Incorporation of 45S5 bioglass via sol-gel in ß-TCP scaffolds: Bioactivity and antimicrobial activity evaluation.

In this work, ß-TCP (ß-tricalcium phosphate) bioresorbable scaffolds were prepared by the gel casting method. Then, they were impregnated with a 45S5 bioglass sol gel solution to improve biocompatibility and promote bioactivity and antimicrobial activity. The ß-TCP scaffolds had an apparent porosity of 72%, and after the incorporation of the bioglass, this porosity was maintained. The elements of the bioglass were incorporated into ß-TCP matrix and there was a partial transformation from the ß-TCP phase to the α-TCP (α-tricalcium phosphate) phase, besides the formation of bioactive calcium and sodium­calcium silicates. The scaffolds ß-TCP with 45S5 bioglass incorporated (ß-TCP/45S5) did not show a reduction in their values of mechanical strength and Weibull modulus, despite the partial transformation to the α-TCP phase. Bioactivity, cell viability, and antimicrobial activity improved significantly for the ß-TCP/45S5 scaffold comparing to the scaffold without the bioglass. The mineralization of carbonated hydroxyapatite was verified in Simulated Body Fluid (SBF). The cell viability, evaluated by the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - MTT in MG63 cells, increased by 178%, and ß-TCP/45S5 scaffold also enhanced cell activity and osteoblast differentiation observed by means of total protein contend and alkaline phosphatase activity, respectively. The formation of growth inhibition zones was also observed in the disk diffusion assay for three tested microorganisms: Staphylococcus aureus, Escherichia coli and Candida albicans. To conclude, the vacuum impregnation method in 45S5 bioglass sol gel solution was effective in penetrating all the interconnected macroporosity of the scaffolds and covering the surface of the struts, which improved their biological properties in vitro, bioactivity and antibacterial activity, without reducing mechanical strength and porosity values. Thus, the ß-TCP/45S5 scaffolds are shown as potential candidates for use in tissue engineering, mainly in bone tissue regeneration and recovery.

Bevacizumab intravítreo en el tratamiento de las oclusiones venosas de la retina / Bevacizumab in the treatment of macular edema complicating retinal vein occlusions

