betadex and Escherichia-coli-Infections

Non-spherical structures are beneficial to advance drug delivery effectiveness compared with common spherical ones, due to increased drug loading capability, improved bonding to a vascular wall, enhanced cellular uptake efficacy and prolonged circulation times. In this study, flower-like Zinc oxide-βcyclodextrin (βCD) nanostructures functionalized by 3-mercaptopropionic acid (MPA) as a non-spherical delivery system was successfully synthesized for aqueous delivery of curcumin (CUR) to enhance its targeting, bioavailability, and release profile. Terminal carboxyl functional groups were used for the conjugation of folic acid (FA) with the aim of active targeting to folate overexpressing breast cancer cells. The in vitro experimental study and mathematical modeling of CUR release revealed a sustained release with Fickian diffusion as the major release mechanism. MTT, colony formation and Annexin-V FITC/PI assays showed the superior anticancer effect of the system compared to free CUR against breast cancer cell line MDA-MB-231 by promoting the apoptotic respond with no cytotoxic effect on HEK293 normal cells. The efficacy of targeting strategy with FA moieties was demonstrated using the augmented cellular uptake of the FA-conjugated system on overexpressed folate receptor alpha (FRα) cells (MDA-MB-468 breast cancer cell line). Furthermore, loading of CUR to the delivery systems significantly lowered the MIC values (2.5 to 5-fold) against S. aureus and E. coli the infections of which are serious problems in cancer patients. According to the results of this study, the system can serve as a promising non-spherical delivery vehicle for enhancing bioavailability and targeting of hydrophobic anticancer agents in the future.

Topics: 3-Mercaptopropionic Acid; Antineoplastic Agents; Apoptosis; beta-Cyclodextrins; Breast Neoplasms; Cell Line, Tumor; Curcumin; Drug Carriers; Escherichia coli; Escherichia coli Infections; Female; Folic Acid; HEK293 Cells; Humans; Nanostructures; Staphylococcal Infections; Staphylococcus aureus; Zinc Oxide

The ileal lesions of Crohn's disease (CD) patients are colonized by adherent-invasive Escherichia coli (AIEC) bacteria. These bacteria adhere to mannose residues expressed by CEACAM6 on host cells in a type 1 pilus-dependent manner. In this study, we investigated different antagonists of FimH, the adhesin of type 1 pili, for their ability to block AIEC adhesion to intestinal epithelial cells (IEC). Monovalent and multivalent derivatives of n-heptyl α-d-mannoside (HM), a nanomolar antagonist of FimH, were tested in vitro in IEC infected with the AIEC LF82 strain and in vivo by oral administration to CEACAM6-expressing mice infected with LF82 bacteria. In vitro, multivalent derivatives were more potent than the monovalent derivatives, with a gain of efficacy superior to their valencies, probably owing to their ability to form bacterial aggregates. Of note, HM and the multi-HM glycoconjugates exhibited lower efficacy in vivo in decreasing LF82 gut colonization. Interestingly, HM analogues functionalized with an isopropylamide (1A-HM) or β-cyclodextrin pharmacophore at the end of the heptyl tail (1CD-HM) exerted beneficial effects in vivo. These two compounds strongly decreased the amount of LF82 bacteria in the feces of mice and that of bacteria associated with the gut mucosa when administered orally at a dose of 10 mg/kg of body weight after infection. Importantly, signs of colitis and intestinal inflammation induced by LF82 infection were also prevented. These results highlight the potential of the antiadhesive compounds to treat CD patients abnormally colonized by AIEC bacteria and point to an alternative to the current approach focusing on blocking proinflammatory mediators.. Current treatments for Crohn's disease (CD), including immunosuppressive agents, anti-tumor necrosis factor alpha (anti-TNF-α) and anti-integrin antibodies, focus on the symptoms but not on the cause of the disease. Adherent-invasive Escherichia coli (AIEC) bacteria abnormally colonize the ileal mucosa of CD patients via the interaction of the mannose-specific adhesin FimH of type 1 pili with CEACAM6 mannosylated proteins expressed on the epithelial cell surface. Thus, we decided to develop an antiadhesive strategy based on synthetic FimH antagonists specifically targeting AIEC bacteria that would decrease intestinal inflammation. Heptylmannoside (HM)-based glycocompounds strongly inhibit AIEC adhesion to intestinal epithelial cells in vitro. The antiadhesive effect of two of these compounds of relatively simple chemical structure was also observed in vivo in AIEC-infected CEACAM6-expressing mice and was associated with a reduction in the signs of colitis. These results suggest a new therapeutic approach for CD patients colonized by AIEC bacteria, based on the development of synthetic FimH antagonists.

