Drifter technique: a new method to obtain metaphases in Hep-2 cell line cultures

A linhagem celular Hep-2 é formada por células de carcinoma da laringe e é muito utilizada em modelos de carcinogênese e mutagenêse. Para avaliar o potencial proliferativo desta linhagem, desenvolvemos uma metodologia citogenética (técnica do sobrenadante) para obtenção de metáfases a partir de células que, ao entrarem em mitose, perdem adesão celular, ficando em suspensão no meio de cultura. Através deste procedimento, foram contadas 2000 células, correspondendo a um índice mitótico (IM) de 22.2% . Apesar de o IM obtido por esta técnica não ter sido estatisticamente diferente do IM obtido por outras metodologias citogenéticas clássicas, a técnica do sobrenadante é vantajosa porque elimina o uso de alguns reagentes utilizados na obtenção de metáfases e também diminui o consumo de reagentes de manutenção desta linhagem.

Transport of amino acids from milk whey by Caco-2 cell monolayer after hydrolytic action of gastrointestinal enzymes

The bioavailability of amino adds from milk whey protein hydrolysates was evaluated using diffusion of the substances through semi-permeable membranes (dialyzability) and transport by Caco-2 cell cultures. The hydrolysates with low degree of hydrolysis (LDH) and high degree of hydrolysis (HDH) were obtained after 120 min of reaction time at 50 degrees C after the initial addition of pepsin, followed by the addition of trypsin, chymotrypsin and carboxypeptidase-A. The proteins and hydrolysates were further subjected to in vitro digestion with pepsin plus pancreatin. HPLC was used to determine the concentrations of dialyzable amino adds (48.4% of the non-hydrolyzed proteins, 63.2% of the LDH sample and 58.3% of the HDH sample), demonstrating the greater dialyzability of the hydrolysates. The LDH and HDH whey protein hydrolysates prepared with pepsin, trypsin, chymotrypsin and carboxypeptidase-A showed only 14.7% and 20.8% of dialyzable small peptides and amino acids, respectively. The efficiency of absorption was demonstrated by the preferential transport of Ile, Lou and Arg through a layer of cells. In the LDH hydrolysate, Tyr was also transported. Prior high- and low-degree hydrolysis of the whey provided transport by 5.7% and 6.6%, respectively, in comparison with 23% for non-hydrolyzed proteins, considering the total amount of these amino adds that was applied to the cells. (C) 2014 Elsevier Ltd. All rights reserved.

Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins.

Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin-B1 was displayed on a reconstituted surface. EphB1-ephrin-B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through alphavbeta3 integrin. EphB1 transfection conferred ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants failed to stimulate ephrin-B1 dependent attachment. These findings lead us to propose that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment.

T-cell reprogramming through targeted CD4-coreceptor and T-cell receptor expression on maturing thymocytes by latent Circoviridae family member porcine circovirus type 2 cell infections in the thymus

Although porcine circovirus type 2 (PCV2)-associated diseases have been evaluated for known immune evasion strategies, the pathogenicity of these viruses remained concealed for decades. Surprisingly, the same viruses that cause panzootics in livestock are widespread in young, unaffected animals. Recently, evidence has emerged that circovirus-like viruses are also linked to complex diseases in humans, including children. We detected PCV2 genome-carrying cells in fetal pig thymi. To elucidate virus pathogenicity, we developed a new pig infection model by in vivo transfection of recombinant PCV2 and the immunosuppressant cofactor cyclosporine A. Using flow cytometry, immunofluorescence and fluorescence in situ hybridization, we found evidence that PCV2 dictates positive and negative selection of maturing T cells in the thymus. We show for the first time that PCV2-infected cells reside at the corticomedullary junction of the thymus. In diseased animals, we found polyclonal deletion of single positive cells (SPs) that may result from a loss of major histocompatibility complex class-II expression at the corticomedullary junction. The percentage of PCV2 antigen-presenting cells correlated with the degree of viremia and, in turn, the severity of the defect in thymocyte maturation. Moreover, the reversed T-cell receptor/CD4-coreceptor expression dichotomy on thymocytes at the CD4(+)CD8(interm) and CD4SP cell stage is viremia-dependent, resulting in a specific hypo-responsiveness of T-helper cells. We compare our results with the only other better-studied member of Circoviridae, chicken anemia virus. Our data show that PCV2 infection leads to thymocyte selection dysregulation, adding a valuable dimension to our understanding of virus pathogenicity.

Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.

This paper describes a method based on experimentally simple techniques--microcontact printing and micromolding in capillaries--to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. The technique uses replica molding in poly(dimethylsiloxane) molds having micrometer-scale relief patterns on their surfaces to form a contoured film of polyurethane supported on a glass slide. Evaporation of a thin (< 12 nm) film of gold on this surface-contoured polyurethane provides an optically transparent substrate, on which SAMs of terminally functionalized alkanethiolates can be formed. In one procedure, a flat poly(dimethylsiloxane) stamp was used to form a SAM of hexadecanethiolate on the raised plateaus of the contoured surface by contact printing hexadecanethiol [HS(CH2)15CH3]; a SAM terminated in tri(ethylene glycol) groups was subsequently formed on the bare gold remaining in the grooves by immersing the substrate in a solution of a second alkanethiol [HS(CH2)11(OCH2CH2)3OH]. Then this patterned substrate was immersed in a solution of fibronectin, the protein adsorbed only on the methyl-terminated plateau regions of the substrate [the tri(ethylene glycol)-terminated regions resisted the adsorption of protein]; bovine capillary endothelial cells attached only on the regions that adsorbed fibronectin. A complementary procedure confined protein adsorption and cell attachment to the grooves in this substrate.

Permeability studies of alkylamides and caffeic acid conjugates from echinacea using a Caco-2 cell monolayer model

Background: Echinacea is composed of three major groups of compounds that are thought to be responsible for stimulation of the immune system-the caffeic acid conjugates, alkylamides and polysaccharides. This study has focussed on the former two classes, as these are the constituents found in ethanolic liquid extracts. Objective: To investigate the absorption of these two groups of compounds using Caco-2 monolayers, which are a model of the intestinal epithelial barrier. Results: The caffeic acid conjugates (caftaric acid, echinacoside and cichoric acid) permeated poorly through the Caco-2 monolayers although one potential metabolite, cinnamic acid, diffused readily with an apparent permeability (P-app) of 1x10(-4) cm/s. Alkylamides were found to diffuse through Caco-2 monolayers with P-app ranging from 3x10(-6) to 3x10(-4) cm/s. This diversity in P-app for the different alkylamides correlates to structural variations, with saturation and N-terminal methylation contributing to decreases in P-app. The transport of the alkylamides is not affected by the presence of other constituents and the results for synthetic alkylamides were in line with those for the alkylamides in the echinacea preparation. Conclusion: Alkylamides but not caffeic acid conjugates are likely to cross the intestinal barrier.

Interaction of peptides, peptidomimetics and other drug candidates with the DI/tripeptide transport system in intestinal epithelial cells, using the in vitro caco-2 cell culture system

