Treatment and prevention of antibiotic associated diarrhea

Mild or severe episodes of antibiotic-associated diarrhea (AAD) are common side effects of antibiotic therapy. The incidence of AAD differs with the antibiotic and varies from 5 to 25%. The major form of intestinal disorders is the pseudomembranous colitis associated with Clostridium difficile which occurs in 10–20% of all AAD. In most cases of AAD discontinuation or replacement of the inciting antibiotic by another drug with lower AAD risk can be effective. For more severe cases involving C. difficile, the treatment of diarrhea requires an antibiotic treatment, with glycopeptides (vancomycin) or metronidazole. Another approach to AAD treatment or prevention is based on the use of non-pathogenic living organisms, capable of re-establishing the equilibrium of the intestinal ecosystem. Several organisms have been used in treatment or prophylaxis of AAD such as selected strains of Lactobacillus acidophilus, L. bulgaricus, Bifidobacterium longum, and Enterococcus faecium. Another biotherapeutic agent, a non-pathogenic yeast, Saccharomyces boulardii has been used. In animal models of C. difficile colitis initiated by clindamycin, animals treated with S. boulardii (at end of vancomycin therapy) had a significant decrease in C. difficile colony-forming units, and of toxin B production. In several clinical randomised trials (versus placebo), S. boulardii has demonstrated its effectiveness by decreasing significantly the occurrence of C. difficile colitis and preventing the pathogenic effects of toxins A and B of C. difficile. It has been shown to be a safe and effective therapy in relapses of C. difficile colitis. A good response has been seen in children with AAD, treated by S. boulardii only. In ICUs prevention of AAD remains based on limitation of antibiotic overuse and spread of C. difficile or other agents of AAD should be prevented by improved hygiene measures (single rooms, private bathrooms for patients, use of gloves and hand washing for personnel). In addition the increasing use of biotherapeutic agents such as S. boulardii should permit the prevention of the major side effect of antibiotics, i.e. AAD in at risk patients.     

Effect of oral Saccharomyces boulardii treatment on the activity of Clostridium difficile toxins in mouse digestive tract.

Human antibiotic-associated diarrhoea and pseudomembranous colitis are partly due to toxin production by Clostridium difficile. It is now well documented that Saccharomyces boulardii protects against C. difficile induced diseases. In an attempt to understand better the mechanism of this protective effect, the action of S. boulardii on a crude toxin preparation was studied in vitro and in vivo. The results showed that the yeast had no effect on the toxins in vitro but was able to protect mice inoculated with these toxins. Furthermore, the observation by scanning electron microscopy that the mucosa of S. boulardii protected mice was not damaged suggests that the yeast mainly acts on the intestinal mucosa.     

The mycotoxin fumonisin B1 alters the proliferation and the barrier function of porcine intestinal epithelial cells.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides (formerly F. moniliforme), a fungus that commonly contaminates maize. FB1 causes toxicological effects in laboratory and domestic animals including pigs. Because the gastrointestinal tract represents the first barrier met by exogenous food compounds, the purpose of this study was to investigate the effects of FB1 on IPEC-1, a porcine intestinal epithelial cell line. We first verified that low concentrations of FB1 did not exert any cytotoxic effect on IPEC-1. Indeed, significant LDH release was only observed for FB1 concentrations greater than 50 and 700 microM on proliferating and nonproliferating cells, respectively. We then demonstrated that FB1 inhibits proliferation of IPEC-1. Fluorescence-activated cell sorting (FACS) analysis of the cell cycle indicated that FB1 blocks the proliferation of intestinal cells in the G0/G1 phase. Similar results were obtained with LLC-PK1, a renal porcine epithelial cell line, which is considered to be a good model for studying FB1 in vitro effects. We have also assessed the effects of FB1 on the integrity of the barrier formed by the intestinal epithelium. We demonstrated that FB1 decreases the transepithelial electrical resistance (TEER) of IPEC-1 in a time- and dose-dependent manner. This effect was only noticed after a long exposure (8-12 days of treatment). FB1 induced the TEER decrease independently of the cell differentiation stage, and this effect was partially reversible. Taken together, our data indicate that FB1 alters the proliferation and the barrier function of intestinal cells. These results may have implications for humans and animals consuming FB1-contaminated food or feed.     