INTRODUCCIÓN: Dentro de las vasculopatías retinianas, las oclusiones venosas de la retina (OVR) representan la segunda causa más frecuente de pérdida de visión luego de la retinopatía diabética. Se han descripto dos tipos diferentes de OVR: la oclusión de vena central de la retina (OVCR), y la oclusión de rama venosa retinal (ORVR). De acuerdo al compromiso oclusivo del lecho capilar de la retina, se las clasifica en isquémicas (o no perfundidas), y no isquémicas (perfundidas), presentando una patogénesis, diagnóstico, pronóstico y tratamiento diferente. Se ha demostrado que los niveles de factor de crecimiento vascular endotelial (VEGF) aumentan significativamente en las oclusiones venosas retinianas, lo que tiene como consecuencia una disfunción de la barrera hematorretinal y el aumento de la permeabilidad vascular, con el consecuente edema macular. La inyección intravítrea de bevacizumab, un anticuerpo monoclonal recombinante que actúa en forma directa contra cualquier forma activa de VEGF, ha demostrado ser efectiva para reducir el espesor macular y mejorar la agudeza visual en el tratamiento de OVR. OBJETIVO: Evaluar los cambios en la agudeza visual y en los espesores maculares de pacientes con oclusiones venosas retinianas tratados con inyecciones intravítreas de bevacizumab. Pacientes y métodos: Se evaluaron retrospectivamente 39 pacientes con diagnóstico de oclusión venosa retiniana (OVR) complicada de edema macular y tratados mediante inyección intravítrea de 1,25 mg/0,05 ml bevacizumab (Avastin®, Roche Diagnostic GmbH, Manheim, Alemania), con un seguimiento mínimo de 18 meses. La agudeza visual se evaluó mediante tabla de Snellen y se convirtió en unidades LogMAR. El espesor macular se evaluó mediante tomografía de coherencia óptica (OCT). Según la presentación clínica se las clasificó en: 1. Oclusión de rama (ORVR), incluyendo las oclusiones hemisféricas y 2. Oclusión de vena central (OVCR), incluyendo oclusión venosa hemicentral. A su vez, mediante angiografía fluoresceínica se las clasificó en oclusiones predominántemente isquémicas, y predominántemente no isquémicas. Las variables cuantitativas se analizaron mediante medidas de tendencia central, dispersión y forma. Los cambios en la agudeza visual se calcularon utilizando la prueba de Wilcoxon. Las diferencias entre variables continuas fueron calculadas mediante la prueba T de Student para variables paramétricas, y Mann Whitney para variables no paramétricas. RESULTADOS: La edad promedio fue de 66,8 años (DE: 13,65; rango= 31-92 años). El 55% de los casos estudiados fueron OVCR y el 37,5% ORVR. La media del total de inyecciones durante los 18 meses de seguimiento fue de 4,11±1,61 (rango= 1 a 6). La terapia con láser de rescate se realizó en 10 (25%) pacientes. Todos los pacientes que debieron ser tratados con láser tuvieron una AV inferior a 1 décima al final del seguimiento. La agudeza visual promedio al inicio del tratamiento fue de 1,092 con una desviación estándar (DE) de 0,36, con un rango de 0,10 y 1,60. Tras el inicio del tratamiento, todos los grupos de pacientes logran un incremento significativo en la AV (P= <0,0001). A los 18 meses de tratamiento la agudeza visual promedio fue de 0,739 con una DE de 0,45 con un rango de entre 0,00 y 1,50. La agudeza visual media de los pacientes con OVCR antes del tratamiento fue de 1,09 (DE: 0,35; rango= máxima 0,50 y mínima 1,60), y a los 18 meses de tratamiento de 0,87 (DE: 0,73; rango= 0,20 a 1,50). En cambio, los pacientes con ORVR antes del tratamiento presentaron una media de 1,10 (DE: 0,31; rango= máxima de 0,40 y mínima de 1,6), y luego del tratamiento una media de 0,53 (DE: 0,38; rango= máxima 0,1 y mínima 1,2). El 54,5% de los casos fue de predominio isquémico o mixtos, y el 43,5% fue a predominio edematosos al inicio del tratamiento. La agudeza visual media de los pacientes con tipo edematoso fue de 0,93 (DE: 0,34; rango= máxima 0,10 y mínima 1,40), mientras que los pacientes con patología isquémica presentaron una media de 1,25 (DE: 0,32; rango= máxima de 0,40 y mínima de 1,6). Ambos grupos respondieron en forma significativa al tratamiento (p< 0.05). Sin embargo, los pacientes con predominio edematoso lograron una mejoría mayor que aquellos con predominio isquémico. (P=0.0054) La agudeza visual media de los pacientes con predominio edematoso a los 18 meses del tratamiento fue de 0,50 (DE: 0,44; rango= máxima 0,0 y mínima 1,40), mientras que los pacientes con predominio isquémico presentaron una media de 0,97 (DE: 0,35; rango= máxima 0,20 y mínima 1,4). La media de los espesores maculares previo al inicio del tratamiento fue de 654.19 ± 272.05 micras (rango= 215 a 1497 micras), mientras que la media luego del tratamiento fue de 449 ± 247,62 micras (rango= 140 a 1005 micras) (p=0,0009). No hubo efectos colaterales ni complicaciones locales ni sistémicas atribuibles a la inyección de bevacizumab durante el seguimiento. CONCLUSIÓN: El bevacizumab fue eficaz en el tratamiento del edema macular secundario a OVR. La respuesta terapéutica fue mayor en las ORVR. En nuestra serie no hubo complicaciones oculares ni sistémicas vinculadas al tratamiento. (AU)

