Inhibition of NK cells by murine CMV-encoded class I MHC homologue m144

In this study the effects of two chitosan salts, namely chitosan hydrochloride and chitosan glutamate (0.5 and 1.5% w/v), on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cell monolayers, using the radioactive marker [14C]-mannitol, were investigated in a slightly acidic (pH 6.2) and neutral (pH 7.4) environment. Both salts are soluble in acidic conditions up to a concentration of 1.5% w/v and solutions of this strength, at a pH of 6.2, caused a pronounced lowering in the TEER of Caco-2 cell monolayers in the order of 70+/-1% (chitosan glutamate) and 77+/-3% (chitosan hydrochloride), 20 min after incubation started. In agreement with the TEER results the transport of the radioactive marker, [14C]-mannitol, was increased 25-fold (chitosan glutamate) and 36-fold (chitosan hydrochloride), respectively, at this pH. However, at a pH of 7.4 both salts are insoluble and prove to be ineffective since no reduction in the TEER values or increase in the transport of [14C]-mannitol were found. The results show that these chitosan salts are potent absorption enhancers in acidic environments. We conclude that there is a need for chitosan derivatives with increased solubility, especially at neutral and basic pH values, for use as absorption enhancers aimed at the delivery of therapeutic compounds in the more basic environment of the large intestine and colon.     

Mechanism of intestinal absorption of ranitidine and ondansetron: transport across Caco-2 cell monolayers

We have investigated the transport of ranitidine and ondansetron across the Caco-2 cell monolayers. The apparent permeability co-efficients (Papp) were unchanged throughout the concentration range studied, indicating a passive diffusion pathway across intestinal mucosa. No metabolism was observed for ranitidine and ondansetron during the incubation with Caco-2 cell monolayers. Papp values for ranitidine and ondansetron (bioavailability of 50 and approximately 100% in humans, respectively) were 1.03 +/- 0.17 x 10(-7) and 1.83 +/- 0.055 x 10(-5) cm/sec, respectively. The Papp value for ranitidine was increased by 15- to 20-fold in a calcium-free medium or in the transport medium containing EDTA, whereas no significant change occurred with ondansetron, indicating that paracellular passive diffusion is not rate determining for ondansetron. Uptake of ondansetron by Caco-2 cell monolayers was 20- and 5-fold higher than that of ranitidine when the uptake study was carried out under sink conditions and at steady state. These results suggest that ranitidine and ondansetron are transported across Caco-2 cell monolayers predominantly via paracellular and transcellular pathways, respectively.     

Microsurgical management of craniopharyngiomas--outcomes in 56 patients

PURPOSE: The tight junctions in the intestinal epithelium represent highly specialized intercellular junctions. Ranitidine, an H2-antagonist, causes a tightening of the tight junctions. Hence, we have investigated the effect of ranitidine and other H2-antagonists on the function of the intestinal tight junctions. METHODS: Effect of the H2-antagonists on the tight junctions has been investigated using the transepithelial electrical resistance (TEER) and the transport of mannitol across the Caco-2 cell monolayers. RESULTS: Four different H2-antagonists caused an increase in the TEER across the Caco-2 cell monolayers, accompanied by a decrease in the permeability for mannitol. The effect was concentration-dependent and saturable. Ranitidine and famotidine, caused a decrease in their own transport rate across the Caco-2 cells. Ranitidine competitively inhibited the increase in TEER caused by famotidine, whereas compounds which represent molecular fragments of ranitidine had no effect. The relative potency of the four H2-antagonists in causing an increase in the TEER correlated inversely with the oral bioavailability of these compounds in humans. CONCLUSIONS: We hypothesize that the H2-antagonists exert their effect on the tight junctions of Caco-2 cells by modulation of interactions among proteins associated with the tight junctional complex.     

Correlation between oral drug absorption in humans, and apparent drug permeability in TC-7 cells, a human epithelial intestinal cell line: comparison with the parental Caco-2 cell line

