Caco 2 Cell

Enterocyte differentiation is a dynamic process during which reinforcement of cell-cell adhesion favours migration along the crypt-to-villus axis. Functional polarization of Caco-2 cells, the most commonly used model to study intestinal differentiation, is assessed by dome formation and tightness of the monolayer and is under the control of the extracellular matrix (ECM). Furthermore, our biochemical and confocal microscopy data demonstrate that the ECM dramatically reinforces E-cadherin targeting to the upper lateral membrane, formation of the apical actin cytoskeleton and its colocalization with E-cadherin in functional complexes. In our model, these effects were produced by native laminin-5-enriched ECM as well as by type IV collagen or laminin 2, which suggests a common pathway of induction through integrin receptors. Indeed, these effects were antagonized by blocking anti-β1-and anti-α6-integrin antibodies and directly induced by a stimulating anti-β1-integrin antibody. These results demonstrate that integrin-dependent cell to ECM adhesion reinforces E-cadherin-dependent cell-cell adhesion in Caco-2 cells and further support the notion that enterocyte differentiation is supported by a molecular crosstalk between the two adhesion systems of the cell.

Enterocyte differentiation is a dynamic process during which reinforcement of cell-cell adhesion favours migration along the crypt-to-villus axis. Functional polarization of Caco-2 cells, the most commonly used model to study intestinal differentiation, is assessed by dome formation and tightness of the monolayer and is under the control of the extracellular matrix (ECM). Furthermore, our biochemical and confocal microscopy data demonstrate that the ECM dramatically reinforces E-cadherin targeting to the upper lateral membrane, formation of the apical actin cytoskeleton and its colocalization with E-cadherin in functional complexes. In our model, these effects were produced by native laminin-5-enriched ECM as well as by type IV collagen or laminin 2, which suggests a common pathway of induction through integrin receptors. Indeed, these effects were antagonized by blocking anti-β1-and anti-α6-integrin antibodies and directly induced by a stimulating anti-β1-integrin antibody. These r...

We previously reported on the occurrence of prominin-1-carrying membrane vesicles that are released into body fluids from microvilli of epithelial cells. This release has been implicated in cell differentiation. Here we have characterized these vesicles released from the differentiated Caco-2 cells. We find that in these vesicles, prominin-1 directly interacts with membrane cholesterol and is associated with a membrane microdomain. The cholesterol depletion using methyl-b-cyclodextrin resulted in a marked increase in their release, and a dramatic change in the microvillar ultrastructure from a tubular shape to a ''pearling" state, with multiple membrane constrictions, suggesting a role of membrane cholesterol in vesicle release from microvilli.

Purpose: The aim of this research is to model the effect of methylation on hydrogen bonding ability, surface area, polar surface area, volume, lipophilicity, charge, and cross-sectional diameters of a series of mono-, di-, and tri- methyl substituted analogs of arginine-glycine-aspartic acid (RGD) and compare these parameters to in vitro transport properties across Caco-2 monolayers. Methods: Molecular modeling was used to investigate the structural parameters that may influence the transport properties of RGD and its methyl analogs at pH 7.4. Log P was experimentally determined using a potentiometric method and compared to cLogP. Transport studies were carried out using Caco-2 cell monolayers. Results: Parameters such as polar and total surface area, volume, and Log P were found to vary with both the number and the sites of methyl substitution on the RGD molecule. The calculated as well as the experimental Log P values were found to be less than minus 2. The calculated maximum cross-sectional diameters ranged from 9 to 12 A. No detectable transport was noted. Conclusions: Results of our study indicate that in the design considerations for the development of new peptidomimetic RGD analogs with enhanced oral bioavailability, an important parameter to consider is the three dimensional conformation of the peptides which influences their hydrogen bonding ability, polarity and molecular geometry.

The aim of this research is to model the effect of methylation on hydrogen bonding ability, surface area, polar surface area, volume, lipophilicity, charge, and cross-sectional diameters of a series of mono-, di-, and tri- methyl substituted analogs of arginine-glycine-aspartic acid (RGD) and compare these parameters to in vitro transport properties across Caco-2 monolayers. Molecular modeling was used to investigate the structural parameters that may influence the transport properties of RGD and its methyl analogs at pH 7.4. Log P was experimentally determined using a potentiometric method and compared to cLogP. Transport studies were carried out using Caco-2 cell monolayers. Parameters such as polar and total surface area, volume, and Log P were found to vary with both the number and the sites of methyl substitution on the RGD molecule. The calculated as well as the experimental Log P values were found to be less than minus 2. The calculated maximum cross-sectional diameters range...

Quantitative Structure-Permeability Relationships (QSPerR) of the intestinal permeability across the (Caco-2) cells monolayer could be obtained by the application of new molecular descriptors. A novel topologic-molecular approach to computer molecular design ( TOMOCOMD-CARDD ) has been used to estimate the intestinal-epithelial transport of drug in Caco-2 cell culture. The Permeability Coefficients in Caco-2 cells (P) for 33 structurally diverse drugs were well described using quadratic indices of the molecular pseudograph's atom adjacency matrix as molecular descriptors. A quantitative model that discriminates the high-absorption compounds from those with moderate-poor absorption was obtained for the training data set, showing a global classification of 87.87%. In addition, two QSPerR models, through a multiple linear regression, were obtained to predict the P [apical to basolateral (AP-->BL) and basolateral to apical (BL-->AP)]. A leave- n -out and leave- one -out cross-...

of Literature 1.2incidence of E.coli in soft cheese 2.2 phenotypic character of E.coli 3.2 in vitro antimicrobial sensitivity 4.2genotypic detection of virulence genes of E.coli 5.2 public health importance 3-19 3 13 13 14 19 3-Materials and methods 20-28 4-Results 29-34 5-Discussion 35-43 6-Conclusion and -Recommendations 44-45

Preliminary clinical evidence suggests that Helicobacter pylori may be associated with diarrhea through its vacuolating toxin (VacA). To establish whether VacA induces intestinal secretion, epithelial damage, or both, purified pH-activated VacA was added to Caco-2 cell monolayers mounted in Ussing chambers, and electrical parameters were monitored. Mucosal addition of VacA induced an increase in short circuit current, consistent with enterotoxic effect. The effect was time-and dose-dependent and saturable. It was not found if the toxin was not pH-activated, added to the serosal side, or preheated. In cells preloaded with the Ca2 ϩ buffering compound BAPTA/AM or with the Cl Ϫ channel inhibitor 5-nitro-2-3-(3phenylpropylamino)benzoic acid, short circuit current did not change, indicating that VacA induces activation of Ca2 ϩ -dependent Cl Ϫ channels. VacA did not show cytopathic effects, as judged by tissue resistance. These results support the hypothesis that H. pylori may be associated with diarrhea through production of VacA.

