A new principle for tight junction modulation based on occludin peptides

The aim of this study was to investigate whether peptides from the extracellular loops of the tight junction protein occludin could be used as a new principle for tight junction modulation. Peptides of 4 to 47 amino acids in length and covering the two extracellular loops of the tight junction protein occludin were synthesized, and their effect on the tight junction permeability in Caco-2 cells was investigated using [C-14] mannitol as a paracellular marker. Lipopeptide derivatives of one of the active occludin peptides (OPs), synthesized by adding a lipoamino acid containing 14 carbon atoms (C-14-) to the N terminus of the peptide, were also investigated. Peptides corresponding to the N terminus of the first extracellular loop of occludin increased the permeability of the tight junctions without causing short-term toxicity. However, the peptides had an effect only when added to the basolateral side of the cells, which could be partly explained by degradation by apical peptidases and aggregate formation. By contrast, the lipopeptide C-14-OP90-103, which protects the peptide from degradation and aggregation, displayed a rapid apical effect. The L- and D-diastereomers of C-14-OP90-103 had distinctly different effects. The D-isomer, which releases intact OP90-103 from the lipoamino acid, displayed a rapid and transient increase in tight junction permeability. The L- isomer, which releases OP90-103 more rapidly, gave a more sustained increase in tight junction permeability. In conclusion, C-14-OP90-103 represents a prototype of a new class of tight junction modulators that act on the extracellular domains of tight junction proteins.

In vitro bioactivity of MOEP grafted ePTFE membranes for craniofacial applications

The bioactivity of three methacryloyloxyethyl phosphate (MOEP) grafted expanded polytetrafluoroethylene (ePTFE) membranes with varying surface coverage as well as unmodified ePTFE was investigated through a series of in vitro tests: calcium phosphate (CaP) growth in simulated body fluid (SBF), serum protein adsorption, and a morphology and attachment study of human osteoblast-like SaOS-2 cells. The graft copolymers were prepared by means of gamma irradiation induced grafting and displayed various surface morphologies and wettabilities depending on the grafting conditions used. Unmodified ePTFE did not induce nucleation of Cal? minerals, whereas all the grafted membranes revealed the growth of Cal? minerals after 7 days immersion in SBF. The sample with lowest surface grafting yield (24% coverage), a smooth graft morphology and relatively high hydrophobicity (theta(adv) = 120 degrees, theta(rec) = 80 degrees) showed carbonated hydroxyapatite growth covering the surface. On the other hand, the samples with high surface grafting yield (76% and 100%), a globular graft morphology and hydrophilic surfaces (theta(adv) = 60 degrees and 80 degrees, theta(rec) = 25 degrees and 15 degrees, respectively) exhibited irregular growth of non-apatitic Cap minerals. Irreversibly adsorbed protein measured after a 1 h immersion in serum solution was quantified by the amount of nitrogen on the surface using XPS, as well as by weight increase. All grafted membranes adsorbed 3-6 times more protein than the unmodified membrane. The sample with the highest surface coverage adsorbed the most protein. Osteoblast-like SaOS-2 cells cultured for 3 h revealed significantly higher levels of cell attachment on all grafted membranes compared to unmodified ePTFE. Although the morphology of the cells was heterogeneous, in general, the higher grafted surfaces showed a much better cell morphology than both the low surface-grafted and the control unmodified sample. The suite of in vitro tests confirms that a judicious choice of grafted monomer such as the phosphate-containing methacrylate monomer (MOEP) significantly improves the bioactivity of ePTFE in vitro. (c) 2005 Elsevier Ltd. All rights reserved.

