In vitro expansion of CD4+CD25highFOXP3+CD127low/- regulatory T cells from peripheral blood lymphocytes of healthy Mycobacterium tuberculosis-infected humans.

CD4+CD25highFOXP3+ regulatory T (Treg) cells have recently been found at elevated levels in the peripheral blood of tuberculosis patients, compared to Mycobacterium tuberculosis latently infected (LTBI) healthy individuals and non-infected controls. Here, we show that CD4+CD25highFOXP3+ T lymphocytes can be expanded in vitro from peripheral blood mononuclear cells (PBMC) of LTBI individuals, but not of uninfected controls by incubating them with BCG in the presence of TGF-beta. These expanded cells from the PBMC of LTBI subjects expressed CTLA-4, GITR and OX-40, but were CD127low/- and have therefore the phenotype of Treg cells. In addition, they inhibited in a dose-dependant manner the proliferation of freshly isolated mononuclear cells in response to polyclonal stimulation, indicating that they are functional Treg lymphocytes. In contrast, incubation of the PBMC with BCG alone preferentially induced activated CD4+ T cells, expressing CD25 and/or CD69 and secreting IFN-gamma. These results show that CD4+CD25highFOXP3+ Treg cells can be expanded or induced in the peripheral blood of LTBI individuals in conditions known to predispose to progression towards active tuberculosis and may therefore play an important role in the pathogenesis of the disease.

Clonación de gametas y transgénesis mediada por fertilización in-vitro en bovinos

En la presente tesis se desarrolló un método de clonación del genoma del espermatozoide y del ovocito bovino mediante la producción de embriones androgenéticos y partenogenéticos haploides. Esta técnica también fue utilizada para generar embriones bovinos que expresan un gen exógeno (transgen) en forma homogénea. Las tasas de desarrollo de los embriones reconstruídos utilizando genomas espermaticos clonados (blastomeras androgenéticas), alcanzaron 85.1 por ciento de clivaje, 9 por ciento de blastocistos y todos los embriones expresaron el transgen (EGFP) durante el desarrollo in-vitro. Las tasas de clivaje y de blastocistos de los embriones reconstruídos utilizando genomas clonados de ovocitos (blastomeras partenogenéticas), alcanzaron 78.4 por ciento y 10.8 por ciento respectivamente. Todos los embriones reconstruidos utilizando blastomeras partenogenéticas que expresaban el transgen mostraron expresión de EGFP y el 96.6 por ciento de ellos en forma homogénea. Posteriormente se desarrolló un nuevo método de transgenesis que permite transfectar cigotos de fertilización in vitro (FIV) y ovocitos activados partenogeneticamente (AP). El 70 por ciento de los embriones clivados y el 50 por ciento de los blastocistos expresaron EGFP cuando complejos pCX-EGFP-liposomas fueron inyectados 16 h post-fertilizacion y utilizando una concentración de 500 ng ADN exógeno.ƒÊl. Al inyectar ovocitos 3 h post-activación partenogénetica se obtuvo una tasa de expresión de 48.4 por ciento. Por otro lado, evaluamos la incidencia de fragmentación del ADN tras la inyección del transgen, demostrando que su expresión afecta la integridad del ADN en blastocistos bovinos de FIV, pero no así las tasas de desarrollo in vitro. En resumen, la presente tesis conforma una base sólida para concluir que es posible la clonación de genomas de ovocitos y espermatozoides con capacidad de generar embriones biparentales que evolucionan hasta estadio de blastocisto. Además, este procedimiento demostró ser una herramienta eficiente para la incorporación de genes exógenos en un embrión. Finalmente se demostró que la inyección intracitoplasmática de liposomas es una estrategia eficiente para introducir ADN exógeno en embriones de FIV y AP.

Efect of Nostoc muscorum Ag. on germination in vitro of Bremia lactucae sporangia in Lactuca sativa L. Regel

p.1-8

Microscopia electrónica de barrido en cultivos in vitro de embriones inmaduros y mesocotilos de plántulas de maíz

p.95-105

Eficiencia in vitro de antagonistas de Rhizoctonia solani y Sclerotium rolfsii y análisis camparativo de distintos modelos de crecimiento

p.59-65

Efect of Nostoc muscorum Ag. on germination in vitro of Bremia lactucae sporangia in Lactuca sativa L. Regel

p.1-8

Influencia de distintas citocininas en el cultivo in vitro de apices caulinares de Fragaria x ananassa y F. virginiana

p.65-70

Aspects of the in vitro bioactivity and antimicrobial properties of Ag+- and Zn2+-exchanged 11 Å tobermorites