Paclitaxel, a widely used chemotherapeutic agent, exhibits limited oral bioavailability and therapeutic efficacy due to its substrate affinity for P-glycoprotein (P-gp), an efflux transporter that restricts drug absorption and promotes clearance. This study investigates the pharmacokinetic profile of paclitaxel when co-administered with natural P-gp inhibitors, including curcumin, quercetin, and piperine, in rodent in vivo models. Following oral administration, plasma concentrations of paclitaxel were quantified using validated LC-MS/MS methods, and key pharmacokinetic parameters such as C_max, T_max, AUC_0-∞, and half-life (t_1/2) were analyzed. Co-administration with natural inhibitors significantly enhanced paclitaxel's systemic exposure, with observed increases in AUC and C_max, indicating improved bioavailability. Among the tested inhibitors, piperine exhibited the most pronounced effect, likely due to its dual role in inhibiting P-gp and cytochrome P450 enzymes. These findings suggest that natural P-gp inhibitors can effectively modulate the pharmacokinetics of paclitaxel, offering a promising strategy to optimize its oral delivery and therapeutic index.

Various bacteria can cause mastitis in cows. Escherichia coli (E. coli) is the most common cause of both clinical and subclinical mastitis on dairy farms, leading to a decrease in milk production and economic losses. Also, E. coli is responsible for more than 81% of urinary tract infections (UTIs) in humans and animals, causes dysuria, and may lead to kidney damage. The purpose of this study was the detection and serotyping of E. coli isolated from bovines' milk and workers' urine in different dairy farms in Gharbia governorate, Egypt. A bacteriological examination of 158 samples (100 milk and 58 workers' urine) showed that the prevalence of E. coli in bovine milk samples was 5% and 6.9% in workers' urine samples. Serological identification of these isolates revealed that the predominant serotypes in samples from milk were E. coli O125 (3%) followed by E. coli O157 and E. coli O55 (1% for each), while in worker's urine samples, E. coli O157 was (3.4%) and E. coli O125 was (1.7%). The same E. coli serogroups were found in both bovine milk and workers' urine, posing a serious threat to both human and animal health due to zoonotic transmission of these important serotypes.

One hundred fifty apparently healthy samples of some seafoods (30 each of Om EL-Khloul "Donax trunculus anatinus", baclawese "Tartufo di mare", boltifish "Oreochromis niloticus", sardine "Sardinella gibbosa" and cuttlefish "Sepia spp.") were randomly purchased during the period from May to August 2006 from Port-Said markets. The samples were examined for enumeration and isolation of halophilic Vibrio species as well as the incidence of Kanagawa positive phenomena in the isolated strains and their public health significance was determined. The incidence of positive samples for halophilic Vibrio species was 96.67% (29), 73.33% ( ), 60.00% (18) 80.00% (24) and 70.00% (21) while the mean values of the total halophilic Vibrio counts were 8.3 X 10 3 , 3.5 X 10 3 , 1.4 X 10 3 , 3.8 X 10 3 and 1.8 X 10 3 CFU/g of Om EL-Khloul "Donax trunculus anatinus", baclawese "Tartufo di mare", boltifish "Oreochromis niloticus", sardine "Sardinella gibbosa" and cuttlefish "Sepia spp." respectively. The numbers of halophilic Vibrio isolates were 69, 52, 41, 59 and 50 while the incidence of Kanagawa positive phenomena in these isolates was 13.04% (9), 9.62% (5), 4.88% (2), 8.47% (5) and 4.00% (2) in the examined samples of Om EL-Khloul "Donax trunculus anatinus", baclawese "Tartufo di mare", boltifish "Oreochromis niloticus", sardine "Sardinella gibbosa" and cuttlefish "Sepia spp." respectively. The bacterial isolates in the examined samples were identified as Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio furnissii, Vibrio fluvialis, Vibrio mimicus and Vibrio metschnikovii. The relationship between the number of the isolated strains and Kanagawa positive phenomena in the examined samples were discussed.

Animals have been used in the evaluation of probiotic potentials of lactic acid bacteria for the development of functional food as only in-vitro tests may not be enough to ascertain the probiotic ability of an organism and its safety in a living host. The action of a probiotic Pediococcus acidilactici strain isolated from Wara, a Nigerian milk product, in the treatment of Diarrhea in wistar rats infected orally with Diarrhoeagenic Enterotoxigenic Escherichia coli as well as the survival and microflora modulation of the probiotic strain in the gastrointestinal tract were evaluated. Five groups of seven rats each were infected and treated as the case may be with the pathogen and probiotic respectively. Each group received specific treatments for 30 days, during which the animals were closely monitored and the faecal samples were analyzed for the trend of E. coli and LAB counts. Also, specific organs of the Gastrointestinal tract (GIT) were examined for any histomorphological disparity...

Maize-meal porridge is commonly consumed meal for the adults as breakfast food and for the children as complementary food. Food-to-food fortification was employed in order to improve the protein content of maize-meal porridge using soy flour and local groundnut paste. The study was aimed at evaluating the nutritional properties and consumer preference of the attributes of the unfortified porridge, legume-fortified porridge, and powdered milk-fortified porridge. The influence of consumers’ knowledge of the type of fortificant added to the porridge was also investigated. Soy-fortified porridges provide comparable ash, crude fibre and fat contents to powdered milk- fortified porridge but with higher protein than powdered milk-fortified porridge. Soy flour raised the protein and ash content of the porridge by 90% and 63% respectively, the groundnut paste raised the protein and ash content by 88% and 41% and the powdered milk by 87% and 65% respectively. The unfortified porridge was the ...

