirmed.
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Bulk physicochemical properties of neurotrophin 4 (NT-4) in electrolyte solutions and its adsorption/desorption on/from mica surfaces have been studied using dynamic light scattering (DLS), microelectrophoresis, a solution depletion technique (enzyme-linked immunosorbent assay, ELISA), and AFM imaging. Our study presents a determination of the diffusion coefficient, hydrodynamic diameters, electrophoretic mobility, and isoelectric point of the NT-4 under various ionic strength and pH conditions. The size of the NT-4 homodimer for an ionic strength of 0.015 M was substantially independent of pH and equal to 5.1 nm. It has been found that the number of electrokinetic charges per NT-4 molecule was equal to zero for all studied ionic strengths at pH 8.1, which was identified as the isoelectric point (iep). The protein adsorption/desorption on/from mica surfaces was examined as a function of ionic strength and pH. The kinetics of neurotrophin adsorption/desorption were evaluated at pH 3.5, 7.4, and 11 by direct AFM imaging and the ELISA technique. A monotonic increase in the maximum coverage of adsorbed NT-4 molecules with ionic strength (up to 5.5 mg/m2) was observed at pH 3.5. These results were interpreted in terms of the theoretical model postulating an irreversible adsorption of the protein governed by the random sequential adsorption (RSA). Our measurements revealed a significant role of ionic strength, pH, and electrolyte composition in the lateral electrostatic interactions among differently charged NT-4 molecules. The transition between adsorption/desorption processes is found for the region of high pH and low surface concentration of adsorbed neurotrophin molecules at constant ionic strength. Additionally, results presented in this work show that the adsorption behavior of neurotrophin molecules may be governed by intrasolvent electrostatic interactions yielding an aggregation process. Understanding polyvalent neurotrophin interactions may have an impact on the reversibility/irreversibility of adsorption, and hence they might be useful for obtaining well-ordered protein layers, targeting the future development of drug delivery systems for treating neurodegenerative diseases.
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Factores de Crecimiento Nervioso/química , Adsorción , Fenómenos Químicos , Humanos , Concentración de Iones de Hidrógeno , Concentración Osmolar , Propiedades de SuperficieRESUMEN
Oral colonization with Candida spp. is not synonymous with a systemic active infection. The aim of the study was to evaluate enzymatic activity of Candida strains isolated from the oral cavity in patients with nephrotic syndrome (NS) and to compare it with the activity determined in urine. We studied 32 children with NS and 26 control healthy children. Children with NS were treated with glucocorticosteroids, cyclosporin A, mycophenolate mofetil or azathioprine. In all children, API-ZYM enzymatic tests were performed to evaluate hydrolytic enzymes of Candida isolated from the oral cavity and in urine. Candida spp. were isolated from the oral cavity in 11 patients with NS (34.4%), all receiving immunosuppressive treatment. All strains produced valine arylamidase, 9 alpha-glucosidase (E16), and 9 N-acetyl-beta-glucosaminidase (E18). A positive correlation between the presence of Candida in the oral cavity and E16 and E18 enzymatic activity in both oral cavity and urine was found. A dose of cyclosporin A had an effect on the enzymatic activity (p < 0.05). We conclude that immunosuppressive treatment of NS in children may predispose to systemic Candida invasion. The results of this study suggest that oral candida infection should be monitored in children with nephrotic syndrome, particularly those treated with immunosuppressive agents.
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Bacteriuria/microbiología , Candida/enzimología , Candidiasis Bucal/microbiología , Boca/microbiología , Síndrome Nefrótico/microbiología , Adolescente , Azatioprina/uso terapéutico , Candida/aislamiento & purificación , Candidiasis Bucal/tratamiento farmacológico , Niño , Preescolar , Ciclosporina/uso terapéutico , Femenino , Glucocorticoides/uso terapéutico , Humanos , Inmunosupresores/uso terapéutico , Masculino , Ácido Micofenólico/uso terapéutico , Síndrome Nefrótico/tratamiento farmacológicoRESUMEN
BACKGROUND: Diabetes and Nephrotic syndrome (NS) promote plaque-related gingivitis and yeast-like fungal infections. The study assesses the impact of Candida spp. and general disease- or treatment-related factors on plaque-related gingivitis severity in children and adolescents with Nephrotic syndrome /diabetes. METHODS: Body mass index (BMI), BMI standard deviation score, and oral cavity (Plaque Index--PLI, Gingival Index--GI, mucosa status, presence and Candida enzymatic activity) were assessed in 96 patients (32 with NS: 30- immunosuppressive treatment, 35--type 1 diabetes, and 29 generally healthy), aged; 3-18 years. Laboratory included cholesterol and triglyceride measurements; in diabetic subjects- glycated haemoglobin, in NS: total protein, albumin, creatinine, haemoglobin, haematocrit, white cell count, urinary protein excretion. Medical records supplied information on disease duration and treatment. A statistical analysis was performed; Kendall Tau coefficient, chi-square test, t-test, and multiple regression analysis ( P < 0.05). RESULTS: Candida spp. often occurred in healthy patients, but oral candidiasis was found only in the NS and diabetes groups (9.37% and 11.43%). Gingivitis occurred more frequently in patients with NS/diabetes. Gingivitis severity was correlated with PLI, age, and yeast enzyme activity in NS--to with immunosuppressive treatment with >1 drug, drug doses, treatment duration, lipid disorders, and BMI; in diabetes, with blood glucose and glycated haemoglobin >8%. CONCLUSION: Poor hygiene control is the main cause of gingivitis. Gingivitis severity is most likely related to age, lipid disorders and increase in body mass. Candida spp., in uncompensated diabetes and in those using immunosuppressive treatment, might intensify plaque-related gingivitis.