PURPOSE: To determine and compare the relationship between in vivo oral absorption in humans and the apparent permeability coefficients (Papp) obtained in vitro on two human intestinal epithelial cell lines, the parental Caco-2 and the TC-7 clone. METHODS: Both cell lines were grown for 5-35 days on tissue culture-treated inserts. Cell monolayers were analysed for their morphology by transmission electron micrography, and for their integrity with respect to transepithelial electrical resistance, mannitol and PEG-4000 transport, and cyclosporin efflux. Papp were determined for 20 compounds exhibiting large differences in chemical structure, molecular weight, transport mechanisms, and percentage of absorption in humans. RESULTS: The TC-7 clone exhibits morphological characteristics similar to those of the parental Caco-2 cell line, concerning apical brush border, microvilli, tight junctions and polarisation of the cell line. The TC-7 clone however appeared more homogenous in terms of cell size. Both cell lines achieved a similar monolayer integrity towards mannitol and PEG-4000. Monolayer integrity was achieved earlier for the TC-7 clone, mainly due to its shorter doubling time, i.e. 26 versus 30 hours for parental Caco-2 cells. When using cyclosporin A as a P-glycoprotein substrate, active efflux was lower in the TC-7 clone than in the parental Caco-2 cells. The Papp and mechanisms of transport (paracellular or transcellular routes, passive diffusion and active transport) were determined for 20 drugs. A relationship was established between the in vivo oral absorption in humans and Papp values, allowing to determine a threshold value for Papp of 2 10(-6) cm/sec, above for which a 100% oral absorption could be expected in humans. Both correlation curves obtained with the two cell types, were almost completely superimposable. These studies also confirmed that the dipeptide transporter is underexpressed in both cell lines. CONCLUSIONS: On the basis of morphological parameters, biochemical activity and drug transport characteristics, the TC-7 clone appeared to be a valuable alternative to the use of parental Caco-2 cells for drug absorption studies.     

Paracellular calcium transport across Caco-2 and HT29 cell monolayers

Intestinal calcium absorption has been shown to include two processes, a saturable transcellular movement and a non-saturable paracellular pathway. The potential utility of cell monolayers for studying transepithelial intestinal calcium transport has already been demonstrated; however, simultaneous evaluation of the contribution of the saturable transcellular and of the non-saturable paracellular processes to the total transepithelial transport has not yet been attempted. The aim of this study was to investigate the contribution both of transcellular and paracellular transport processes to the total transepithelial calcium transport in two cell culture monolayers. Caco-2 cells and a clone derived from HT-29 cells (HT29-Cl.19A), two cell lines derived from colon adenocarcinomas which are known to be able to exhibit typical enterocytic differentiation, were used. Cell monolayers were grown on a permeable support and used after 15 days of culture when these cells express enterocytic differentiation and high transepithelial resistance. Isotopic transport rate measurements were performed in the absence of a chemical gradient. The paracellular route was evaluated using [3H]mannitol. Calcium and [3H]mannitol transport rates across cell monolayers were not significantly different. Augmentation of calcium uptake by 200 mM sorbitol did not significantly increase calcium or mannitol transepithelial transport; however, calcium accumulation in the cells was increased by about 200%. Modulation of the monolayer permeability by addition of 10 nM vasoactive intestinal polypeptide (VIP) or 0.5 mM carbachol treatment, which respectively increased and decreased the transepithelial resistance, consequently modified calcium and mannitol transport in a parallel manner. Our results show that Caco-2 and HT29-Cl.19A cell monolayers are good models for studying the calcium paracellular transport pathway.     

Transcellular retrograde labeling of radial glial cells with WGA-HRP and DiI in neonatal rat and hamster

Topographically distinct populations of radial glial cells in the diencephalon and mesencephalon of neonatal rats and hamsters were transcellularly labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and with the lipophilic tracer DiI. A comparison of the histological distribution of the two tracers is suggestive of two different mechanisms of transcellular labeling. Intraocular injections of WGA-HRP resulted in the uptake of exogenously applied WGA-HRP by retinal ganglion cells, followed by anterograde axonal transport and exocytosis within the optic target nuclei. In addition to the transneuronal labeling, which is typical of such injections, we observed the transcellular labeling of the processes and somata of radial glial cells that were topographically associated with the terminal fields of the labeled axons. Similar transcellular labeling of radial glial cells associated with the axon terminal fields of the colliculogeniculate projection to the medial geniculate nucleus was observed following injections of WGA-HRP in the inferior colliculus. The transcellular labeling within the radial glial cells was discontinuous and somatopetally concentrated, indicating the existence of a retrograde active transport mechanism within the radial glial processes subsequent to its uptake following release of tracer from axons. This type of labeling can be referred to as transcellular retrograde glioplasmic transport. In contrast, DiI was used as a tracer through its capacity to diffuse within the plasmalemma. Topographically distinct populations of radial glial cells were transcellularly labeled following placements of DiI in the retina, inferior colliculus, or dorsal thalamus of fixed brains. The radial processes of labeled radial glial cells consistently extended into regions that also contained labeled axons. It is likely that the transcellular radial glial labeling with DiI occurred via transmembranous diffusion. These data indicate that a close structural and functional relation exists between axons and glial cells in the developing brain.     