General Synthesis Information. Reactions were run in screw capped glass vials (4 mL) stirred with Teflon®-coated magnetic stir bars. Moisture and air-sensitive reactions were performed in flame-dried round bottom flasks, fitted with rubber septa or glass gas adapters, under a positive pressure of nitrogen. Moisture and air-sensitive liquids or solutions were transferred via nitrogen-flushed syringe. Concentration of solvents was accomplished by rotary evaporation using a Büchi rotary evaporator at temperatures between 35 °C and 50 °C. Experiments were monitored by thin layer chromatography (TLC). Materials. Unless otherwise noted, materials were obtained from commercial suppliers and used without purification. Removal of solvent under reduced pressure refers to distillation with a Büchi rotary evaporator attached to a vacuum pump (~3 mmHg). Products obtained as solids or high boiling oils were dried under vacuum (~1 mmHg). Whatman 250 micron aluminum backed UV F254 precoated silica gel flexible plates. Subsequent to elution, ultraviolet illumination at 254 nm allowed for visualization of UV active materials. Staining with panisaldehyde, basic potassium permanganate solution, or Molisch's reagents allowed for further visualization. The retardation factor (Rf) is the ratio of the distance traveled by the compound to the distance traveled by the eluent. Physical Data. Proton nuclear magnetic resonance spectra ( 1 H NMR) were recorded on Avance 300 or Avance 500 MHz nuclear magnetic resonance spectrometers. Chemical shifts for 1 H NMR spectra are reported as in units of parts per million (ppm) relative to tetramethylsilane ( 0.0) using the residual solvent signal as an internal standard or tetramethylsilane itself: chloroform-d ( 7.26, singlet). The number of protons (n) for a given resonance is indicated by nH. IR spectra were recorded on Bruker Alpha spectrometer and mass analyses (ESI) were performed on Finnegan MAT 1020 mass spectrometer operating at 70 eV. General procedure for the Zn(OTf) 2 -catalyzed Ferrier reaction. To a stirred solution of 3,4,6-tri-O-acetyl-D-glucal 1 (1 equiv) and acceptor (1.2 equiv) in anhydrous 1,2-dichloroethane (2 mL/mmol) under an atmosphere of argon was added Zn(OTf) 2 (5-10 mol%) at room temperature. The reaction mixture was stirred until the complete consumption of the starting material (glycal). The solvent was filtered, filtrate was concentrated in vacuo, the crude residues were redissolved in dichloromethane and loaded on a silica gel column. The product was purified by silica gel chromatography using Hexane/EtOAc to afford the 2,3-unsaturated -glycosides as major product in excellent yields. All the Ferrier products were confirmed by IR, 1 H NMR, 13 C NMR and MS/HRMS spectroscopy. Characterization data of compounds 3a-3n. Benzyl 4,6-di-O-acetyl-2,3-dideoxy--D-erythro-hex-2enopyranoside (3a) Yield: 116 mg (98%); viscous liquid.

This study reports the isolation of lactic acid bacteria from 13 honey samples produced in Malaysia, Libya and Saudi Arabia and their antibacterial activity against three Gram negative pathogenic bacteria. Approach: A modified MRS agar with 0.8% CaCO 3 and MRS with 1% glucose was found to facilitate isolation of LAB compared to MRS, tomato juice agar and modified tomato juice agar. 32 isolates were confirmed LAB by catalase test and Gram staining. Six isolates were screened for antibacterial activity and identified as strains of Lactobacillus acidophilus 1 by API CH50. Results: All the isolates showed very good inhibitory activity against target Gram negative bacteria as indicated by the diameter of inhibition zone: Salmonella Typhimurium (23-30 mm), Escherichia coli (7-18 mm) and Enterobacter aerogenes (10-18 mm) after 24 h incubation at 30°C. Supernatants of L. acidophilus 1 strains showed good antibacterial activity against all target bacteria. Heating the supernatants at 90 and 121°C for 1 h enhanced the antibacterial activity against all target bacteria except supernatants H006-A and H010-G against S. Typhimurium. Antibacterial activity of supernatants were maintained after pH adjustment to 3, but at pH5 supernatants H006-A, H008-D and H010-G lost the activity against S. Typhimurium and E. coli within 48 h of incubation while at pH 6 all supernatants lost activity except against E. aerogenes. Enzymes treatments of supernatants with RNase II and Proteinase K for 1 h inhibited all target bacteria except supernatants H008-D, H008-E and H006-A which were relatively sensitive to both enzymes against S. Typhimurium and E. coli. Conclusion/Recommendations: In conclusion, honey from different sources contains strains of L. acidophilus 1 that produced compounds with good antibacterial activity which may be responsible for the antibacterial properties of honey.

ABSTRACTWe undertook a study of the mechanism by which Dr-positive bacteria invade epithelial cells. Our findings show that Dr-positive bacteria enter via a zipper-like mechanism that is independent of the Dr-induced mobilization of F-actin and of the signaling molecules that control Dr-induced F-actin rearrangements. We also observed that Dr-positive IH11128 bacteria entered cells that were positive for the caveola marker VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) to the same extent as those that were not (parental Caco-2 cells). Using fluorescence labeling and confocal laser scanning microscopy, we provide evidence that during the adhesion step, the α5β1 integrin, which plays a pivotal role in Afa/Dr diffusely adheringEscherichia colibacterial entry, is mobilized around adhering Dr-positive bacteria. We show that the receptor for Afa/Dr adhesins, glycosylphosphatidylinositol-anchored CD55; the raft marker, ganglioside GM1; and VIP21/caveolin are all recruited around adhering Dr-...