An uptake system was developed using Caco-2 cell monolayers and the dipeptide, glycyl-[3H]L-proline, as a probe compound. Glycyl-[3H]L-proline uptake was via the di-/tripeptide transport system (DTS) and, exhibited concentration-, pH- and temperature-dependency. Dipeptides inhibited uptake of the probe, and the design of the system allowed competitors to be ranked against one another with respect to affinity for the transporter. The structural features required to ensure or increase interaction with the DTS were defined by studying the effect of a series of glycyl-L-proline and angiotensin-converting enzyme (ACE)-inhibitor (SQ-29852) analogues on the uptake of the probe. The SQ-29852 structure was divided into six domains (A-F) and competitors were grouped into series depending on structural variations within specific regions. Domain A was found to prefer a hydrophobic function, such as a phenyl group, and was intolerant to positive charges and H+ -acceptors and donors. SQ-29852 analogues were more tolerant of substitutions in the C domain, compared to glycyl-L-proline analogues, suggesting that interactions along the length of the SQ-29852 molecule may override the effects of substitutions in the C domain. SQ-29852 analogues showed a preference for a positive function, such as an amine group in this region, but dipeptide structures favoured an uncharged substitution. Lipophilic substituents in domain D increased affinity of SQ-29852 analogues with the DTS. A similar effect was observed for ACE-NEP inhibitor analogues. Domain E, corresponding to the carboxyl group was found to be tolerant of esterification for SQ-29852 analogues but not for dipeptides. Structural features which may increase interaction for one series of compounds, may not have the same effect for another series, indicating that the presence of multiple recognition sites on a molecule may override the deleterious effect of anyone change. Modifying current, poorly absorbed peptidomimetic structures to fit the proposed hypothetical model may improve oral bioavailability by increasing affinity for the DTS. The stereochemical preference of the transporter was explored using four series of compounds (SQ-29852, lysylproline, alanylproline and alanylalanine enantiomers). The L, L stereochemistry was the preferred conformation for all four series, agreeing with previous studies. However, D, D enantiomers were shown in some cases to be substrates for the DTS, although exhibiting a lower affinity than their L, L counterparts. All the ACE-inhibitors and β-lactam antibiotics investigated, produced a degree of inhibition of the probe, and thus show some affinity for the DTS. This contrasts with previous reports that found several ACE inhibitors to be absorbed via a passive process, thus suggesting that compounds are capable of binding to the transporter site and inhibiting the probe without being translocated into the cell. This was also shown to be the case for oligodeoxynucleotide conjugated to a lipophilic group (vitamin E), and highlights the possibility that other orally administered drug candidates may exert non-specific effects on the DTS and possibly have a nutritional impact. Molecular modelling of selected ACE-NEP inhibitors revealed that the three carbonyl functions can be oriented in a similar direction, and this conformation was found to exist in a local energy-minimised state, indicating that the carbonyls may possibly be involved in hydrogen-bond formation with the binding site of the DTS.

Investigation of the absorption pathways of some poorly absorbed drugs using human intestinal (CAC0-2) cell monolayers

Absorption across the gastro-intestinal epithelium is via two pathways; the transcellular and paracellular pathway. Caco-2 cells, when cultured on polycarbonate filters, formed a confluent monolayer with many properties of differentiated intestinal epithelial cells, As a model of human gastro-intestinaJ tract epithelia they were used to elucidate and characterise the transepithelial transport of two protein kinase C inhibitors, N-(3-chlorophenyl)-4-[2-(3-hydroxypropylamino)-4-pyridyl]-2-pyrimidinamin (CHPP) and N-benzoyl-staurosporine (NBS), and the polypeptide, human calcitonin. Lanthanum ions are proposed as a paracellular pathway inhibitor and tested with D-mannitol permeability and transepithelial electrical resistance measurements. The effect La3+ has on the carrier-mediated transport of D-glucose and Sodium taurocholate as well as the vesicularly transcytosed horseradish peroxidase was also investigated. As expected, 2 mM apical La3+ increases transepithelial electrical resistance 1.S-fold and decreases mannitol permeability by 63.0 % ± 1.37 %. This inhibition was not repeated by other cations. Apical 2 mM La3+ was found to decrease carrier-mediated D-glucose and taurocholate permeability by only 8.7 % ± 1.6 %, 26.3 % ± 5.0 %. There was no inhibitory effect on testosterone or PEG 4000 permeability observed with La3+. However, for horseradish peroxidase and human calcitonin permeability was decreased by 98.7 % ± 11.7%, and 96.2 % ± 0.8 % respectively by 2 mM La3+. Indicating that human calcitonin could also be transported by vesicular transcytosis. The addition of 2 mM La3+ to the apical surface of Caco-2 monolayers produces a paracellular pathway inhibition. Therefore, La3+ could be a useful additional tool in delineating the transepithelial pathway of passive drug absorption.