Permeability enhancement in Caco-2 cell monolayers by sodium salicylate and sodium taurodihydrofusidate: assessment of effect-reversibility and imaging of transepithelial transport routes by confocal laser scanning microscopy

The effects of sodium salicylate and sodium tauro-24,25-dihydrofusidate (STDHF) on the aqueous permeability of confluent monolayers of Caco-2 cells were studied. Measurements of transepithelial electrical resistance (TEER) showed a concentration-dependent effect of both compounds after apical incubation for 1 hr. Reductions in TEER resulting from EC50 concentrations (2.8 mM for STDHF; 173 mM for salicylate) were reversible within 5.75 hr. The transpithelial fluxes of two hydrophilic model compounds, sodium fluorescein F (molecular weight 376) and a fluorescein isothiocyanate-labeled dextran (mean molecular weight 4000) was significantly increased by STDHF (2.8 mM). Sodium salicylate (173 mM) only enhanced the transport of sodium fluorescein significantly. At the EC50 concentrations, confocal laser scanning microscopy (CLSM) visualized both fluorescent tracers mainly in the paracellular route. With higher enhancer concentrations (373 mM sodium salicylate and 8 mM STDHF), both transport markers appeared intracellularly as a result of cell death. STDHF rapidly extracted an exogenous lipophilic membrane probe, 5-(N-hexadecanoyl)aminofluorescein (HEDAF), from the apical part of Caco-2 plasma membranes, indicating qualitatively that STDHF interacts with the lipid portion of cell membranes. These results suggest that both sodium salicylate and STDHF can be used to reversibly increase paracellular permeability of Caco-2 cell monolayers, whereby STDHF appears to be advantageous compared to sodium salicylate. By adapting the Costar cell culture system to CLSM, we have shown that this technique is suitable to study membrane interactions qualitatively and for visualizing transport routes of hydrophilic tracers through nonfixed, filter-grown monolayers.     

A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model

Caco-2 cell monolayers mimic intestinal absorptive epithelium and represent a very useful tool for studying transepithelial transport. The literature on Caco-2 cells is controversial regarding transepithelial resistance and permeabilities of different marker compounds across monolayers. This paper discusses probable causes for these discrepancies. First, we present the role of culture conditions, such as the nature of the support or the passage number, on cell biology and transport properties. Further, we compare the presence of transport proteins in Caco-2 cells to mammalian intestinal tissue and discuss their implication for drug absorption. We also examine the advantages and disadvantages of systems such as Transwell and side-by-side diffusion chambers. A summary of comparisons between permeabilities across Caco-2 monolayers and mammalian intestinal tissues is provided. We conclude that the origin of Caco-2 cells and the culture conditions are in part responsible for the discrepancies encountered in the literature.     

Parental sex effects in bipolar affective disorder pedigrees

Measurements of transepithelial electrical resistance (TEER), the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) test and monitoring of poly(ethylene glycol) (PEG) transport have been used to study the effects of the non-ionic surfactants Solulan C24 and Solulan 16, either free in solution or as an integral part of niosome bi-layers, on intestinal epithelial cells from man (Caco-2 cell monolayers). The effects on epithelial integrity and on the transport of the hydrophilic drug metformin depend on the concentration of the surfactants. At concentrations above 1% the effect on TEER of the surfactant in niosomal form and free in solution were equivalent whereas cell viability was preserved to a higher concentration of Solulans when the Solulans were present in the niosomal form. It was concluded that the toxic effect of niosomes arises from free surfactant present in the niosome suspension.     

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PURPOSE: Using polarized bovine brain microvessel endothelial cells (BBMEC) monolayers as in vitro model of the blood brain barrier and Caco-2 monolayers as a model of the intestinal epithelium, the present work investigates the effects of Pluronic P85 block copolymer (P85) on the transport of the P-gycoprotein (P-gp)- dependent probe, rhodamine 123 (R123). METHODS: The permeability and cell efflux studies are performed with the confluent cell monolayers using Side-Bi-Side diffusion cells. RESULTS: At concentrations below the critical micelle concentration, P85 inhibits P-gp efflux systems of the BBMEC and Caco-2 cell monolayers resulting in an increase in the apical to basolateral permeability of R123. In contrast, at high concentrations of P85 the drug incorporates into the micelles, enters the cells and is then recycled back out to the apical side resulting in decrease in R123 transport across the cell monolayers. Apical to basolateral permeability of micelle-incorporated R123 in BBMEC monolayers was increased by prior conjugation of P85 with insulin, suggesting that modified micelles undergo receptor-mediated transcytosis. CONCLUSIONS: Pluronic block copolymers can increase membrane transport and transcellular permeability in brain microvessel endothelial cells and intestinal epithelium cells. This suggests that these block copolymers may be useful in designing formulations to increase brain and oral absorption of select drugs.     