Pax9 and Jagged1 act downstream of Gli3 in vertabrate limb development

From early in limb development the transcription factor Gli3 acts to define boundaries of gene expression along the anterior-posterior (AP) axis, establishing asymmetric patterns required to provide positional information. As limb development proceeds, posterior mesenchyme expression of Sonic hedgehog (Shh) regulates Gli3 transcription and post-translational processing to specify digit number and identity. The molecular cascades dependent on Gli3 at later stages of limb development, which link early patterning events with final digit morphogenesis, remain poorly characterised. By analysing the transcriptional consequences of loss of Gli3 in the anterior margin of the E11.5 and E12.5 limb bud in the polydactylous mouse mutant extra-toes (Gli3(Xt/Xt)), we have identified a number of known and novel transcripts dependent on Gli3 in the limb. In particular, we demonstrated that the genes encoding the paired box transcription factor Pax9, the Notch ligand Jagged1 and the cell surface receptor Cdo are dependent on Gli3 for correct expression in the anterior limb mesenchyme. Analysis of expression in compound Shh;Gli3 mutant mouse embryos and in both in vitro and in vivo Shh signaling assays, further defined the importance of Shh regulated processing of Gli3 in controlling gene expression. In particular Pax9 regulation by Shh and Gli3 was shown to be context dependent, with major differences between the limb and somite revealed by Shh bead implantation experiments in the chick. Jagged1 was shown to be induced by Shh in the chick limb and in a C3H10T1/2 cell based signaling assay, with Shh;Gli3 mutant analysis indicating that expression is dependent on Gli3 derepression. Our data have also revealed that perturbation of early patterning events within the Gli3(Xt/Xt), limb culminates in a specific delay of anterior chondrogenesis which is subsequently realised as extra digits. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Gravity spun polycaprolactone fibers for applications in vascular tissue engineering:proliferation and function of human vascular endothelial cells

Poly(ε-caprolactone) (PCL) fibers produced by wet spinning from solutions in acetone under low-shear (gravity-flow) conditions resulted in fiber strength of 8 MPa and stiffness of 0.08 Gpa. Cold drawing to an extension of 500% resulted in an increase in fiber strength to 43 MPa and stiffness to 0.3 GPa. The growth rate of human umbilical vein endothelial cells (HUVECs) (seeded at a density of 5 × 104 cells/mL) on as-spun fibers was consistently lower than that measured on tissue culture plastic (TCP) beyond day 2. Cell proliferation was similar on gelatin-coated fibers and TCP over 7 days and higher by a factor of 1.9 on 500% cold-drawn PCL fibers relative to TCP up to 4 days. Cell growth on PCL fibers exceeded that on Dacron monofilament by at least a factor of 3.7 at 9 days. Scanning electron microscopy revealed formation of a cell layer on samples of cold-drawn and gelatin-coated fibers after 24 hours in culture. Similar levels of ICAM-1 expression by HUVECs attached to PCL fibers and TCP were measured using RT-PCR and flow cytometry, indicative of low levels of immune activation. Retention of a specific function of HUVECs attached to PCL fibers was demonstrated by measuring their immune response to lipopolysaccharide. Levels of ICAM-1 expression increased by approximately 11% in cells attached to PCL fibers and TCP. The high fiber compliance, favorable endothelial cell proliferation rates, and retention of an important immune response of attached HUVECS support the use of gravity spun PCL fibers for three-dimensional scaffold production in vascular tissue engineering. © Mary Ann Liebert, Inc.

Dissolution rate enhancement, in vitro evaluation and investigation of drug release kinetics of chloramphenicol and sulphamethoxazole solid dispersions

Formulation of solid dispersions is one of the effective methods to increase the rate of solubilization and dissolution of poorly soluble drugs. Solid dispersions of chloramphenicol (CP) and sulphamethoxazole (SX) as model drugs were prepared by melt fusion method using polyethylene glycol 8000 (PEG 8000) as an inert carrier. The dissolution rate of CP and SX were rapid from solid dispersions with low drug and high polymer content. Characterization was performed using fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). FTIR analysis for the solid dispersions of CP and SX showed that there was no interaction between PEG 8000 and the drugs. Hyper-DSC studies revealed that CP and SX were converted into an amorphous form when formulated as solid dispersion in PEG 8000. Mathematical analysis of the release kinetics demonstrated that drug release from the various formulations followed different mechanisms. Permeability studies demonstrated that both CP and SX when formulated as solid dispersions showed enhanced permeability across Caco-2 cells and CP can be classified as well-absorbed compound when formulated as solid dispersions. © 2013 Informa Healthcare USA, Inc.