Although the transmission of coxsackievirus B3 occurs mainly via the oral route, little is known about the primary replication and persistence of this agent in the intestine. To address this question, BALB/c mice were inoculated by gavage with coxsackievirus B3, Nancy strain. The mice were killed from 1 hr to 90 days after infection. The viral markers were detected in the small intestine using RT-PCR, cell culture and detection of VP1 protein. Coxsackievirus B3 was detected positive by the three methods from hr 2 to day 45 after infection. By using monoclonal antibodies directed towards VP1, CD40 and CD26, the virus was shown to be present in the lymphocytes of the mucosa as soon as 2 hr after infection; in contrast, no virus was detected in the epithelial cells lining the intestinal lumen. Further experiments were performed to evaluate the capacity of coxsackievirus B3 to establish a persistent infection in two intestinal cell lines. In contrast to HT29 cells, the CaCo-2 cells were shown to develop a persistent infection for up to 20 passages, as demonstrated by the detection of viral RNA and VP1 protein. This study provides further evidence that, after infection by the oral route, the viral particles are concentrated in the lymphocytes of the mucosal layer. In addition, the results suggest that coxsackievirus B3 is capable of establishing a persistent infection in the small intestine that may act as a reservoir of viral particles for the delayed spread of the virus to other target organs.

Background The rhizome of Zingiber cassumunar and the seed of Nigella sativa are two ingredients in Thai traditional medicine to relieve dysmenorrhea and adjust the menstrual cycle. Mixture of these two herbs produces three esters, namely (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-yl linoleate (1), (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-yl oleate (2) and (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-yl palmitate (3). The aim of this study is to examine in vitro absorption of these esters and evaluate their transport across the membrane. Methods In vitro transport of these three esters was observed in Caco-2 cell monolayers. The ester compounds 1, 2 and 3 at a concentration of 10 μM were hydrolyzed by porcine liver esterase. Results All esters transported across the Caco-2 cell without enzymatic hydrolysis. The apparent permeability coefficients P app of compound 1 at 53 μM and 106 μM were 13.94 (0.60) × 10-6 and 14.33 (0.17) × 10-6cm/s respectively, while those of compound 2 were 9.45 (0.29) × 10...

Two unrelated adult males, aged 36 (patient 1) and 25 whereas complex II was decreased only in the pretherapy muscle of patient 2. Flavin adenine dinucleotide (FAD) and (patient 2) years, presented with subacute carnitinedeficient lipid storage myopathy that was totally and partly flavin mononucleotide (FMN) concentrations in muscle and isolated mitochondria, and the activity of mitochondrial responsive to riboflavin supplementation in the two patients, respectively. Plasma acyl-carnitine and urinary FAD pyrophosphatase, showed that patient 1 had low levels of FAD (46%) and FMN (49%) in mitochondria, with a organic acid profiles indicated multiple acyl coenzyme A dehydrogenase deficiency, which was mild in patient 1 and significant increase (P < 0.01) in mitochondrial FAD pyrophosphatase (273%) compared with controls. Patient severe in patient 2. The activities of short-chain and medium-chain acyl coenzyme A dehydrogenases in 2 had similar low levels of FAD and FMN in both total muscle (FAD and FMN 22% of controls) and mitochondria mitochondrial fractions were decreased, especially in patient 2. This was in agreement with Western blotting (FAD 26%; FMN 16%) and normal activity of mitochondrial FAD pyrophosphatase. All of these results. Flavin-dependent complexes I and II were studied by immunoblotting and densitometric quantification of biochemical parameters were either totally or partly corrected after riboflavin therapy. two-dimensional electrophoresis with comparable results. Complex I was present in normal amounts in both patients, Keywords: acyl-carnitine profile; flavo-coenzyme; mitochondrial FAD pyrophosphatase; multiple acyl coenzyme A dehydrogenase deficiency; OXPHOS enzymes Abbreviations: CoA ϭ coenzyme A; FAD ϭ flavin adenine dinucleotide; FMN ϭ flavin mononucleotide; OXPHOS ϭ oxidative phosphorylation; MAD ϭ multiple acyl coenzyme A dehydrogenase deficiency; MCAD ϭ medium-chain acyl coenzyme A dehydrogenase; SCAD ϭ short-chain acyl coenzyme A dehydrogenase

The association of enterotoxigenic Escherichia coli expressing colonization factor antigen I (CFA/I) with the cultured human colon adenocarcinoma cell, a model of the mature enterocyte of the small intestine, is dependent on the binding of CFA/I to a brush border-associated component. Binding of the purified radiolabeled [125I]CFA/I- and 14C-labeled CFA/I-positive bacteria could be displaced by an increasing concentration of unlabeled CFA/I. Moreover, we showed that expression of the specific CFA/I binding developed as a function of cell differentiation in Caco-2 cells, whereas expression of the nonspecific binding did not. Expression of the brush border differentiation-associated component acting as a binding site for CFA/I was up-regulated by glucose. Indeed, the enterocyte-like HT-29 glc- cell subpopulation not expressing the CFA/I binding site when cultured in dialyzed serum and hexose-free medium regained the ability to bind CFA/I when the cells were returned to culture medium ...

In the present study, the role of direct procaryote-eucaryote interactions in the virulence of Bacillus cereus was investigated. As a model of human enterocytes, differentiated Caco-2 cells were used. Infection of fully differentiated Caco-2 cells with B. cereus in the exponential phase of growth, in order to minimize the concentration of spores or sporulating microorganisms, shows that a strain-dependent cytopathic effect develops. Interestingly, addition of 3-h-old cultures of some strains resulted in complete detachment of the cultured cells after a 3-h infection whereas no such effect was found after a 3-h infection with 16-h-old cultures. Infection of enterocyte-like cells with B. cereus leads to disruption of the F-actin network and necrosis. Even though the effect of secreted factors cannot be ruled out, direct eucaryote-procaryote interaction seems to be necessary. In addition, we observed that some B. cereus strains were able to be internalized in Caco-2 cells. Our findings...