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Candidiasis Bucal/microbiología , Diabetes Mellitus Tipo 1/complicaciones , Gingivitis/microbiología , Síndrome Nefrótico/complicaciones , Adolescente , Glucemia/análisis , Proteínas Sanguíneas/análisis , Índice de Masa Corporal , Niño , Preescolar , Colesterol/sangre , Creatinina/sangre , Placa Dental/microbiología , Índice de Placa Dental , Diabetes Mellitus Tipo 1/sangre , Femenino , Gingivitis/etiología , Hemoglobina Glucada/análisis , Hematócrito , Hemoglobinas/análisis , Humanos , Inmunosupresores/uso terapéutico , Recuento de Leucocitos , Masculino , Síndrome Nefrótico/tratamiento farmacológico , Índice Periodontal , Proteinuria/orina , Albúmina Sérica/análisis , Triglicéridos/sangreRESUMEN
Invasive aspergillosis (IA) is a severe infection with a 70% mortality rate. Aspergillus fumigatus is responsible for over 90% of those infections. The diagnosis of invasive aspergillosis is based on clinical sample culture and detection of fungal hyphae in histopathological examination. Additional tests may include the detection of the galactomannan antigen and of fungal genetic material in serum and bronchoalveolar washings. The present study was to assess the use of these two rapid tests in the diagnosis of invasive aspergillosis: serological one - to detect the galactomannan antigen (ELISA assay), and real-time PCR, and to establish a possible correlation between these two methods.
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Adsorption of immunoglobulin G (IgG) from aqueous NaCl solutions of the concentration 10(-3)-0.15M on mica was studied. Initially, the kinetics was evaluated at pH 3.5 by direct AFM imaging. A monotonic increase in the maximum coverage of IgG with NaCl concentration was observed. These results were interpreted in terms of the theoretical model postulating an irreversible adsorption of the protein governed by the random sequential adsorption (RSA) model. Additionally, IgG adsorption and desorption was studied under in situ conditions, with streaming potential measurements. These measurements revealed that the maximum coverage of irreversibly adsorbed IgG varies from 0.37mgm(-2) for 10(-3)M, NaCl to 1.2mgm(-2) for 0.15M, NaCl. The significant role of ionic strength was attributed to the lateral electrostatic repulsion among adsorbed IgG molecules, positively charged at this pH value. These experimental results confirmed that monolayers of irreversibly bound IgG can be produced by adjusting ionic strength of the protein solution. In further experiments the stability and acid base properties of such monolayers were studied using the streaming potential method. It revealed that the monolayers were stable against pH cycling for the range from 3.5 to 9.5. The isoelectric point of mica supported IgG monolayers was 5.9, similar to derived from the micro-electrophoretic measurements in the bulk (5.8). Beside significance for basic sciences, the results indicate that thorough characteristics of IgG can be acquired via streaming potential measurements using microgram quantities of the protein.