Comparative accuracy of glucose monitors

The objective of this study was to rationalize the shape of membrane permeability-lipophilicity curves, when considering, in addition to the usual transcellular route, a parallel diffusion pathway through aqueous pores as present in biological membranes. The theoretical influence of different pH in donor and acceptor compartment and the molecular weight on the permeability curves was studied. We combined and extended two previously proposed absorption models, namely one describing diffusion through a simple membrane (two stagnant aqueous and two organic layers in series, no pores) as the sum of the two distribution steps at both membrane interfaces, and a second theoretical model considering the sum of different diffusional resistances through stagnant layers and membrane, respectively. Under certain conditions the equivalence of the two-step distribution model and the diffusional resistance model can be demonstrated. Incorporation of an aqueous diffusion pathway leads to an extended two-step distribution model. This theoretical membrane permeation model will permit a more physicochemical-based interpretation of permeation data and shows that combined log D values and molecular weight are important determinants for membrane transport processes through, e.g. Caco-2 monolayers and the mucosal GI membranes. We have demonstrated that the well-known sigmoidal permeability-lipophilicity relationship should be considered as a molecular weight-dependent set of sigmoidal relationships.     

Domain evolution in the alpha-amylase family

Recently, we have characterized glial cultures derived from very early neurogenesis (E3) and found them to consist largely of early glioblastic or astroblastic cells with the capacity to differentiate into astrocytes given sufficient time in culture or with advancing age, i.e., cell passage. This study examines and compares the characteristics of astrocyte-enriched cultures derived from advanced embryonic ages (E15) in the chick embryonic cerebral hemispheres. We report several remarkable findings. 1) Mature astrocytes (GFAP+, vimentin-) appear as early as 5 days in vitro (DIV) in primary culture (P0). 2) Also apparent in primary cultures were extensive populations of neurons (neurofilament+; NF+) growing atop or in close proximity to mature astrocytes. 3) NF+ neurons disappeared after the first cell passage, and GFAP+ astrocytes were greatly diminished within two cell passages thereafter. 3) High concentrations of NGF were expressed, presumably by glial cells, in primary cultures through 14 DIV, declining to a low plateau through 27 DIV and remaining low, but measurable in subsequent cell passages. 4) At later cell passages (> 5) immature phenotypes of these same cell types continued to be expressed in E15CH cultures, i.e., positive staining for GFAP and vimentin and GFAP, GS, and NGF can all be detected on Western blots. We conclude from these findings that 1) mitotic multipotential neural cells are present within cerebral hemispheres even at late stages of development (E15); 2) neuroblasts and astroblasts have a reciprocal relationship requiring the presence of both cell types in order for mature expression of their phenotypes; 3) the NGF profile parallels the appearance and disappearance of neurons in E15 chick embryonic cerebral hemisphere primary cultures, strongly suggesting that this trophic factor may be involved in the mutually beneficial relationship between astrocytes and neurons.     

Permeability characteristics of novel mydriatic agents using an in vitro cell culture model that utilizes SIRC rabbit corneal cells