Four human Lactobacillus acidophilus strains were tested for their ability to adhere onto human enterocyte like Caco-2 cells in culture. The LA 1 strain exhibited a high calcium independent adhesive property. This adhesion onto Caco-2 cells required a proteinaceous adhesion promoting factor, which was present in the spent bacterial broth culture supernatant. LA 1 strain also strongly bound to the mucus secreted by the homogeneous cultured human goblet cell line HT29-MTX. The inhib- itory effect of LA 1 organisms against Caco-2 cell adhesion and cell invasion by a large variety of diarrhoeagenic bacteria was investi- gated. As a result, the following dose dependent inhibitions were obtained: (a) against the cell association of enterotoxigenic, diffusely adhering and enteropathogenic Escherichia coli, and Salmonella typhimurium; (b) against the cell invasion by enteropathogenic Eschericha coli, Yersinia pseudotuberculosis, and Salmonella typhimurium. Incubations of L acidophilus LA 1 before and together with enterovirulent E coli were more effective than incubation after infection by E coli.

The influence of cell culture conditions and previous drug exposure on P-glycoprotein (P-gp) expression levels in Caco-2 cells was determined. In this study, the expression of P-gp is demonstrated (i) visually by confocal laser scanning microscopy (CLSM), (ii) functionally by transport studies with substrates of the efflux pump, and (iii) quantitatively by flow cytometry (FCM) analysis using specific monoclonal antibodies (anti P-gp MRK 16 as an external antibody and P-GlycoCheck C219 as an internal antibody). Trypsinization of the cells after reaching confluence led to a decrease of P-gp expression levels, while trypsinization before reaching confluence led to an increase after long-term cultivation. Culturing the cells on polycarbonate filters did not elicit a significant change of P-gp expression over time in culture, whereas in plastic flasks (polystyrene) a decrease was detected. Using CLSM a strong fluorescence on the apical side of Caco-2 cell monolayers was observed, as a result of incubation with MRK 16 as primary and IgG Cy5 as secondary antibody. Previous drug exposure of the cells showed that verapamil, celiprolol, and vinblastine induced the P-gp expression, while metkephamid (MKA) decreased the P-gp expression level as compared to the control. Permeation studies consolidated the theory that P-gp is expressed in the Caco-2 cells examined. For talinolol and MKA, a higher transport from basolateral to apical side than from apical to basolateral could be measured. Incubation of the cell monolayer with MRK 16 reduced the secretion process to the apical side, but did not influence [ 3 H]mannitol flux. Caco-2 cells seem to be a suitable cell line model for P-gp-mediated secretion studies. However, the variability of the P-gp expression requires careful control when this model is to be used in quantitative structure/secretion studies.

Several in vivo and in vitro studies provide convincing evidence for the anticarcinogenic properties of phytic acid (PA) (Inositol hexaphosphate, IP 6 ). The objectives of this investigation were to elucidate the effects of PA on suppression of colonic aberrant crypt foci (ACF) and to study the inhibitory effect of IP 6 on human colon carcinoma, CaCo-2 cell line. Fisher 344 male, weanling rats were divided into 3 groups of 15 each and were fed control diet (AIN 93 G-C) and C+(1 or 2 g/100 g PA in water) for 13 weeks. Rats received 2 s.c. injections of azoxymethane (AOM) in saline at 16 mg/kg body weight at 7 and 8 weeks of age. There was a 36% and 42% reduction in ACF in 1 and 2 g/100 g PA groups compared to control group ðPo0:001Þ. Cytotoxic effect of IP 6 was evaluated on Caco-2 cell line at concentrations of 0.25-4 g mol/l using lactate dehydrogenase (LDH) assay (LDH released from the cytosol of damaged cells into the supernatant), histone-associated DNA fragmentation assay using a cell death detection ELISA s kit and microscopic analysis. The cells were treated with 0.25, 0.5, 1.0, 2.0, 4 g mol/l of IP 6 and incubated for 24 and 48 h. After 24 h of incubation, LDH release ranged from 13.55% to 44.70%. Histone-associated DNA fragmentation (an assay based on the quantitative sandwich-enzyme-immunoassayprinciple directed against DNA and histones which allows the specific determination of mono-and oligonucleosomes in the cytoplasmic fraction of cell lysates) was also dose dependent. CaCo-2 cells grown on slide flaskets in the presence of 0.25 g mol/l IP 6 showed membrane blebbing (zeiosis) characteristic of apoptitic activity. Results of this study indicate that IP 6 might exert anticarcinogenic activity through induction of apoptosis.

The presence of inhibitors of drug eux transporters, such as P-glycoprotein (P-gp), in grapefruit juice (GFJ) was con®rmed based on the uptake of [ 3 H]-vinblastine (VBL) by Caco-2 cells. 2 The uptake of [ 3 H]-VBL by Caco-2 cells was signi®cantly increased by the ethyl acetate extract of GFJ as well as by cyclosporin A. The extract was separated on a Cosmosil column and the eluate with 60% methanol increased [ 3 H]-VBL uptake, while the activity to inhibit CYP3A4 was greatest in the 70 and 80% eluates. 3 These results show that the major inhibitor of eux transport of VBL is dierent from that of CYP3A4. 4 Further separation of the 60% methanol eluate aorded dihydroxybergamottin (DHBG). Both ethyl acetate extract of GFJ and DHBG increased steady-state [ 3 H]-VBL uptake by LLC-GA5-COL300 cells. Besides DHBG, other furanocoumarins contained in GFJ, such as bergamottin, FC726, bergaptol and bergapten, increased the steady-state uptake of [ 3 H]-VBL by Caco-2 cells. 5 The order of inhibitory potency of these compounds was FC7264DHBG4bergamottin4ber-gapten4bergaptol. While, the IC 50 values for inhibition of CYP3A4 were 0.075, 0.45, 1.0, 1.0 and 420 mM, respectively. Bergaptol speci®cally inhibited VBL eux. 6 DHBG was thus identi®ed as a candidate for inhibitors of VBL transport, together with other furanocoumarins. Moreover, partly involvement of the P-gp inhibition was suggested. 7 Therefore, the inhibition of eux transport of drugs as well as of drug metabolism by CYP3A4 could be an important cause of drug-GFJ interaction.