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Silicatos de Aluminio/química , Electroquímica/métodos , Inmunoglobulina G/aislamiento & purificación , Microscopía de Fuerza Atómica , Adsorción , Animales , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/química , Cinética , Concentración Osmolar , Albúmina Sérica , Ovinos , Electricidad Estática , Propiedades de SuperficieRESUMEN
Solid organ transplant recipients are at high risk of fungal infections, because of ongoing immunosuppressive treatment. There are three post organ transplant phases: early, intermediate, and late, all of them at risk of Candida infections. Since conventional tests are insufficient, specific secondary diagnostic tests are still being explored. Serological tests are currently the most common choice. The present study was to determine the usefulness of mannan antigen and anti-mannan antibody detection in diagnosing invasive candidiasis in liver or kidney transplant recipients. The levels of mannan and anti-mannan antibodies were assessed with Platelia Candida Ag Plus, and Platelia Candida Ab Plus (Biorad, Marne-la-Coquette, France) commercial tests, according to manufacturer's guidelines. Sixty six serum samples were obtained from 25 patients (9 liver transplant recipients, 7 kidney transplant recipients, and 9 patients prepared for a kidney transplant), 29 serum samples from 15 patients tested positive for mannan antigen. Serum samples were obtained from 14 patients tested positive for anti- mannan antibodies. Fungal antigen detection in blood serum in patients under immunosuppression, especially with neutropenia, suggests that antifungal treatment should be administered. Serological tests, especially mannan and anti-mannan ones, are very useful for confirmation or exclusion of invasive candidiasis in high-risk patients.
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Adsorption of human serum albumin (HSA) on mica at pH 3.5 (0.15M NaCl) was studied using in situ streaming potential measurements, AFM imaging and XPS. Results obtained by streaming potential were consistent with AFM measurements and theoretical predictions based on the random sequential adsorption model. These results in combination with complementary data derived from XPS allowed one to determine both the kinetics of adsorption and the maximum coverage of irreversibly bound HSA, which was close to 1.6 mg m(-2) (dimensionless coverage 0.45). It was concluded that HSA adsorption on mica at pH 3.5 occurred under side-on, electrostatically controlled mechanisms with no tendency to multilayer formation. Such irreversible bound HSA monolayers of well-defined coverage can find applications for performing efficient immunological tests, designing biomaterials surfaces and biosensors.
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Silicatos de Aluminio , Albúmina Sérica/química , Adsorción , Algoritmos , Electroquímica , Electroforesis en Gel de Poliacrilamida , Flujometría por Láser-Doppler , Microscopía de Fuerza Atómica , Peso Molecular , Espectroscopía de Fotoelectrones , Soluciones , Tensión SuperficialRESUMEN
Adsorption of human serum albumin (HSA) on mica from 0.15 M NaCl solutions and various pH values was studied using in situ streaming potential measurements, AFM imaging, and XPS. The results obtained by the streaming potential were consistent with AFM measurements and theoretical predictions based on the random sequential adsorption model. This allowed one to determine both the kinetics of adsorption and the maximum coverage of HSA as a function of pH. At pH 3.5, the maximum coverage of HSA was 0.45 (which corresponds to 1.4 mg m(-2) neglecting hydration). This decreased monotonically with the increase in pH, attaining 0.30 (pH 5.1) and 0.25 (pH 7.4). At pH >10.5, the adsorption of HSA on mica was negligible. Further experimental studies performed for HSA monolayers of well-controlled coverage revealed their stability against pH cycling. It was found in these experiments that at pH <4 and >8 the electrokinetic properties of HSA monolayers approached the reference data pertinent to the bulk. However, for an intermediate pH range, deviations from the bulk reference data were observed, suggesting a dipolar (heterogeneous) charge distribution over adsorbed HSA molecules. This caused a slight shift in the isoelectric point of the monolayer determined to be 4.7 compared to the bulk value of 5.1. However, for the HSA coverage below 0.2, the effect of the substrate was significant, making the zeta potential more negative and shifting the apparent isoelectric point to more acidic values. It was suggested that these results obtained for planar and smooth interfaces could be used as reference data for interpreting albumin adsorption on colloid carrier particles.
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Silicatos de Aluminio/química , Técnicas Electroquímicas , Albúmina Sérica/química , Adsorción , Humanos , Concentración de Iones de Hidrógeno , Cinética , Cloruro de Sodio/química , Soluciones , Propiedades de SuperficieRESUMEN
Adsorption and desorption of human serum albumin (HSA) from aqueous solutions on mica were studied using AFM and in situ streaming potential measurements. A quantitative interpretation of these experiments was achieved in terms of the theoretical model postulating a 3D adsorption of HSA molecules as discrete particles. These measurements, performed for various ionic strength, allowed one to determine the coverage of HSA as a function of the zeta potential of mica. This allowed one to determine the amount of irreversibly bound HSA as a function of the ionic strength. It was found that the coverage of irreversibly adsorbed HSA increased from 0.52 mg m(-2) for I=1.3×10(-3) M to 1.6 mg m(-2) for I=0.15M (pH=3.5). The significant role of ionic strength was attributed to the lateral electrostatic repulsion among adsorbed HSA molecules, positively charged at this pH value. This was quantitatively interpreted in terms of the effective hard particle concept previously used for colloid particles. The experimental results confirmed that monolayers of irreversibly bound HSA of a well-controlled coverage can be produced by adjusting the ionic strength of the suspension.