The purpose of this study was to evaluate the permeability characteristics of a previously reported in vitro corneal model that utilizes SIRC rabbbit corneal cells and to investigate the permeability of three novel esters of phenylephrone chemical delivery systems (CDS) under different pH conditions using this in vitro model. The SIRC rabbit corneal cell line was grown on transwell polycarbonate membranes, and the barrier properties were assessed by measuring transepithelial electrical resistance (TEER) using a voltohmmeter. The permeabilities of esters of phenylephrone CDS across the SIRC cell layers were measured over a pH range 4.0-7. 4. The esters tested include phenylacetyl (1), isovaleryl (2), and pivalyl (3). The SIRC rabbit corneal cell line, when grown on permeable filters, formed tight monolayers of high electrical resistance with TEER values increasing from 71.6 +/- 20.8 Omega.cm2 at day 3 in culture to 2233.42 +/- 15.2 Omega.cm2 at day 8 in culture and remained constant through day 14 in culture. The transepithelial permeability coefficients (Papp) at pH 7.4 ranged from 0.58 x 10(-6) cm/s for the hydrophilic marker, mannitol, to 43. 5 x 10(-6) cm/s for the most lipophilic molecule, testosterone. The Papp at pH 7.4 for phenylephrine was 4.21 x 10(-6) cm/s. The Papp values and the lag times of the three esters of phenylephrone were pH dependent. The Papp for 1, 2, and 3 at pH 7.4 were 14.76 x 10(-6), 13.19 x 10(-6), and 12.86 x 10(-6) cm/s, respectively and the permeabilities decreased at conditions below pH 7.4. The lag times at pH 7.4 were 0.10, 0.17, and 0.12 h for 1, 2, and 3, respectively, and the values increased at lower pH conditions. The TEER values of SIRC cell line observed at day 8 to day 14 in the present investigation are similar to the resistance value reported for rabbit cornea (2 kOmega.cm2). All the esters showed significantly (p < 0.05) higher permeabilities than phenylephrine at pH 7.4. The rate and extent of transport of the drugs across the cell layers were influenced by the fraction of ionized and un-ionized species and the intrinsic partition coefficient of the drug. The results indicate that the permeability of ophthalmic drugs through ocular membranes may be predicted by measuring the permeability through the new in vitro cell culture model.     

Chromosomal abnormalities in HPV-16-immortalized oral epithelial cells

Human papilloma virus (HPV) type 16 has an established association with anogenital carcinoma, and to some extent with human oral squamous cell carcinoma. We hypothesize that HPV type 16 is capable of inducing chromosomal and cell cycle changes in cultured oral epithelial cells. Normal human oral epithelia] cells were immortalized with recombinant retrovirus containing the E6/E7 open reading frames of HPV type 16. These cells have been in culture for more than 350 passages and over 4 years. Flow cytometry demonstrated an average of 42% nuclear aneuploidy in HPV 16-immortalized cells; 16% in normal controls (probably tetrasomy). Cytogenetic analysis demonstrated significant progression of chromosomal abnormalities. Cells at early passage (p10) showed trisomy 20, with no other major changes. At passage 18, trisomy 1q and monosomy 13 were seen in addition to trisomy 20. At passage 61 there were two distinct cell populations ('a' and 'b'), with multiple chromosomal changes including trisomy 5q,14,20 in one line and 7p,9q,llq in the other. Both populations had monosomy 3p, with monosomy 8p in one population and monosomy 13 in the other. At passage 136, the cells were essentially identical to population 'b' of passage 61. At this passage, mutation of the p53 gene was detected at codon 273 of exon 8, with G to T conversion (Arg to Leu). This was absent in the normal cells from which this line was developed. Passage 262 contained the two major cell populations, each with a sub-group with additional chromosomal changes such as 10p monosomy. Cells from passages 217 and 305 were injected into nude mice a year apart. Both failed to produce tumors, as did normal cells. In conclusion, we present an HPV type 16-immortalized oral epithelial cell line (IHGK) with extensive and progressive chromosomal abnormalities, invasive growth in culture and yet no tumor formation in nude mice. We suggest that the question as to whether HPV alone can induce transformation is still open.     

Characterization of P-glycoprotein mediated transport of K02, a novel vinylsulfone peptidomimetic cysteine protease inhibitor, across MDR1-MDCK and Caco-2 cell monolayers