Uptake and transport of orally-deliverable drugs across caco-2 cell monolayers:the effect of lipid formulations

The aim of this thesis is to investigate the physicochemical parameters which can influence drug loading within liposomes and to characterise the effect such formulations have on drug uptake and transport across in vitro epithelial barrier models. Liposomes composed of phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC) and cholesterol (0, 4, 8, 16 µM) were prepared and optimised in terms of drug loading using the hand-shaking method (Bangham et al., 1965). Subsequently, liposomes composed of 16 µM PC or DSPC and cholesterol (4 µM) were used to monitor hydroxybenzoate release and transport from Iiposomes. The MIT (3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and crystal violet assays were employed to determine toxicity of the Iiposome. formulations towards the Caco-2 cell line, employed to model the epithelial barrier in vitro. Uptake and transport of mannitol, propranolol, glutamine and digoxin was measured in the presence and absence of Iiposome formulations to establish changes in absorption resulting from the presence of lipid formulations. Incorporation of the four hydroxybenzoates was shown to be influenced by a number of factors, including liposome composition and drug conformation. Methyl hydroxybenzo.ate (MP) was incorporated into the bilayer most effectively with percentage incorporation of 68% compared to 45% for butyl hydroxybenzoate (BP), despite its increased Iipophilicity. This was attributed to the decreased packing ability of BP within the hydrocarbon core of the lipid bilayer compared to MP. Release studies also suggested that the smaller MP was more strongly incorporated within the lipid bilayer with only 8% of the incorporated solute being released after 48-hours compared to 17% in the case of BP. Model transport studies were seen to reflect drug release profiles from the liposome bilayers with significantly (p < 0.01) higher amounts of BP partitioning from the liposome compared to MP, Caco-2 cell viability was maintained above 86% in the presence of all Iiposome formulations tested indicating the liposome formulations are non-toxic towards Caco-2 cells. Paracellular (apical-to-basolateral) transport of mannitol was significantly increased in the presence of DSPC, PC / DSPC:Cholesterol (16:4 µM; 1000 µg). Glutamine uptake and transport via the carrier-mediated route was Significantly (p < 0.01) increased in the presence of PC I DSPC:Cholesterol (16:0; 16:4 µM). Digoxin apical-to-basolateral transport was significantly increased (p < 0,01) in the presence of PC / DSPC:Cholesterol (16:0; 16:4 µM); thus reducing digoxin efflux via P-glycoprotein. In contrast, PC:ChoJesterol (16:0; 16:4 µM) significantly (p < 0.01) decreased propranolol uptake via the passive transcellular route. Bi-directional transport of propranolol was significantly (p < 0,01) decreased in the presence of PC/DSPC:Cholesterol (16:0; 16:4 µM). The structure of a solute is an important determinant for the incorporation and release of a solute from liposome formulations. PC, DSPC and cholesterol liposome formulations are nontoxic towards Caco-2 cell monolayers and improved uptake and transport of mannitol, glutamine. and digoxin across Caco-2 cell monolayers; thus providing a potential alternative delivery vehicle.