11 Å tobermorite, Ca5Si6O16(OH)2 · 4H2O, is a layer lattice ion exchange mineral whose potential as a carrier for Ag+ and Zn2+ ions in antimicrobial, bioactive formulations has not yet been explored. In view of this, the in vitro bioactivity of Ag+- and Zn2+-exchanged 11 Å tobermorites and their bactericidal action against S. aureus and P.aeruginosa are reported. The in vitro bioactivity of the synthetic unsubstituted tobermorite phase was confirmed by the formation of bone-like hydroxycarbonate apatite (HCA) on its surface within 48 h of contact with simulated body fluid. The substitution of labile Ag+ ions into the tobermorite lattice delayed the onset of HCA-formation to 72 h; whereas, the Zn2+-substituted phase failed to elicit an HCA-layer within 14 days. Both Ag+- and Zn2+-exchanged tobermorite phases were found to exhibit marked antimicrobial action against S. aureus and P.aeruginosa, two common pathogens in biomaterial-centred infections.

Aspects of the in vitro bioactivity of hydraulic calcium (alumino)silicate cement

In response to a burgeoning interest in the prospective clinical applications of hydraulic calcium (alumino)silicate cements, the in vitro bioactivity and dissolution characteristics of a white Portland cement have been investigated. The formation of an apatite layer within 6 h of contact with simulated body fluid was attributed to the rapid dissolution of calcium hydroxide from the cement matrix and to the abundance of pre-existing Si-OH nucleation sites presented by the calcium silicate hydrate phase. A simple kinetic model has been used to describe the rate of apatite formation and an apparent pseudo-second-order rate constant for the removal of HPO42- ions frorn solultion has been calculated (k(2) = 5.8 x 10(-4) g mg(-1)). Aspects of the chemistry of hydraulic cements are also discussed with respect to their potential use in the remedial treatment of living tissue. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 90A: 166-174, 2009

Synthesis of a 1,4-benzodiazepine containing palladacycle with in vitro anticancer and cathepsin B activity

The reaction of the five-membered C,N-palladacycle [(L)PdCl](2), where LH = 1-methyl-5-phenyl-1H-1,4-benzodiazepin-2(3H)-one, with 1,2-ethanebis(diphenylphosphine), dppe, leads to the formation of the bridged palladacycle. [Pd(2)L(2)(mu-dppe)Cl(2)] 3, which was characterised in solution by (1)H and (31)P NMR spectroscopy and in the solid state by X-ray crystallography. Complex 3 was tested in vitro against a number of cell lines. For example, it inhibited K562 leukaemia cells with an IC(50) value of 4.3 microM (1 h exposure) and displayed cathepsin B inhibitory action with an IC(50) value of 3 microM.

Intracellular fate of bioresponsive poly(amidoamine)s in vitro and in vivo

Linear poly(amidoamine)s (PAAs) have been designed to exhibit minimal non-specific toxicity, display pH-dependent membrane lysis and deliver genes and toxins in vitro. The aim of this study was to measure PAA cellular uptake using ISA1-OG (and as a reference ISA23-OG) in B16F10 cells in vitro and, by subcellular fractionation, quantitate intracellular trafficking of (125)I-labelled ISA1-tyr in liver cells after intravenous (i.v.) administration to rats. The effect of time after administration (0.5-3h) and ISA1 dose (0.04-100mg/kg) on trafficking, and vesicle permeabilisation (N-acetyl-b-D-glucosaminidase (NAG) release from an isolated vesicular fraction) were also studied. ISA1-OG displayed approximately 60-fold greater B16F10 cell uptake than ISA23-OG. Passage of ISA1 along the liver cell endocytic pathway caused a transient decrease in vesicle buoyant density (also visible by TEM). Increasing ISA1 dose from 10mg/kg to 100mg/kg increased both radioactivity and NAG levels in the cytosolic fraction (5-10 fold) at 1h. Moreover, internalised ISA1 provoked NAG release from an isolated vesicular fraction in a dose-dependent manner. These results provide direct evidence, for the first time, of PAA permeabilisation of endocytic vesicular membranes in vivo, and they have important implications for potential efficacy/toxicity of such polymeric vectors.