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Silicatos de Aluminio , Albúmina Sérica/química , Adsorción , Humanos , Cinética , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Concentración OsmolarRESUMEN
The most frequent etiological factors of fungal infections in patients receiving total parenteral nutrition (TPN) belong to Candida genus of the yeastlike fungi. In the TPN patients the several infectious complications can develop: venous catheter infection, catheter candidemia (fungemia), fungal endocarditis or fungal ophtalmitis. The capability of hydrolytic (proteolytic, lipolytic) enzymes secretion as well as biofilm formation on artificial surfaces are the most important factors of fungal strains pathogenity. In the study from clinical materials of 37 patients receiving total parenteral nutrition 31 strains of Candida glabrata (56.4%), 13 strains of Candida albicans (23.6%), 3 Candida tropicalis strains, 2 Candida krusei strains, 2 Candida lusitaniae strains and 1 strain of Candida inconspicua were identified. The phenotypic analysis of isolated strains were performed using API YM (bioMerieux) tests for the enzymatic activity determination. Simultaneously the proteolytic and lipolytic activity analysis were performed. Candida albicans isolates secreted 10 out of 19 enzymes and Candida glabrata 7. The secretion of proteases was demonstrated in 12 C. albicans strains. All Candida glabrata isolates from examined and from control group secreted proteolitic enzymes. Candida glabrata is the dominant species in clinical materials of patients receiving total parenteral nutrition. The numerous isolation of C. glabrata from clinical materials of patients receiving total parenteral nutrition might be connected with the selection of azole resistant strains and also to ability of creatin biofilm on the biomaterial surfaces.
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Candida/clasificación , Candida/patogenicidad , Candidiasis/microbiología , Nutrición Parenteral Total/efectos adversos , Biopelículas , Candida/enzimología , Humanos , Péptido Hidrolasas/metabolismo , Especificidad de la EspecieRESUMEN
Long-term antibiotic therapy is one of the main risk factors for mycosis. The urinary D-arabinitol/L-arabinitol (D-/L-ARA) ratio (a biomarker of several Candida species) was determined by gas chromatography with an electron capture detector in samples from 51 infants undergoing long-term antibiotic therapy. Although 47 of these children had higher D-/L-ARA ratios than healthy controls (P<0.0003), their values nonetheless remained within upper-normal limits (D-/L-ARA ratio of <3.6). Four children with suspected invasive candidiasis had above-normal ratios that normalized with fluconazole treatment.
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Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Candidiasis/diagnóstico , Alcoholes del Azúcar/orina , Antibacterianos/administración & dosificación , Femenino , Humanos , Lactante , Masculino , Factores de TiempoRESUMEN
UNLABELLED: In recent years substantial progress has been seen in the development of approaches and methods for serological diagnosis of the mycosis. The aim of our study was to assess the clinical usefulness of serological tests for fungal infection in pts who underwent prolonged antibiotic therapy after valve replacement. MATERIAL AND METHODS: The group consisted of 27 pts after valve replacement with prolonged antibiotic therapy (6-12 weeks) due to infective endocarditis (22 pts) or sternal infection (5 pts). The samples of blood were taken 10 days after antibiotics were started and next every 10 days. Serological techniques for the diagnosis of Candidiasis consisted of antibody detection to: the cell wall polysaccharide antigen (test A), cytoplasmic protein antigen (test B) and the germ tube antigens (test C). RESULTS: Patients were divided in 3 groups due to the kinetics of the antibody responses to the Candida antigen: 1. pts with normal level of antibody during antibiotic treatment--they didn't required antifungal therapy, 2. pts with increasing level of antibody needed antifungal treatment, 3. pts with increasing kinetics of antibody responses during antibiotic and antifungal treatment needed the change of antifungal therapy, 4. Control group (pts before cardiac surgery without any sign of fungal infection). Antibody to the cytoplasmic antigen was detected infrequently. The presence of antibody to the cytoplasmic antigen was correlated with the patient clinical outcome and prognosis and necessity for change of antifungal therapy. One pt died after valve replacement due to valve dysfunction because of fungal endocarditis. CONCLUSIONS: Serological tests for Candidiasis can be a useful marker of fungal infection, prognosis and treatment in pts required prolonged antibiotic therapy after valve replacement.