Comparative anti-anaerobic activity of Men 10700, a penem antibiotic

The in vitro activity of Men 10700, a new penem, has been compared with that of metronidazole, clindamycin, ciprofloxacin, co-amoxiclav, imipenem and three third generation cephalosporins against 120 strains of anaerobes. The organisms tested comprised Clostridium perfringens, Clostridium difficile, Bacteroides fragilis and speciated members of the genera Fusobacterium, Veillonella and Peptostreptococcus. Men 10700 showed activity similar to that of imipenem, and was more potent than metronidazole against all species except C. difficile and P. anaerobius. The spectrum of activity of Men 10700 suggests this agent may be useful for treating infections caused by anaerobes.     

Resistance determinants in strains of Clostridium difficile from two geographically distinct populations

Ninety-three clinical isolates of Clostridium difficile, comprising 65 from Royal Gwent Hospital, Newport and 28 from Southmead Hospital, Bristol were examined to determine the prevalence of genes coding for macrolide resistance and to explore differences in susceptibility patterns. Antibiogram testing produced similar results for both sets of strains with respect to amoxicillin, tetracycline, erythromycin and cefotaxime. Results differed for rifampicin, where 53% of the Bristol isolates were resistant, compared with 3% of the Newport isolates. Clindamycin disc susceptibility testing produced similar resistance rates. However, clindamycin MIC determinations revealed that 53% of the Bristol strains exhibited high-level resistance (MIC > 256 mg/L), whereas strains from Newport had clindamycin MICs ranging from 0.25 to 3 mg/L. erm (B) was present in 15 of the strains from Bristol and in none of the Newport strains. erm (F) and erm (Q) were not detected in either population. The two geographically distinct populations of C. difficile differed considerably in their susceptibility to antibiotics. The possibility that C. difficile may serve as a conservator for resistant determinants subsequent to exposure to antimicrobial agents, has important implications for infection control.     

Experimental approach for an in vitro toxicity assay with non-aggregated quantum dots

Engineered nanoparticles are increasingly used in consumer products. While the potential of these products hold great promise, it is not known what potential toxic effects these nanomaterials may have on human health. There is a need to develop affordable, systematic, short-term in vitro assays aimed at allowing rapid assessment of potential toxicity. The method reported in this paper describes a system in which the intestinal lining is mimicked (Caco-2 human intestinal cell line) and provides an environment in which quantum dots (QDs), and possibly other nanomaterials, can be applied. Transepithelial electrical resistance (TEER) measurements assessed whether the epithelial integrity was breached because of QD exposure. QDs were suspended in calcium/magnesium-free phosphate buffered saline to study non-aggregated QDs. To maintain cell integrity, normal cell culture conditions were retained below the epithelium to provide necessary nutrients and ions. Toxicity studies completed here show that the nanosized QDs coated with hydrophilic thioglycolate capping ligands purchased for these experiments caused disruption in the epithelium monolayer and cell death at 0.1 mg/L of QDs. This toxicity was caused by the nano-size of the QDs rather than the cadmium ions or the sodium thioglycolate capping ligands. Aggregated QDs did not cause toxicity as measured by TEER.     

Correlation of in vitro cytotoxicity with paracellular permeability in mortal rat intestinal cells

INTRODUCTION: The rat small intestinal cell line, IEC-18, was used as an in vitro model to differentiate between acute cytotoxicity (AC) and paracellular permeability (PP) of selected chemicals. METHODS: This study compares the low resistance rat intestinal mortal cell line, IEC-18 (transepithelial electrical resistance, TEER=160+/-10 Omega cm(2)) with the high resistance human intestinal cell line, Caco-2 (TEER=900+/-100 Omega cm(2)). The two cell lines differ in state of differentiation, TEER and paracellular permeability characteristics. The IEC-18 cell line is originated from the ileum and resembles more closely the small intestine than the Caco-2. Cytotoxicity was carried out using MTT cell viability assay in 96-well plates for 24-h exposure time. PP was measured using TEER (membrane integrity indicator) and PP markers such as [(3)H]-D-mannitol, lucifer yellow (LY) and FITC-dextran (fluorescein-dextran) on cells grown on inserts. RESULTS: The data showed that there is a high correlation (R(2)=0.99) between MTT and TEER using IEC-18 cell for 24-h exposure time. IEC-18 is as sensitive as Caco-2 for both MTT and TEER measurements. Decrease in TEER is inversely proportional with increase in PP of tight junction indicators. There is a good correlation between IC(50)'s MTT, TEER and Registry of Cytotoxicity (RC) data. DISCUSSION: Based on the results from the experiments, IEC-18 can be used as an in vitro model to differentiate between concentrations needed for AC and those required for PP.     