Interaction of quinolone antibiotics with the human intestinal CACO-2 cell model

The transport of a group of quinolone antibiotics across the human intestinal model, Caco-2 cells, was investigated. It was found that the transport of the quinolones generally correlated with the lipophilicity of the compounds, indicating the passive diffusional transcellular processes were involved. However, it was observed that the transport in both directions apical-to-basolateral and basolateral-to-apical was not equivalent, and polarised transport occurred. For all the quinolones studied except, BMS-284756-01, it was found that the basolateral-to-apical transport was significantly greater than the apical-to-basolateral transport. This finding suggested that the quinolones underwent a process of active secretion. The pKas and logPs for the quinolones were determined using potentiometric titrations. The measured logP values were compared with those determined using theoretical methods. The theoretical methods for calculating logP including the Moriguchi method correlated poorly with the measured logP values. Further investigations revealed that there may be an active transporter involved in the apical-to-basolateral transport of quinolones as well. This mechanism was sensitive to competing quinolones, but, it was unaffected by the metabolic inhibitor combination of sodium azide (15mM) with 2-deoxy-D-glucose (50mM). The basolateral-to-apical transport of quinolones was found to be sensitive to inhibition by a number of different inhibitors. The metabolic inhibitors, sodium azide (15mM) with 2-deoxy-D-glucose (50mM) and 2,4-dinitrophenol (1mM), were able to reduce the basolateral-to-apical transport of quinolones. A reduction in temperature from 37°C to 2°C caused an 80-fold decrease in the transport of gatifloxacin in both directions, however, this effect was not sufficient to abolish the greater basolateral-to-apical secretion. As with apical-to-basolateral transport, it was found that quinolones competed with gatifloxacin for basolateral-to-apical transport, both ofloxacin (100μM) and norfloxacin (100μM) significantly (P<0.003) decreased the basolateral-to-apical transport of gatifloxacin; however, ciprofloxacin (100μM and 300μM) had no effect. A number of inhibitors of various transport systems were also investigated. It was found that the anion transport inhibitor, probenecid (100 μM) had a significant inhibitory effect on the basolateral-to-apical transport of ciprofloxacin (P=0.039), while the cation transport inhibitor cimetidine (100μM and 500μM) had no effect. The organic anion exchange inhibitor 4,4'diisothiocyanostilbene-2-2' -disulphonic acid DIDS (400μM) also had a significant inhibitory effect (P=O.O 13). The PgP inhibitor and anion exchange inhibitor verapamil (400Mμ) was able to completely abolish the basolateral-to-apical secretion of gatifloxacin and bring it into line with the apical-to-basolateral flux. In conclusion, the apical-to-basolateral and basolateral-toapical transport of quinolones involved an active component. The basolateral-to-apical secretion was abolished by a verapamil (400μM), a bisubstrate for PgP and the anion transporter.

Efficacy of Stanozolol on bone regeneration: in vitro and in vivo study

La ricerca di nuove strategie per la rigenerazione ossea rappresenta un focus di interesse centrale per migliorare la gestione di casi clinici complessi nell’ambito della chirurgia orale e maxillo-facciale. Uno degli approcci più utilizzati in tale contesto si basa sull’utilizzo di molecole con proprietà osteoinduttive e molte sostanze sono state fino ad oggi sperimentate. E’ noto in letteratura che gli androgeni svolgono un ruolo chiave nella regolazione della morfogenesi ossea e nel mantenimento della sua omeostasi durante il corso della vita. Questo lavoro di tesi nasce dall’ipotesi che la somministrazione locale di tali ormoni, eventualmente combinata a materiali da innesto, possa favorire la guarigione di difetti ossei. Stando a questa premessa, sono stati valutati gli effetti dello steroide sintetico Stanozololo sulla rigenerazione ossea in diversi settings sperimentali. La tesi è strutturata secondo un percorso che segue le fasi della ricerca, attraverso sperimentazioni in vitro e in vivo; ogni capitolo può essere approcciato come uno studio a sé stante, corrispondente ad una determinata tappa dell’iter sperimentale. Sulla base di questi intenti, viene fornito inizialmente un quadro d’insieme circa gli effetti degli androgeni sull’osso. A seguire, è presentata una sperimentazione in vitro nella linea cellulare SaOS-2. Infine, è proposta un’innovativa metodologia di analisi per lo studio della rigenerazione ossea nel modello di ratto, ove viene testata la somministrazione locale di Stanozololo combinato a materiale da innesto.