INTRODUCTION: Within the retinal vascular disease,retinal venous occlusion (RVO) represents the second most common cause of vision loss after diabetic retinopathy. In turn, it is estimated that the RVO affects approximately 1.6% of people worldwide. It has been described two different types of RVO: the central retinal vein occlusion (CRVO), which includes the hemicentral retinal vein occlusion (HCRVO), and the branch retinal vein occlusion (BRVO), which includes Major BRVO (occlusion of a retinal vein that drains one of the quadrants or more), Minor BRVO (occlusion of a retinal vein that drains less than a quadrant) and Hemispheric retinal vein occlusions (half or more of the retina). According to the occlusive pattern of the retinal capillary bed, this disease is classified as ischemic (or non perfused); when the areas of noncapillary perfusion, seen under fluorescein angiography, have an extension of more than 10 disc diameters; and non-ischemic (o perfused), presenting a pathogenesis, diagnosis, prognosis and different treatment. Non-ischemic form is generally more benign, with a less dramatic visual impairment, a greater chance of spontaneous recovery, a better treatment response, and generally has no risk of neovascularization. Instead; ischemic form is much more severe, causing a more dramatic visual acuity decline, with little chance of clinical improvement, an increased risk of blindness and an increased risk of neovascularization. Although the physiopathogenesis of OVR is not accurately known, it is suggested that the thickening and stiffness of arteries and arterioles caused by arteriosclerosis leads to compression of the retinal veins at the sites where adventitia is shared with arteries (in its intraneural route to the optic nerve and the arteriovenous crossings), leading eventually to a turbulent blood flow, damage to endothelial cells, thrombus formation and eventual vein occlusion. This theory is supported by studies showing pathological structural changes in veins and arteries of patients with VO. Moreover, other factors related to venous deregulation, such as inadequate vasoconstriction and increased vascular permeability, could also play an important role in the retinal venous occlusive phenomena. In some patients, especially young people and adults under 50 years old, inflammatory disorders and hypercoagulable states may contribute to the hysiopathogenesis of the RVO. It has been shown that the presence of vascular endothelial growth factor (VEGF) increased significantly the chances to develop retinal vein occlusions. The result is a blood­retinal barrier dysfunction and an increased vascular permeability, with consequent macular edema. It has also been reported that VEGF plays an important role in the pathogenesis of macular edema in both ORV and CRVO.10 While there is insufficient level 1 evidence to support OVR treatment, multiple therapeutic possibilities have been described, such as anticoagulation, intravitreal fibrinolysis, hemodilution, laser photocoagulation, laser-induced anastomosis, radial optical neurotomy, adventiciotomía, intravitreal injection of triamcinolone acetonide, intravitreal injection of dexamethasone, and injection intravitreal antiangiogenic monoclonal antibodies, such as ranibizumab, bevacizumab, and aflibercept, alone or associated with photocoagulation, have shown efficacy and variables complications rates. Intravitreal injection of bevacizumab, a recombinant monoclonal antibody that acts directly against any active form of VEGF, has been shown to be effective in reducing macular thickness and improving visual acuity in the treatment of OVR. However, the need for multiple injections has been reported since the effects of intravitreal bevacizumab (as well as the other antiangiogenics) are short-lived. On one hand, Bevacizumab has the disadvantage that it is administered off-label; in addition each dose must be extracted from a vial for oncological use, which represents a risk of contamination if strictly rules of asepsis are not taken. On the other hand, the cost of each dose is lower than that of other products (ranibizumab, aflibercept) already approved for intraocular use. This allows that more patients have access to a prolonged treatment in underdeveloped or developing countries, such as Argentina. PURPOSE: To evaluate changes in visual acuity and macular thickness after intravitreal bevacizumab for the treatment of macular edema in patients with retinal vein occlusions (RVO). PATIENS AND METHODS: 39 consecutive patients with macular edema complicating RVO and treated with intravitreal injections of bevacizumab 1.25 mg/0.05 ml alone or associated with scattered peripheral laser photocoagulation, with a minimun follow-up of 18 months, were evaluated. Snellen visual acuity in LogMar units and macular thinckness measured by optical coherence tomography (OCT) were the end points. According to clinical and fluorescein angiographic presentation RVO were classified in branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO), and in non ischemic and ischemic. Wilcoxon test for paired variables, Mann Whitney for independant variables, and Student test for continuous variables, were used for statistical analysis. RESULTS: With a mean age of 66.8 years (SD: 13.65; range= 31-92 years), 62.5% of cases were CRVO and 37.5% BRVO. Mean number of injections was 4.11±161 (range= 1-6). Laser was performed in 25% of patients that did not respond to bevacizumab. At 18 months of follow-up, improvement of visual acuity and macular thickness was statistically significant (p=0.0001 and p=0.0009 respectively). BRVO showed better response to treatment than CRVO. Best visual results were obtained at first month (median= 1.00 P25= 0.5 P75= 1.30) and at 9 month (median= 0.8 P25= 0.5 P75= 1.20) after first injection, but no further improvement was observed beyond 9 months of treatment (p=0.84). No significant visual and macular thickness differences were obtained between patients treated with bevacizumab alone (p=0.116) and bevacizumab and laser (p=0.846). No ocular or systemic attributable-to-treatment side effects were observed. CONCLUSIONS: Bevacizumab was effective in improving visual acuity and reducing macular thinckness in patients with macular edema complicating RVO, especially in BRVO. No ocular or systemic complications were observed during follow-up. (AU)