The development of functional in vitro toxicity tests

In vitro toxicity tests which detect evidence of the formation of reactive metabolites have previously relied upon cell death as a toxicity end point. Therefore these tests determine cytotoxicity in terms of quantitative changes in specified cell functions. In the studies involving the CaC0-2 cell model, there was no significant change in the transport of [3H] L-proline by the cell after eo-incubation with either dapsone or cyclophosphamide (50µM) and rat liver microsomal metabolite generating system. The pre incubation of the cells with N-ethylmalemide to inhibit Phase II sulphotransferase activity, prior to the microsomal incubations, resulted in cytotoxcity in all incubation groups. Studies involving the L6 cell model showed that there was no significant effect in the cell signalling pathway producing the second messenger cAMP, after incubation with dapsone or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. There was also no significant affect on the vasopressin stimulated production of the second messenger IP3, after incubation with the hydroxylamine metabolite of dapsone, although there were some morphological changes observed with the cells at the highest concentration of dapsone hydroxylamine (100µM). With the test involving the NG115-401 L-C3 cell model, there was no significant changes in DNA synthesis in terms of [3H] thymidine incorporation, after eo-incubation with either phenytoin or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. In the one compartment erythrocyte studies, there were significant decreases in glutathione with cyclophosphamide (50µM) (0.44 ± 0.04 mM), sulphamethoxazole (50µM) (0.43 ± 0.08mM) and carbamazepine (50µM) (0.47 ± 0.034 mM), when eoincubated with the rat microsomal system, compared to the control (0.52 ± 0.07mM). There was no significant depletion in glutathione when the erythrocytes were eoincubated with phenytoin and the rat microsomal system. In the two compartment erythrocyte studies, there was a significant decrease in the erythrocyte glutathione with cyclophosphamide (50µM) (0.953 ± 0110mM) when co-incubated the rat microsomal system, compared to the control (1.124 ± 0.032mM). Differences were considered statistically significant for p<0.05, using the Student's two tailed 't' test with Bonferroni's correction. There was no significant depletion of glutathione with phenytoin, carbamazepine and sulphamethoxazole when co-incubated with the rat microsomalsystem, compared to the control.

Polarised secretion of leukaemia inhibitory factor

Leukaemia inhibitory factor (LIF) is a cytokine that is active on a wide variety of cells. Multiple LIF transcripts have been described. The transcripts LIF-D and LIF-M encode different signal peptides, which in mouse have been associated with differential localisation of the mature protein. LIF-D is associated with a freely diffusible protein, whereas the LIF-M is associated with the extracellular matrix. The polarity of LIF secretion has yet to be described and could illuminate the mechanisms of LIF localisation. Here the polarised endogenous secretion of human LIF and IL-6 in Caco-2 cells was characterised under normal culture conditions and following induction with IL-1b. Whether the apical or basolateral membrane was stimulated influenced the pattern of secretion (LIF: Unstimulated, 59% basolateral. Dual stimulation, 68% basolateral. Basolateral stimulation, 79% basolateral. Apical stimulation, 53% basolateral). IL-6 displayed a similar dependence on the site of stimulation but was predominantly secreted at the membrane that was stimulated. To determine the effect of the alternate signal peptides on the polarity of LIF secretion, LIF was epitope tagged with FLAG. Epitope-tagging with FLAG was used to separate endogenous from exogenous protein expression. However, despite the normal biological activity of LIF-FLAG and detection of the FLAG in a western blot, detection of the LIF-FLAG under non-reducing conditions was not observed, and therefore it was unsuitable for secretion studies. Untagged LIF was expressed exogenously in Madin-Darby canine kidney (MDCK) cells under the control of a tetracycline response promoter that allowed a variety of LIF expression levels to be tested. Exogenous murine LIF was secreted predominantly from the apical (60%) membrane of MDCK cells irrespective of the signal peptide expressed.