Clostridium sordellii cytotoxin induces phosphorylation of an 80,000 mol. wt protein in McCoy cultured cells

The cytotoxins from Clostridium difficile (toxin B) and Clostridium sordellii (toxin L) induce rounding of cultured cells. The cellular effects induced by these two cytotoxins are clearly distinct, suggesting that both toxins use a similar, but not identical mechanism for cell intoxication. We have employed the technique of two-dimensional PAGE for the separation of ³²P-labelled cell lysates of McCoy cultured cells to investigate changes in the phosphorylation status of cellular proteins after treatment with toxin B and with toxin L. The two-dimensional electrophoresis patterns suggest the implication of an 80,000 mol. wt cellular protein (named pp80c) in the cytopathic action of the cytotoxin from C. sordellii. This protein shows immunoreactivity with non-muscle caldesmon. Toxin B, however, does not affect the phosphorylation of pp80c, but alters the phosphorylation of another cellular protein, pp77, indicating another mechanism for cell intoxication. In addition, our experiments suggest that the mechanism of action of okadaic acid, a phosphatase inhibitor which causes cell rounding similar to that induced by C. sordellii, and these two cytotoxins are different.     

唐古特大黄多糖促进肠上皮细胞的增殖、移行和分化

目的　观察唐古特大黄多糖对正常人肠上皮细胞(HIEC)增殖、移行和分化的影响,探讨其肠粘膜可能的修复机制。方法　HIEC细胞接种后24h,加入不同浓度的大黄多糖,加药后24h,MTT法观察细胞增殖情况,HE染色法和碱性磷酸酶活性的测定观察细胞分化情况,细胞接种后待长成单细胞层后,在细胞层上作一圆形损伤区,并加入大黄多糖,分别于加药后0、12、24、36、48h倒置显微镜下观察细胞移行情况并拍照,采用图像分析定量测定缺损区随时间变化的情况。结果　大黄多糖可剂量依赖性地促进HIEC细胞的增殖、移行和分化,与正常对照组比较,细胞增殖、移行以及碱性磷酸酶活性差异均具有显著性(P< 0 .05或P<0. 01)。结论　大黄多糖可能通过促进肠上皮细胞的增殖移行和分化来促进肠粘膜的损伤修复。     

Protective effects of lactoferrin against intestinal mucosal damage induced by lipopolysaccharide in human intestinal Caco-2 cells

Indirect evidence suggests that lactoferrin (Lf), a major iron-binding protein in human milk, induces enterocyte growth and proliferation, depending on its concentration and affects the function and permeability of the intestinal mucosa. The bacterial endotoxin (lipopolysaccharide, LPS) is known to cause mucosal hyperpermeability in vivo. However, protective effects of Lf against LPS-mediated intestinal mucosal damage and barrier function in epithelial cells are not yet fully clarified. The aim of this study was to investigate whether Lf can reduce the cellular injury and alter epithelial hyperpermeability caused by LPS in human intestinal Caco-2 cells. When cell viability was measured by a WST-1 assay (tetrazolium salt-based assay), the protective effects against LPS-induced damage to Caco-2 cells were observed at doses of 800 and 1000 microg/ml Lf. The barrier function of Caco-2 monolayer tight junctions was assessed by measuring transepithelial electrical resistance (TEER) and permeability of FITC-labeled dextran 4000 (FD-4). The treatment of Caco-2 cells with Lf at doses of 400 and 1000 microg/ml significantly increased TEER as compared to treatment with LPS alone for 2 h (p<0.05). Further, at doses of 400 and 1000 microg/ml, Lf inhibited the enhancement of LPS-mediated permeability in Caco-2 cell monolayer. The results of this study suggest that Lf may have protective effects against LPS-mediated intestinal mucosal damage and impairment of barrier function in intestinal epithelial cells.     