The presence of inhibitors of drug efflux transporters, such as P‐glycoprotein (P‐gp), in grapefruit juice (GFJ) was confirmed based on the uptake of [3H]‐vinblastine (VBL) by Caco‐2 cells. The uptake of [3H]‐VBL by Caco‐2 cells was significantly increased by the ethyl acetate extract of GFJ as well as by cyclosporin A. The extract was separated on a Cosmosil column and the eluate with 60% methanol increased [3H]‐VBL uptake, while the activity to inhibit CYP3A4 was greatest in the 70 and 80% eluates. These results show that the major inhibitor of efflux transport of VBL is different from that of CYP3A4. Further separation of the 60% methanol eluate afforded dihydroxybergamottin (DHBG). Both ethyl acetate extract of GFJ and DHBG increased steady‐state [3H]‐VBL uptake by LLC‐GA5‐COL300 cells. Besides DHBG, other furanocoumarins contained in GFJ, such as bergamottin, FC726, bergaptol and bergapten, increased the steady‐state uptake of [3H]‐VBL by Caco‐2 cells. The order of inhibitory p...

~What is the difference between an obstacle and an opportunity? Our attitude toward it. Every opportunity has a difficulty, and every difficulty has an opportunity~ J.Sidlow Baxter. This dissertation is presented to you not only on my own accord. It would not have been possible without the assistance of various contributors. I would like to acknowledge the following individuals:  Firstly, my Heavenly Father. Without you precious talents that you granted on me, gracious love, guidance and protection none of this would have been possible.  To my parents, Elna and Hennie van Niekerk. Thank you for your continuous love, guidance, encouragement and believing in me. Without you support over all these years, this would have been an impossible task to finish.  To my brother, Cornèl van Niekerk. Thank you for your support and being my partner in crime.  Murray-Hancke Oberholster, closer to a real sister I could not get. Thank you for every little thing you do for me, I do not deserve a friend like you, you cared for me and put up with my mixed emotions at times, but still you kept on encouraging me.  Lauren Cilliers, your friendship, support and help through these two years meant more to me than you will ever believe. I will always remember and appreciate you.  My fellow students, especially Corneli Jacobzs, Alex Laux, Carmen Annandale, Anja Haasbroek and Sarika du Plessis, thank you that you were always willing to lend a helping hand, give advice and contributed to a positive working environment.  Prof. Sias Hamman, my study leader. I would like to thank you for the opportunity that you gave me to do my masters degree and guiding me with so much compassion and dedication. The respect and admiration that I have for your superb academic ability as a leader in pharmaceutical research is indescribable. It was a privilege to work under your supervision and guidance.  Dr. Dewald Steyn and Dr. Liezl Badenhorst, thank you for your support and being always willing to help during these two years. I appreciate it.  Prof. Jan du Preez, thank you for your assistance with the HPLC characterisation of my samples.  Prof. Suria Ellis, for assisting me with my statistical analysis.

Mutations in the HFE gene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake , FASEB J 15, 1276-1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, b-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and b-2 microglobulin (b2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE-b2m to the apical membrane. The amount of HFE-b2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or b2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE-b2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.

Diarrheagenic Escherichia coli (ETEC) bearing CFA/I or CFA/II adhesive factors specifically adhere onto the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. Heat-killed Lactobacillus acidophih~s strain LB, that adheres onto Caco-2 cells, inhibits diarrheagenic Escherichia coli adhesion in a concentration-dependent manner. Since the L. acidophilus does not express ETEC-CFA adhesive factors, it can be postulated that the heat-killed L. acidophilus LB cells inhibit diarrheagenic E. coli attachment by steric hindrance of the human enterocytic ETEC receptors.

The effect of N-trimethylchitosan chloride on the intestinal permeability of capreomycin sulfate, a polypeptide antibiotic, using an in vitro model, was evaluated. To improve the mucoadhesivity and permeation enhancer properties of N-trimethylchitosan chloride, it ...

ABSTRACTWe undertook a study of the mechanism by which Dr-positive bacteria invade epithelial cells. Our findings show that Dr-positive bacteria enter via a zipper-like mechanism that is independent of the Dr-induced mobilization of F-actin and of the signaling molecules that control Dr-induced F-actin rearrangements. We also observed that Dr-positive IH11128 bacteria entered cells that were positive for the caveola marker VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) to the same extent as those that were not (parental Caco-2 cells). Using fluorescence labeling and confocal laser scanning microscopy, we provide evidence that during the adhesion step, the α5β1 integrin, which plays a pivotal role in Afa/Dr diffusely adheringEscherichia colibacterial entry, is mobilized around adhering Dr-positive bacteria. We show that the receptor for Afa/Dr adhesins, glycosylphosphatidylinositol-anchored CD55; the raft marker, ganglioside GM1; and VIP21/caveolin are all recruited around adhering Dr-...

In the present study, comparative pharmacokinetics study between [Di (4-amino N-acetyl) phenoxy] methyl ketone (DPMK) and paracetamol in rats were investigated. DPMK and paracetamol were orally given to experimental rats in dose 500 mg/kg/b.wt. At various time intervals, blood (1 h -24 h) and urine (6 h -84 h) sample was collected from each rat. The drug concentrations were measured in blood supernatants and urine samples by using validated UV-visible spectrophotometric method. Various pharmacokinetic parameters were calculated by non compartmental model for DPMK and paracetamol. DPMK reached a higher concentration Cmax =22.26 ± 1.85 µg/mL after oral administration in rat as compared to standard paracetamol Cmax = 5.18 ± 0.57 µg/mL. There was a significant increased in the AUC (0-∞) = 85.82 ± 8.67 µg h/mL and AURC (0-∞) = 338.86 ± 53.76 mg of DPMK as compared to the paracetamol AUC (0-∞) = 20.51 ± 1.79 µg h/mL and AURC (0-∞)= 42.97 ± 1.58 mg. No significant differences were found in elimination rate constant during the PK study of DPMK and paracetamol. The apparent volume distribution (Vss = 7.24 ± 1.51 lit) and total clearance (CL = 171 ± 0.24 lit/h) for DPMK were found to be less as compared to the paracetamol (Vss = 29.40 ± 4.17 lit, CL = 7.43 ± 0.89 lit/h). Furthermore, the relative bioavailability of DPMK was found to be in the range 4.18-7.92 for both blood and urine sample.