The anti-inflammatory action of tetracyclines

For six decades tetracyclines have been successfully used for their broad spectrum antibiotic effects. However, non-antibiotic effects of tetracyclines have been reported. The anti-inflammatory effects of tetracycline drugs have been investigated in the context of a range of inflammatory diseases including sepsis and a number of neurodegenerative diseases. This thesis investigates the effects of a range of clinically important tetracyclines (oxytetracycline, doxycycline, minocycline and tigecycline) on the ability of the J774.2 cell line to produce nitric oxide when stimulated with the bacterial cell wall component, LPS. The proteome of J774.2 cells was analysed in response to LPS stimulation (1 µg/ml) with and without prior treatment with minocycline (50µg/ml), this allows the unbiased analysis of the cellular proteome in response to minocycline and LPS, protein spots of interest were excised and identified by nano-electrospray ionisation-linear ion trap mass spectroscopy. All of the tetracyclines that were investigated inhibited LPS-induced nitric oxide production in a dose dependent manner and this was due to the inhibition of inducible nitric oxide synthase expression. This is the first report to show that tigecycline inhibits inducible nitric oxide expression and nitric oxide production. Using two-dimensional gel electrophoresis and total protein staining eleven proteins were identified as being modulated by LPS. Of these eleven proteins; expression of some, but not all was modulated when the cells received a prior treatment with minocycline suggesting that minocycline does not completely block LPS-induced macrophage activation but probably specifically acts on particular inflammatory signaling pathways in macrophages. Three protein spots with a similar molecular weight but different pI values identified in this proteomic study were identified as ATP synthase ß chain. These different protein spots probably correspond to different phosphorylation states of the protein, suggesting that minocycline affects the balance of protein kinase and protein phosphatase activity in the immune response.

Pluronic F127-modified liposome-containing tacrolimus-cyclodextrin inclusion complexes:improved solubility, cellular uptake and intestinal penetration

Objective The aim of this study was to investigate Pluronic F127-modified liposome-containing cyclodextrin (CD) inclusion complex (FLIC) for improving the solubility, cellular uptake and intestinal penetration of tacrolimus (FK 506) in the gastrointestinal (GI) tract. Methods Molecular modelling was performed to screen the optimal CD for the solubilization of FK 506. FLIC was prepared by thin-lipid film hydration with the inclusion complex solutions followed by membrane extrusion. Dilution tests were conducted in simulated gastric fluids and phosphate-buffered solution of sodium taurocholate to investigate the solubility improvement of FK506. The cellular uptake of nanocarriers was studied in Caco-2 cells, and intestinal mucous membrane penetration in the GI tract was evaluated in Sprague-Dawley rats. Key findings The results showed that β-CD had the strongest binding energy with the guest molecule among the CDs. The prepared FLIC has an average diameter of 180.8 ± 8.1 nm with a spherical shape. The solubility and cellular uptake of FK 506 was greatly improved by the incorporation of CD complexes in the Pluronic F127-modified liposomes. Intestinal mucous membrane penetration was also significantly improved by the preparation of FLIC. Conclusion With improved drug solubility and intestinal mucous membrane penetration, FLIC shows a promising oral delivery system for FK 506. © 2013 Royal Pharmaceutical Society.

A novel concentration dependent amino acid ion pair strategy to mediate drug permeation using indomethacin as a model insoluble drug

Assessment of oral drug bioavailability is an important parameter for new chemical entities (NCEs) in drug development cycle. After evaluating the pharmacological response of these new molecules, the following critical stage is to investigate their in vitro permeability. Despite the great success achieved by prodrugs, covalent linking the drug molecule with a hydrophobic moiety might result in a new entity that might be toxic or ineffective. Therefore, an alternative that would improve the drug uptake without affecting the efficacy of the drug molecule would be advantageous. The aim of the current study is to investigate the effect of ion-pairing on the permeability profile of a model drug: indomethacin (IND) to understand the mechanism behind the permeability improvement across Caco-2 monolayers. Arginine and lysine formed ion-pairs with IND at various molar ratios 1:1, 1:2, 1:4 and 1:8 as reflected by the double reciprocal graphs. The partitioning capacities of the IND were evaluated using octanol/water partitioning studies and the apparent permeabilities (P app) were measured across Caco-2 monolayers for the different formulations. Partitioning studies reflected the high hydrophobicity of IND (Log P = 3) which dropped upon increasing the concentrations of arginine/lysine in the ion pairs. Nevertheless, the prepared ion pairs improved IND permeability especially after 60 min of the start of the experiment. Coupling partitioning and permeability results suggest a decrease in the passive transcellular uptake due to the drop in IND portioning capacities and a possible involvement of active carriers. Future work will investigate which transport gene might be involved in the absorption of the ion paired formulations using molecular biology technologies. © 2014 Elsevier B.V. All rights reserved.