应用Het-1A细胞系研究CDDO-Me对食管黏膜上皮屏障的保护作用#br#

摘要：目的　观察CDDO-Me对食管黏膜屏障的保护效应，探讨CDDO-Me作为黏膜屏障增强剂的可行性。方法　分别以不同pH值的酸（pH4、pH5、pH6）处理Het-1A细胞系，处理时间为5、10、30、60 min，检测各组Het-1A细胞系跨上皮电阻抗（TEER）值的变化；以含不同浓度的CDDO-Me（0、0.25、0.5、1、2.5、5 μmol/L）预刺激Het-1A细胞系，再以酸处理Het-1A细胞系，观察TEER值的变化。结果　pH为4、5、6时，不同处理时间对Het-1A细胞的TEER作用比较差异均有统计学意义（P＜0.01），随着处理时间（10、30、60 min）的延长，TEER下降幅度越大。处理时间为10、30、60 min时，各组间差异均有统计学意义，以pH4组60 min时TEER下降最明显（P＜0.01）。CDDO-Me预刺激后以pH4酸处理Het-1A细胞系，12、24、48 h时，不同浓度CDDO-Me预刺激组TEER比较差异均有统计学意义（P＜0.05），24 h时，随着刺激浓度的增加，各组TEER逐渐升高，以5 μmol/L组最明显（P＜0.05）；浓度为0.5、1、2.5、5 μmol/L时，各组间差异均有统计学意义（P＜0.05）。结论　酸损害食管上皮屏障功能，与酸度和作用时间有关；CDDO-Me对于酸对食管上皮屏障的损害有保护作用。     

中药兔耳风提取物ainsliadimer A对坏死性小肠结肠炎的作用和机制研究

目的：Ainsliadimer A是提取于中药兔耳风的一种内酯类化合物，前期研究已经发现其具有抑制NF-kB信号活性和抑制肿瘤生长的功能。已知的NF-kB信号通路在介导坏死性小肠结肠炎的炎症反应中发挥重要作用，因此，拟研究ainsliadimer A对坏死性小肠结肠炎的作用并对机制进行探讨。方法：通过LPS刺激人肠上皮细胞（FHC）、大鼠小肠隐窝上皮细胞（IEC-6）诱导肠细胞炎症模型，在不同浓度ainsliadimer A处理后，qPCR检测IL-6、TNF-α的mRNA水平，ELISA检测上清中IL-6水平，检测其对炎症因子水平的影响；对FHC和IEC-6细胞处理不同浓度ainsliadimer A后，CCK-8法和划痕实验分别检测其对细胞增殖、迁移的影响，评估其对坏死性小肠结肠炎的肠细胞修复功能；通过免疫印迹检测ainsliadimer A处理后，细胞内NF-kB信号的活性，初步探讨ainsliadimer A的作用机制。结果：Ainsliadimer A不同浓度（5，10 μmol·L^-1）皆可有效降低FHC和IEC-6细胞中IL-6、TNF-α的水平，同时，显著恢复LPS引起的肠细胞增殖、迁移抑制，免疫印迹显示ainsliadimer A显著降低了NF-kB信号的活性。结论：Ainsliadimer A可有效降低LPS引起的炎症因子表达和释放，且恢复了肠细胞的迁移和增殖，表现出明显的肠修复作用；机制研究初步揭示其通过下调NF-kB信号活性来降低炎症因子水平，缓解炎症反应，研究在细胞水平证实了ainsliadimer A对坏死性小肠结肠炎的缓解作用，为坏死性小肠结肠炎的药物研发和治疗提供了新线索。     

The Effect of Monomers and of Micellar and Vesicular Forms of Non-ionic Surfactants (Solulan C24 and Solulan 16) on Caco-2 Cell Monolayers

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.     

In vitro study of the pulmonary translocation of nanoparticles: a preliminary study

Recent studies indicate that inhaled ultrafine particles can pass into the circulation. To study this translocation in an in vitro model three types of pulmonary epithelial cells were examined. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. TEER was too low in A549 cells. In these preliminary experiments, TEER values of 1007 ± 300 and 348 ± 62 Ω cm² were reached for the Calu-3 cell line, using permeable membranes of 0.4 and 3 μm pore size, respectively. Growing primary rat type II pneumocytes on 0.4 μm pores, a TEER value of 241 ± 90 Ω cm² was reached on day 5; on 3 μm pores, no acceptable high TEER value was obtained. Translocation studies were done using 46 nm fluorescent polystyrene particles. When incubating polystyrene particles on membranes without a cellular monolayer, significant translocation was only observed using 3 μm pores: 67.5% and 52.7% for carboxyl- and amine-modified particles, respectively. Only the Calu-3 cell line was used in an initial experiment to investigate the translocation: on 0.4 μm pores no translocation was observed, on 3 μm pores 6% translocation was observed both for carboxyl- and amine-modified particles.     