Topics: Adhesins, Escherichia coli; Administration, Oral; Animals; Antigens, CD; Bacterial Adhesion; Bacterial Load; beta-Cyclodextrins; Cell Adhesion Molecules; Crohn Disease; Drug Discovery; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Feces; Fimbriae Proteins; Fimbriae, Bacterial; Glycoconjugates; GPI-Linked Proteins; Intestinal Mucosa; Intestines; Mannosides; Mice

Research on mastitis in dairy cows caused by Escherichia coli has reported the emergence of strains capable of inducing chronic mastitis and that these strains adhered to and internalized into bovine mammary epithelial cells better than strains of E. coli isolated from acute mastitis. To understand mechanisms and strategies used by chronic E. coli strains to survive intracellularly internalization studies using bovine mammary epithelial cells treated with inhibitors of caveolae-mediated endocytosis (CME) and receptor-mediated endocytosis (RME), double immunofluorescence labeling confocal laser and fluorescence microscopy were conducted. Internalization studies showed that strains chronic E. coli strains persisted intracellularly longer than acute E. coli strains. Treatment of bovine mammary epithelial cells CME or RME inhibitors resulted in lower numbers of intracellular E. coli strains associated with chronic or acute mastitis than untreated controls. In addition, when selective CME inhibitors were used significantly fewer chronic E. coli were detected intracellularly than acute E. coli or untreated controls. Confocal laser microscopy showed that chronic E. coli strains colocalized preferentially with caveolae whereas acute strains did so with early endosomes, an early step of RME. These results suggest that strains of E. coli associated with chronic mastitis exploit lipid rafts/CME to internalize into and move through mammary epithelial cells. By exploiting this endocytosis pathway, chronic E. coli strains avoid bactericidal mechanisms such as endosome acidification and endosome-lysosome fusion, thus allowing intracellular survival. Data from this study helps to explain how these strains are capable of causing chronic E. coli mastitis.

Topics: Animals; beta-Cyclodextrins; Cadaverine; Cattle; Caveolae; Cell Line; Cell Survival; Endocytosis; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Female; Filipin; Mastitis, Bovine; Microscopy, Confocal

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes outbreaks of bloody diarrhea and the hemolytic-uremic syndrome. EHEC intimately adheres to epithelial cells, effaces microvilli and induces attaching-effacing (AE) lesions. Detergent-resistant microdomains (lipid rafts) serve as membrane platforms for the recruitment of signaling complexes to mediate host responses to infection. The aim of this study was to define the role of lipid rafts in activating signal transduction pathways in response to AE bacterial pathogens. Epithelial cell monolayers were infected with EHEC (MOI 100:1, 3 h, 37 degrees C) and lipid rafts isolated by buoyant density ultracentrifugation. Phosphoinositide 3-kinase (PI3K) localization to lipid rafts was confirmed using PI3K and anti-caveolin-1 antibodies. Mice with cholesterol storage disease Niemann-Pick, type C were used as in vivo models to confirm the role of lipid rafts in mediating signaling response to AE organisms. In contrast to uninfected cells, PI3K was recruited to lipid rafts in response to EHEC infection. Metabolically active bacteria and cells with intact cholesterol-rich microdomains were necessary for the recruitment of second messengers to lipid rafts. Recruitment of PI3K to lipid rafts was independent of the intimin (eaeA) gene, type III secretion system, and production of Shiga-like toxins. Colonization of NPC(-/-) colonic mucosa by Citrobacter rodentium and AE lesion formation were both delayed, compared with wild-type mice infected with the murine-specific AE bacterial pathogen. C. rodentium-infected NPC(-/-) mice had reduced colonic epithelial hyperplasia (64+/-8.251 vs 112+/-2.958 microm; P<0.05) and decreased secretion of IFN-gamma (17.6+/-17.6 vs 71+/-26.3 pg/ml, P<0.001). Lipid rafts mediate host cell signal transduction responses to AE bacterial infections both in vitro and in vivo. These findings advance the current understanding of microbial-eukaryotic cell interactions in response to enteric pathogens that hijack signaling responses mediated through lipid rafts.

Topics: Animals; beta-Cyclodextrins; Cell Line; Citrobacter rodentium; Escherichia coli Infections; Escherichia coli O157; Host-Pathogen Interactions; Humans; Membrane Microdomains; Mice; Mice, Inbred BALB C; Models, Animal; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Virulence

Enteropathogenic Escherichia coli (EPEC) is a significant cause of paediatric diarrhoea worldwide. Virulence requires adherence to intestinal epithelial cells, mediated in part through type IV bundle-forming pili (BFP), and the EPEC protein Tir. Tir is inserted into the enterocyte plasma membrane (PM), resulting in the formation of actin-rich pedestals. Tir is translocated by the type III secretion system (TTSS), through a pore comprised of EPEC proteins inserted into the PM. Here, we demonstrate that in the absence of BFP, EPEC adherence, effector translocation and pedestal formation are dependent on lipid rafts. Lipid raft disruption using methyl-beta-cyclodextrin (MbetaCD) decreased adherence by an EPEC BFP-deficient strain from 85% to 1%. Translocation of the effectors Tir and EspF was blocked by MbetaCD treatment, although the TTSS pore still formed. MbetaCD treatment after Tir delivery decreased pedestal formation by EPEC from 40% to 5%, but not by the related pathogen E. coli O157:H7 which uses a different Tir-based mechanism. In contrast, EPEC expressing the BFP can circumvent the requirement for membrane cholesterol. This suggests that lipid rafts play a role in virulence of this medically important pathogen.

Topics: Bacterial Adhesion; beta-Cyclodextrins; Carrier Proteins; Cell Membrane; Cholesterol; Diarrhea; Enterocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Fimbriae, Bacterial; Humans; Intracellular Signaling Peptides and Proteins; Membrane Microdomains; Protein Transport; Receptors, Cell Surface