In vitro-in vivo correlations of self-emulsifying drug delivery systems combining the dynamic lipolysis model and neuro-fuzzy networks

The aim of the current study was to evaluate the potential of the dynamic lipolysis model to simulate the absorption of a poorly soluble model drug compound, probucol, from three lipid-based formulations and to predict the in vitro-in vivo correlation (IVIVC) using neuro-fuzzy networks. An oil solution and two self-micro and nano-emulsifying drug delivery systems were tested in the lipolysis model. The release of probucol to the aqueous (micellar) phase was monitored during the progress of lipolysis. These release profiles compared with plasma profiles obtained in a previous bioavailability study conducted in mini-pigs at the same conditions. The release rate and extent of release from the oil formulation were found to be significantly lower than from SMEDDS and SNEDDS. The rank order of probucol released (SMEDDS approximately SNEDDS > oil formulation) was similar to the rank order of bioavailability from the in vivo study. The employed neuro-fuzzy model (AFM-IVIVC) achieved significantly high prediction ability for different data formations (correlation greater than 0.91 and prediction error close to zero), without employing complex configurations. These preliminary results suggest that the dynamic lipolysis model combined with the AFM-IVIVC can be a useful tool in the prediction of the in vivo behavior of lipid-based formulations.

In vitro release of bovine serum albumin from alginate/HPMC hydrogel beads

In recent years, the use of swelling polymeric matrices for the encapsulation and controlled release of protein drugs has received significant attention. The purpose of the present study was to investigate the release of albumin, a model protein from alginate/hydroxypropyl-methylcellulose (HPMC) gel beads. A hydrogel system comprised of two natural, hydrophilic polymers; sodium alginate and HPMC was studied as a carrier of bovine serum albumin (BSA) which was used as a model protein. The morphology, bead size and the swelling ratio were studied in different physical states; fully swollen, dried and reswollen using scanning electron microscopy and image analysis. Finally the effect of different alginate/HPMC ratios on the BSA release profile in physiological saline solution was investigated. Swelling experiments revealed that the bead diameter increases with the viscosity of the alginate solution while the addition of HPMC resulted in a significant increase of the swelling ratio. The BSA release patterns showed that the addition of HPMC increased the protein-release rate while the release mechanism fitted the Peppas model. Alginate/HPMC beads prepared using the ionic gelation exhibited high BSA loading efficiency for all formulations. The presence of HPMC increased the swelling ability of the alginate beads while the particle size remained unaffected. Incorporation of HPMC in the alginate gels also resulted in improved BSA release in physiological saline solution. All formulations presented a non-Fickian release mechanism described by the Peppas model. In addition, the implementation of non-parametric tests showed significant differences in the release patterns between the alginate/HPMC and the pure alginate beads, respectively.

A preliminary investigation of the in vitro bioactivity of white Portland cement

Freshly-mixed and partially-cured ordinary Portland cement (OPC) pastes have been shown to exhibit good biological compatibility with a range of cells and tissue-types; particularly those associated with bone formation. Formulations based on OPC have been used as dental restoratives and are now being investigated for their potential use in orthopaedic repair. Despite the current clinical interest in OPCs, very little is known about their chemistry in the physiological environment. In this respect, research to investigate aspects of the interactions between a white Portland cement (WPC) paste and simulated body fluid (SBF) has been carried out in vitro. Exposure to SBF has been found to promote the precipitation of a layer of 'bone-like' hydroxyapatite on the surface of WPC paste which underpins its ability to integrate with living tissue. The dissolution of portlandite and formation of calcite were also observed on contact with SBF.

Evaluating the process of borosilicate glass capillaries used for fabrication of in-vitro fertilization (IVF) micro-pipettes

In this paper we investigate a number of gas flames for fire polishing borosilicate glass capillaries used in the manufacturing of IVF micro-pipettes. Hydrofluoric acid (HF) was also used as an alternative to finish the pipette end. Glass micro tools in the IVF industry are drawn from hollow glass capillaries of diameter 1 mm. These capillaries are cut manually to a length of 100 mm from hollow glass rods resulting in sharp and chipped edges. These capillaries are held in a customised holder having padding of soft silicone or rubber. Sharp and uneven edges of these capillaries pick up particles of rubber or soft silicone shavings, rendering them ineffective for IVF treatments. The working range of borosilicate glass is 800-1,200 degrees C. The experiments involved analysis of fire polishing process for borosilicate glass capillaries using candle, butane, propane, 2350 butane propane, oxyacetylene gas flames, finding the optimum distance of the capillary relative to the flame, optimum time for which the capillary should be held in the flame and optimum region of the flame which gives the required temperature range. The results show that 2350 butane propane gas mix is optimum for fire polishing of borosilicate glass capillaries. The paper is concluded by comparing the results of fire polishing with the results of acid polishing, in which HF of 1.6% concentration is used to etch the ends of the borosilicate glass pipettes.