Physical and biological properties of a novel siloxane adhesive for soft tissue applications

The aim of this study was to investigate the adhesive properties of an in-house amino-propyltrimethoxysilane-methylenebisacrylamide (APTMS-MBA) siloxane system and compare them with a commercially available adhesive, n-butyl cyanoacrylate (nBCA). The ability of the material to perform as a soft tissue adhesive was established by measuring the physical (bond strength, curing time) and biological (cytotoxicity) properties of the adhesives on cartilage. Complementary physical techniques, X-ray photoelectron spectroscopy, Raman and infrared imaging, enabled the mode of action of the adhesive to the cartilage surface to be determined. Adhesion strength to cartilage was measured using a simple butt joint test after storage in phosphate-buffered saline solution at 37°C for periods up to 1 month. The adhesives were also characterised using two in vitro biological techniques. A live/dead stain assay enabled a measure of the viability of chondrocytes attached to the two adhesives to be made. A water-soluble tetrazolium assay was carried out using two different cell types, human dermal fibroblasts and ovine meniscal chondrocytes, in order to measure material cytotoxicity as a function of both supernatant concentration and time. IR imaging of the surface of cartilage treated with APTMS-MBA siloxane adhesive indicated that the adhesive penetrated the tissue surface marginally compared to nBCA which showed a greater depth of penetration. The curing time and adhesion strength values for APTMS-MBA siloxane and nBCA adhesives were measured to be 60 s/0.23 MPa and 38 min/0.62 MPa, respectively. These materials were found to be significantly stronger than either commercially available fibrin (0.02 MPa) or gelatin resorcinol formaldehyde (GRF) adhesives (0.1 MPa) (P <0.01). Cell culture experiments revealed that APTMS-MBA siloxane adhesive induced 2% cell death compared to 95% for the nBCA adhesive, which extended to a depth of approximately 100-150 μm into the cartilage surface. The WST-1 assay demonstrated that APTMS-MBA siloxane was significantly less cytotoxic than nBCA adhesive as an undiluted conditioned supernatant (P <0.001). These results suggest that the APTMS-MBA siloxane may be a useful adhesive for medical applications. © VSP 2005.

Genomic evaluation during permeability of indomethacin and its solid dispersion

Drug resistance was first identified in cancer cells that express proteins known as multidrug resistance proteins that extrude the therapeutic agents out of the cells resulting in alteration of pharmacokinetics, tissue distribution, and pharmacodynamics of drugs. To this end studies were carried out to investigate the role of pharmacological inhibitors and pharmaceutical excipients with a primary focus on P-glycoprotein (P-gp). The aim of this study was to investigate holistic changes in transporter gene expression during permeability upon formulation of indomethacin as solid dispersion. Initial characterization studies of solid dispersion of indomethacin showed that the drug was dispersed within the carrier in amorphous form. Analysis of permeability data across Caco-2 monolayers revealed that drug absorption increased by 4-fold when reformulated as solid dispersion. The last phase of the work involved investigation of gene expression changes of transporter genes during permeability. The results showed that there were significant differences in the expression of both ATP-binding cassette (ABC) transporter genes as well as solute carrier transporter (SLC) genes suggesting that the inclusion of polyethylene glycol as well as changes in molecular form of drug from crystalline to amorphous have a significant bearing on the expression of transporter network genes resulting in differences in drug permeability. © 2011 Informa UK, Ltd.

Modulation du développement du cancer de l'intestin et du côlon par des nutriments des produits laitiers

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.