PURPOSE: Here we characterized the transport properties of morpholine-urea-phenylalanine-homophenylalanine-vinylsulfone-phenyl (K02), a newly developed peptidomimetic cysteine protease inhibitor, across monolayers of P-gp-expressed MDRI transfected MDCK cells (MDR1-MDCK) and Caco-2 cells. METHODS: MDR1-MDCK, MDCK and Caco-2 cells, grown to confluence on Transwell insert membranes, were used to investigate transcellular transport of [14C]-K02. RESULTS: The basolateral to apical (B-A) flux of 10 microM [14C]-K02 across MDR1-MDCK cells was markedly greater than its apical to basolateral (A-B) flux (ratio = 39). This specific B-A transport was temperature dependent and saturable, with an apparent Michaelis-Menten constant and maximum velocity of 69.1 +/- 19.5 microM and 148.9 +/- 16.3 pmol/min/cm2, respectively. This B-A flux was significantly inhibited by cyclosporine (IC50 = 17.1 +/- 0.7 microM), vinblastine (IC50 = 75.9 +/- 13.0 microM) and verapamil (IC50 = 236 +/- 63 microM). In Caco-2 cell monolayers, the B-A flux was reduced about 50% compared to that in MDR1-MDCK and the A-B flux was increased about 8-fold. The apparent Michaelis-Menten constant and maximum velocity values for the B-A transport were 71.8 +/- 45.9 microM and 35.3 +/- 9.0 pmol/min/ cm2. This B-A flux was also significantly inhibited by P-gp substrates/ inhibitors. Western blots showed that the P-gp expression in MDR1-MDCK cells was about 10-fold that in Caco-2 cells. CONCLUSIONS: K02 is transported by P-gp in both MDR1-MDCK and Caco-2 cells, and the in vitro interactions between K02 and various P-gp substrates may provide strategies to overcome the bioavailability barrier by intestinal P-gp.     

Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.     

Degradation of carboxy-terminal-tagged cytoplasmic proteins by the Escherichia coli protease HflB (FtsH)

PURPOSE: In general, the intestinal epithelium is quite refractory to viral and non-viral methods of gene transfer. In this report, various cyclodextrin formulations were tested for their ability to enhance adenoviral transduction efficiency in two models of the intestinal epithelium: differentiated Caco-2 cells and rat jejunum. METHODS: Transduction efficiency of replication-deficient adenovirus type 5 vectors encoded with either the E. coli beta-galactosidase or the jellyfish green fluorescent protein gene was assessed by X-gal staining or visualization of fluorescence 48 hours after infection. In vivo experiments were performed using an intestinal loop ligation technique. RESULTS: Several formulations of neutral and positively charged beta cyclodextrins significantly enhanced adenoviral-mediated gene transfer in the selected models. The cyclodextrin formulations studied increased adenoviral transduction in the intestine by enhancing both viral binding and internalization. Viral binding was significantly increased on cell membranes treated with positively charged cyclodextrins, as seen with confocal microscopy and rhodamine-labeled virus. Permeability studies and TEER readings revealed that the most successful formulations gently disrupt cell membranes. This enhances internalization of viral particles and results in increased levels of gene expression. CONCLUSIONS: These formulations can be of value in gene transfer to cells and tissues in which adenoviral infection is limited due to a lack of fiber and alpha(v) integrin receptors. They are simple to prepare and do not affect the ability of the virus to transduce target cells.     

Killing effects of 5-fluorouracil on human biliary tract cancer cell lines

Previous studies have established that a partially quaternized derivative of chitosan, N-trimethyl chitosan chloride (TMC), can be used as an absorption enhancer for large hydrophilic compounds across mucosal surfaces. This study evaluates and compares the effects of the degree of quaternization of TMC, in a neutral environment, on the permeability of intestinal epithelial cells in vitro, where normal chitosan salts are ineffective as absorption enhancers. The effects of TMC-H [61.2% quaternized, (0.05-1.5% w/v)], TMC-L [12.3% quaternized, (0.5-1.5% w/v)], and chitosan hydrochloride [0.5-1.5% w/v] on the transepithelial electrical resistance (TEER) and permeability, for the hydrophilic model compound [14C]mannitol, of intestinal epithelial Caco-2 cell monolayers, were investigated at pH values of 6.20 and 7.40. The viability of the monolayers was checked with the trypan blue exclusion technique. At a pH of 6.20, all the polymers caused a pronounced reduction (37-67% at 0.5% w/v concentrations) in the TEER of Caco-2 cells. On the contrary, at a pH of 7.40, only TMC-H was able to decrease the TEER values, even in a concentration as low as 0.05% w/v (35% reduction). Comparable results were obtained with the permeation of [14C]mannitol. Large increases in the transport rate (18-23-fold at 0.5% w/v concentrations) were found at pH 6.20, whereas only TMC-H was able to increase the permeation of [14C]mannitol at pH 7.40 (31-48-fold at 0.05-1.5% w/v concentrations of TMC-H). For all the polymers studied, no deleterious effects to the cells could be demonstrated with the trypan blue exclusion technique. It is concluded that highly quaternized TMC is a potent absorption enhancer and the potential use of this polymer, especially in neutral and basic environments where normal chitosan salts are not effective, is expected to be an important contribution to the development of effective delivery systems for hydrophilic compounds such as peptide drugs.