Estudio del polimorfismo del gen de TNF-α, de dicha citocina, y de MCP-1 en pacientes con queratopatía climática esferoidea / Studies of TNF-α gene polymorphism, TNF-α and MCP-1 cytokines in patients with spheroidal climatic keratopathy

Objetivo. Investigar polimorfismo de nucleótidos únicos (SNP) en la posición -308 (G/A) del gen TNF-α y la participación de las citocinas TNF-α y MCP-1 en pacientes con queratopatía climática esferoidea (QCE) y en controles sanos. Materiales y métodos. Participaron 15 pacientes con QCE y 15 individuos sanos del departamento El Cuy, Provincia de Río Negro. Todos ellos, luego de firmar el consentimiento informado, recibieron un examen oftalmológico completo y se recolectaron muestras de sangre y lágrima para realizar diferentes estudios. EL ADN genómico fue obtenido de sangre de todos los individuos mediante el método de salting out y posteriormente amplificado y estudiado mediante reacción en cadena de la polimerasa (PCR) con el sistema de amplificación refractaria a la mutación (ARMS). También se investigaron concentraciones de algunas citocinas proinflamatorias en lágrimas y en sobrenadante de cultivo de células epiteliales corneales humanas (CECH) tratadas o no con radiación ultravioleta B (RUV-B). Resultados. Los resultados de SNP en la posición -308 (G/A) del gen TNF-α (frecuencia alélica y genotípica) indicaron ausencia de diferencias significativas entre pacientes y controles sanos. Fenotípicamente ambos grupos de individuos serían bajos o intermedios productores in vitro de la citocina TNF-α. Sin embargo en las lágrimas de pacientes con QCE se detectaron concentraciones significativamente superiores de TNF-α, IL-1ß y MCP-1 (citocinas proinflamatorias) que en lágrimas de individuos controles sanos (p<0,0001) En la periferia y limbo de la córnea las células dendríticas (CD) incrementaron significativamente con el progreso de la enfermedad (p<0,05). La contribución del epitelio corneal en el proceso inflamatorio fue investigada utilizando CECH expuestas o no a 10 mJ/cm2 de RUV-B. A pesar de la presencia de gelatinasas, IL-6 e IL-8 en sobrenadantes de cultivos obtenidos a las 48 horas (datos no mostrados) no observamos niveles detectables de TNF-α, IL-1ß ni MCP-1. Conclusión. Este trabajo aporta nuevos datos para aumentar los conocimientos sobre los mecanismos inmunológicos involucrados en la etiopatogenia y progresión de la QCE. Demostramos que las citocinas proinflamatorias MCP-1 y TNF-α están significativamente elevadas en lágrimas de individuos con QCE, como se observó previamente con IL-1ß. MCP-1 sería la responsable del aumento de CD en córnea periférica y limbo de estos pacientes a medida de que la enfermedad avanza. El hallazgo de que estas citocinas no pudieron ser detectadas en cultivos de CECH estresadas con RUV-B implica que otras células son las responsables de su producción o que además de RUV-B otros factores son necesarios para iniciar esta cascada de eventos que se observan en esta hipersensibilidad corneal humana(AU)