Purpose. To investigate the effects of passaging on the intrinsic membrane transport parameters of compounds absorbed by means of passive and carrier-mediated processes in the Caco-2 cell line. Methods. Caco-2 cells at low (28–36) and high (93–108) passage numbers were used to evaluate the transport characteristics of model compounds for paracellular diffusion (mannitol), transcellular diffusion (progesterone) and carrier-mediated transport (cephalexin, cephradine, phenylalanine, proline, and taurocholic acid) using side-by-side diffusion chambers. Intrinsic intestinal transport parameters were determined by correcting the effective permeability for potential biases introduced by the microporous filter and aqueous boundary layer. Intrinsic maximal flux (J max, Michaelis constant (K m) and carrier permeability (P c) were determined as a function of passage number. Results. Compared to the low passaged cells, the high passaged Caco-2 cells were characterized by less morphological hete...

In the present investigation, the uptake and transport kinetics of valacyclovir (VACV), 5-aminolevulinic acid (5-ALA), and benzylpenicillin (BENZ) were studied in stably transfected Madin-Darby canine kidney (MDCK)/human peptide transporter 1 (hPepT1)-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein (low, medium, and high expression) and in Caco-2 cells to delineate hPepT1-mediated transport kinetics. These compounds were selected due to the fact that they are known PepT1 substrates, yet also have affinity for other transporters. Caco-2 cells, traditionally used for studying peptide-based drug transport, were included for comparison purposes. The time, pH, sodium, and concentration dependence of cellular uptake and permeability were measured using mock, clonal hPepT1-MDCK, and Caco-2 cells. A pH-dependent effect was observed in the hPepT1-expressing clones and Caco-2 cells, with an increase of 1.96-, 1.84-, and 2.05-fold for VACV, 5-ALA, and BENZ uptake, respectively, at pH 6 versus 7.4 in the high-expressing hPepT1 cells. BENZ uptake was significantly decreased in Caco-2 and MDCK cells in Na ϩdepleted buffer, whereas VACV uptake only decreased in Caco-2 cells. Concentration-dependent uptake studies in the mock-corrected hPepT1-MDCK and Caco-2 cells demonstrated hPepT1 affinity ranking of VACV Ͼ 5-ALA Ͼ BENZ. The apical-to-basal apparent permeability coefficient (P app ) values of VACV, 5-ALA, and BENZ in mock-corrected hPepT1-MDCK cells showed solely hPepT1-mediated transport in contrast to Caco-2 cells. Lower K m values and higher P app in Caco-2 cells compared with hPepT1-MDCK cells suggested the involvement of multiple transporters in Caco-2 cells. Thus, hPepT1-MDCK cells corrected for endogenous transporter expression may be a more appropriate model for screening compounds for their affinity to hPepT1.

Outbreaks of various forms of diarrhoea, particularly cholera and typhoid, are a frequent occurrence in low income countries. Poor water sanitation and hygiene practices are frequently implicated for all diarrhoeal out breaks in the low income countries. The present study assessed (i) the probiotic potential of selected strains of lactic acid bacteria in terms of their inhibition activities against Escherichia coli O157:H7 and Salmonella typhi , and the removal of heavy metals in wastewater. The selected lactic acid bacteria had probiotic properties, namely resistance to low pH (pH2 and 3), tolerance to bile salts and sodium chloride, and significant antimicrobial activities against strains of E. coli O157:H7 and S. typhi ( p &lt; 0.001). Strains of lactic acid bacteria were shown to modulate the concentration of aqueous Cu, Mn, NH4 + and Zn with a significant net decrease between Day 7 or 10 (p ≤ 0.05). The selected lactic acid bacteria were also shown to deploy biofilms on surface...

Polychlorinated biphenyls (PCBs) dissolved in dietary fat are absorbed in the gastrointestinal tract by the enterocytes in combination with the fatty acids proceeding from the lipid hydrolysis in the gut lumen. The effect of fatty acid absorption on the uptake and transport of 14 PCBs in enterocytes was studied using monolayers of the human intestinal Caco-2 cell line as a model system. The diffusive resistance of the unstirred water layer and the facilitating role of mixed bile salt micelles on the PCB uptake were examined by varying the thickness of the unstirred water layer adjacent to the apical membrane. In additional experiments, the polarity of the PCB uptake and transport in Caco-2 cells was determined. The solubility of PCBs in the mixed bile salt-fatty acid micelles was 2.7to 4.8fold higher than the solubility in plain bile salt micelles. Both the uptake and transport of PCBs in Caco-2 cells were significantly higher (up to 10-fold) when the PCBs were presented mixed with fatty acids. Reducing the thickness of the unstirred water layer resulted in an increased uptake of PCBs. The PCB uptake in Caco-2 cells exceeded the uptake as expected from monomer diffusion only, indicating that bile salt micelles facilitate the PCB transport over the unstirred water layer. Concentrations of dichlorobiphenyls accumulating in the basolateral medium stayed unexpectedly low, suggesting that Caco-2 cells might possess the capability of metabolizing lower chlorinated biphenyls. Uptake of PCBs into the Caco-2 cells was not significantly different whether the PCBs were presented at the apical side or at the basolat-era1 side. However, transport of PCBs over the cell monolayer was significantly higher when the PCBs were presented at the apical side as compared to the basolateral side, suggesting that the unidirectional transport of lipids and lipoproteins affected the PCB transport as well. I Our studies indicate that monolayers of the Caco-2 cell line offer a useful model system for studying the intestinal uptake and transport processes of hydrophobic xenobiotics such as polychlorinated biphenyls-Dulfer, W. J., J. P. Groten, and H. A. J. Covers. Effect of fatty acids and the aqueous diffusion barrier on the uptake and transport of polychlorinated biphenyls in Caco-2 cel1s.

ABSTRACTWe undertook a study of the mechanism by which Dr-positive bacteria invade epithelial cells. Our findings show that Dr-positive bacteria enter via a zipper-like mechanism that is independent of the Dr-induced mobilization of F-actin and of the signaling molecules that control Dr-induced F-actin rearrangements. We also observed that Dr-positive IH11128 bacteria entered cells that were positive for the caveola marker VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) to the same extent as those that were not (parental Caco-2 cells). Using fluorescence labeling and confocal laser scanning microscopy, we provide evidence that during the adhesion step, the α5β1 integrin, which plays a pivotal role in Afa/Dr diffusely adheringEscherichia colibacterial entry, is mobilized around adhering Dr-positive bacteria. We show that the receptor for Afa/Dr adhesins, glycosylphosphatidylinositol-anchored CD55; the raft marker, ganglioside GM1; and VIP21/caveolin are all recruited around adhering Dr-...

The possibility of developing bioadhesive drug delivery systems on the basis of molecules which selectively bind to the small intestinal epithelium by specific, receptor-mediated mechanisms was investigated using a lectin isolated from tomato fruits (Lycopersicum esculentum). The tomato lectin (TL) was found to bind specifically onto both isolated, fixed pig enterocytes and monolayers of human Caco-2 cell cultures with a

BackgroundPhosphatidylcholine (PC) is a major lipid of the gastrointestinal mucus layer. We recently showed that mucus from patients suffering from ulcerative colitis has low levels of PC. Clinical studies reveal that the therapeutic addition of PC to the colonic mucus using slow release preparations is beneficial. The positive role of PC in this disease is still unclear; however, we have recently shown that PC has an intrinsic anti-inflammatory property. It could be demonstrated that the exogenous application of PC inhibits membrane-dependent actin assembly and TNF-α-induced nuclear NF-κB activation. We investigate here in more detail the hypothesis that the exogenous application of PC has anti-inflammatory properties.MethodsPC species with different fatty acid side chains were applied to differentiated and non-differentiated Caco-2 cells treated with TNF-α to induce a pro-inflammatory response. We analysed TNF-α-induced NF-κB-activation via the transient expression of a NF-κB-luci...

Amphiphilic polymers for dual drug delivery have been a focus of research in recent years. We have previously developed and characterized Lauroyl sulphated chitosan (LSCS). Here biological characterizations like mucoadhesion, cytotoxicity, calcium binding, tight junction opening and enzymatic degradation studies were performed to understand its applicability. In vitro drug release properties of both hydrophilic insulin and hydrophobic curcumin were carried out. The biological activity and stability of released insulin were also studied. The stability studies of encapsulated curcumin and uptake studies have also been carried out. LSCS showed strong mucoadhesion and 100% of non-toxicity. LSCS could transiently open tight junctions between Caco-2 cells and thus increase the paracellular permeability. LSCS enhanced calcium binding properties and decreased enzymatic degradation rate retaining insulin activity. LSCS could protect curcumin from photodegradation and could also enter into the cells. From release studies, LSCS was found to be a suitable candidate for both drugs.

The aim of the study was to investigate the effect of paclitaxel loaded nanocarrier systems (chitosan nanoparticles or quantum dots) on breast cancer. Paclitaxel loaded chitosan nanoparticles and quantum dots were prepared with the particle size of 153.6 nm and 302.8 nm respectively. The activities nanocarrier systems were determined using 7-12-dimethylbenz a antracene (DMBA)   induced breast cancer model in six-week-old female, nonpregnant, Wistar albino rats. Paclitaxel loaded nanocarrier systems were administered intraperitoneally to tumor bearing rats and their tumor volumes were measured. At the end of the experiment rats were sacrificed and their tissue sections were analyzed. Both nanocarrier systems (chitosan nanoparticles or quantum dots) successfully reduced the tumors size. This study provides some information about the preparation techniques of paclitaxel loaded nanocarriers and some possible alternatives or strategies for the treatment of breast cancer using these nanocarriers.

cats fed the probiotic bacterium also displayed significantly lower cutaneous hypersensitivity response to histamine injection, lower RBC sedimentation rate, and lower IL-6 & TNF-a production in response to LPS stimulation. In contrast, plasma and salivary IgA levels were significantly elevated in AHC7 treated cats. None of these parameters were altered in the placebo diet group. There was no significant treatment difference in daily food intake, weekly body weight, serum a-acid glycoprotein, plasma IgG and IgM, or IFN-g, IL-4 and IL-10 production. In conclusion, Bifidobacterium animalis AHC7 fed for 12 weeks (at 8x109 cfu per day) altered the feline gastrointestinal microbiota and significantly attenuated a number of pro-inflammatory biomarkers. This study provides further support for the concept that modification of the gastrointestinal microbiota can dramatically alter immunological activity at sites outside the gastrointestinal tract and these effects are not restricted to certain mammalian species.

Okadaic Acid (OA) the major diarrheic shellfish poisoning (DSP) toxin is known as a tumor promoter and seems likely implicated in the genesis of digestive cancer. Little is known regarding genotoxicity and carcinogenicity of Domoic Acid (DA), the major Amnesic Shellfish Poisoning (ASP) toxin. Both OA and DA occur in seafood and are of human health concerns. Micronuclei (MN) arise from abnormalities in nuclear division during mitosis due to a failure of the mitotic spindle or by complex chromosomal configurations that pose problems during anaphase. In order to evaluate the ability of okadaic acid (OA) and domoic acid (DA) to induce DNA damage we performed the micronucleus assay using the Caco-2 cell line. To discriminate between a clastogenic or aneugenic effect of OA and DA, the micronucleus assay was conducted by cytokinesis-block micronucleus assay using cytochalasin B with Giemsa staining and/or acridine orange staining, in parallel to fluorescence in situ hybridization (FISH) using a concentrated human pan-centromeric chromosome paint probe. Our results showed that OA and DA significantly increased the frequency of MN in Caco-2 cells. The MN caused by OA are found in mononucleated cells and binucleated cells, whereas those caused by DA are mainly in binucleated cells. The results of FISH analysis showed that OA induced centromere-positive micronuclei and DA increased the percentage of MN without a centromeric signal. In conclusion, both OA and DA bear mutagenic potential as revealed in Caco-2 cells by induction of MN formation. Moreover, OA induced whole chromosome loss suggesting a specific aneugenic potential, whereas DA seems simply clastogenic. At present, one cannot rule out possible DNA damage of intestinal cells if concentrations studied are reached in vivo, since this may happen with concentrations of toxins just below regulatory limits in case of frequent consumption of contaminated shell fishes.