Purpose. To investigate Single Nucleotide Polymorphism (SNP) at -308 position (G/A) of TNF-α gen and involving of TNF-α and MCP-1 cytokines in Climatic Droplet Keratopathy (CDK) patients and healthy controls. Materials and methods. Fifteen patients with CDK and fifteen healthy controls from departamento El Cuy, province of Rio Negro were involved in this study. After informed consent was obtained from all participants, they had a complete eye examination and then tear and blood samples were collected to perform different assays. DNA was obtained from blood of all individuals using the method of "salting out" and then amplified and studied performing the polymerase chain reaction (PCR) with Amplification-refractory Mutation System (ARMS). Furthermore, some cytokines concentrations were measured in tears and supernatants from human corneal epithelial cells (HCEs) exposed or not to UVR-B radiation. Results. Analysis from SNP at position -308 (G/A) of TNF-α gen (allelic and genotypic frequency) showed no significant differences between patients and healthy controls. Phenotypically both groups of individuals would be low or intermediate in vitro producers of TNF-α cytokine. However, in tears from CDK's patients we detected significantly higher concentrations of TNF-α, IL-1ß and MCP-1 (pro-inflammatory cytokines) than in healthy control subjects tears (p<0.0001). At the corneal peripheral / limbus area, dendritic cells (DCs) increased significantly with the progression of the disease (p<0.05). The corneal epithelium contribution to the inflammatory process was investigated using HCEs exposed or not to 10 mJ/cm2 of UV radiation­B (UVR-B). Despite the presence of gelatinases, IL-6 and IL-8 in culture supernatants obtained after 48 hours (data not shown), detectable levels of TNF-α, IL-1ß and MCP-1 were not detected. Conclusion. This study provides new insights to increase our knowledge about the immunological mechanisms involved in the etiopathogenesis and progression of CDK. We showed that pro-inflammatory cytokines MCP-1 y TNF-α were significantly increased in tears from CDK's patients, as previously described with IL-1ß. MCP-1 would be responsible for the increasing of DCs on the corneal peripheral / limbus area of these subjects as the disease progresses. The fact that these cytokines could not be detected in cultures of HCEs stressed with UVR-B implies that other cells are responsible for their production or, in addition to UVR-B, other factors are necessary to initiate the cascade of events observed in this human corneal hypersensitivity. (AU)

Poly (epsilon-caprolactone)/propolis extract: microencapsulation and antibacterial activity evaluation.

Spherical and homogenous microparticles of poly(epsilon-caprolactone) (PCL), containing propolis were prepared by the emulsification-solvent evaporation technique. Using this method of preparation, a solid formulation of propolis, free of ethanol and suitable for manipulation and storage, was obtained from an ethanolic extract of propolis. The incorporation efficiency of propolis in the microparticles was almost 30% and around 60% of the substance was released in 48 h. In vitro propolis microparticles exhibited similar halo zones in the Petri plate test against Streptococcus mutans (GS5) with a 10-fold lower concentration than the free propolis extract showing that the encapsulated propolis in microparticles is more efficient as antibiotic.

Biodegradation of the herbicide Diuron in soil by indigenous actinomycetes.

Three actinomycete strains isolated from soil treated with 2,4-D were able to degrade the herbicide Diuron in vitro. Strain CCT 4916 was the most efficient, degrading up to 37% of applied Diuron (100 mg Kg-1 soil) in 7 days, as measured by HPLC and UV/VIS spectroscopy. All strains showed protease and urease activity; intracellular activity of metapyrocatechase and pyrocatechase were not found. Actinomycete strain CCT 4916 produced manganese peroxidase, which could be potentially related to degradation of Diuron.

Stability and chemical modification of xylanase from Aspergillus sp. (2M1 strain).

The 2M1 strain of Aspergillus sp., which showed high extracellular xylanolytic activities in a pre-screening, was studied. Oat-spelt, birch, eucalyptus and pine xylans were used as xylanolytic inductors. The following activities were found at 50 degrees C in the presence of 1% xylan: 120 units/ml (oat-spelt xylan), 132 units/ml (birch xylan), 107 units/ml (eucalyptus xylan), 67 units/ml (pine xylan) and 137 units/ml (larch-wood xylan). Xylanase induced by pine xylan exhibited a higher stability than those induced by the other xylans. The stability was improved by addition of glycerol. In the crude extract, reagents which were found to affect xylanase activity were 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide for amidation of carboxylic groups and N-bromosuccinimide at a concentration of 0.5 mM for indole oxidation. Methylene Blue, butane-2,3-dione, N-acetylimidazole, chloramine-T and iodoacetate had little effect on the enzyme activity (more than 97% of the original activity remained).

Lignin biodegradation by the ascomycete Chrysonilia sitophila.

The lignin biodegradation process has an important role in the carbon cycle of the biosphere. The study of this natural process has developed mainly with the use of basidiomycetes in laboratory investigations. This has been a logical approach since most of the microorganisms involved in lignocellulosic degradation belong to this class of fungi. However, other microorganisms such as ascomycetes and also some bacteria, are involved in the lignin decaying process. This work focuses on lignin biodegradation by a microorganism belonging to the ascomycete class, Chrysonilia sitophila. Lignin peroxidase production and characterization, mechanisms of lignin degradation (lignin model compounds and lignin in wood matrix) and biosynthesis of veratryl alcohol are outstanding. Applications of C. sitophila for effluent treatment, wood biodegradation and single-cell protein production are also discussed.