Rho激酶在氢气保护LPS诱导的Caco-2细胞上皮屏障功能障碍中的机制研究

目的 探讨Rho激酶（ROCK）在氢气改善离体脓毒症肠屏障功能中的作用。方法 常规培养人结肠上皮细胞Caco-2,随机分为6组（n=3）：对照组、富氢培养基组、LPS处理组、富氢培养基+LPS组、Rho激酶抑制剂组、Rho激酶抑制剂+LPS组。富氢培养基组给予0.6mmol/L富氢培养基;LPS 和Y-27632处理浓度分别为100μg/ml、25μmol/L。建立Transwell小室模型,定期检测跨上皮电阻值（TEER）,当TEER达到800Ω?cm2后给予处理,于6h、12h、24h检测TEER值,24h时检测FITC-右旋糖苷通过率。细胞接种于6孔板,融合达80%~90%后给予处理,实时聚合酶链式反应（RT-PCR）技术检测ZO-1 mRNA和ROCK mRNA表达情况;蛋白免疫印迹技术（Western Blot）检测ZO-1蛋白和ROCK蛋白表达水平。结果 与对照组比较,富氢培养基组12h、24h TEER值升高（P＜0.05）,FITC-右旋糖苷通过率、ZO-1和ROCK mRNA及蛋白表达水平无显著变化（P＞0.05）;ROCK抑制剂组各时间点TEER值升高（P＜0.05）,FITC-右旋糖苷通过率无显著变化（P＞0.05）,ZO-1 mRNA表达增加,ROCK mRNA表达减少（均P＜0.05）;LPS处理组各时间点TEER值降低,FITC-右旋糖苷通过率增高,ZO-1 mRNA和蛋白表达均下降,ROCK mRNA和蛋白表达均增加（P＜0.05）。与LPS处理组比较ROCK抑制剂+LPS组TEER值增高,FITC-右旋糖苷率降低,ZO-1 mRNA表达增加（P＜0.05）。与LPS处理组比较,富氢培养基+LPS组各时间点TEER值增高,FITC-右旋糖苷通过率降低,各时间点ZO-1蛋白表达上升,ROCK蛋白表达下降（均P＜0.05）。结论 氢气可保护脓毒症肠屏障功能,改善肠上皮屏障完整性,减少肠壁通透性,增加肠细胞间紧密连接蛋白表达,这些保护机制可能与氢气抑制LPS诱导的Rho激酶过度表达有关。     

自微乳化系统对细胞紧密连接蛋白的影响

目的从分子细胞水平考察正、负电荷自微乳化系统对细胞紧密连接蛋白复合体的影响。方法建立了模拟小肠上皮细胞结构的Caco-2细胞模型;测定对跨膜电阻和细胞间转运物质甘露醇转运评价制剂对细胞完整性的影响。采用免疫荧光法评价两种自微乳化系统不同稀释倍数对细胞紧密连接蛋白ZO-1和细胞骨架肌动蛋白(actin)的影响。结果负电荷自微乳化系统在不同稀释倍数对跨膜电阻都无显著性影响。正电荷处方在考察的3种稀释倍数显著性降低了跨膜电阻(P<0.05)。细胞单分子层经正电荷自微乳制剂处理2 h，再经过48 h培养，较高稀释倍数跨膜电阻能够完全恢复；50倍稀释的处方不能完全恢复(81.3%)。两种制剂在不同稀释倍数都能显著提高甘露醇的渗透系数(2.9～64.6倍)(P<0.05)，作用与自微乳稀释倍数具有相关性。免疫荧光结果表明，经制剂处理后，影响了actin和ZO-1细胞分布，呈现不连续性。正电荷处方由于静电吸引，可能对细胞膜产生压力，比负电荷处方更能影响actin的分布。对细胞紧密连接影响具有制剂稀释倍数的依赖性。结论自微乳化系统能够提高甘露醇的细胞间转运。通过影响actin和ZO-1的细胞膜分布的作用机制打开细胞紧密连接。     

Vitamin D Analogues Tacalcitol and Calcipotriol Inhibit Proliferation and Migration of T98G Human Glioblastoma Cells

The active form of vitamin D (1α,25‐dihydroxyvitamin D) acts as a steroid hormone and binds to the vitamin D receptor. This receptor is expressed in most cell types including cells in the central nervous system (CNS). Vitamin D has several functions in the body including effects on brain development, neuroprotection and immunological regulation. It has been shown that vitamin D has antiproliferative activities in different cancer cell lines. Tacalcitol and calcipotriol are synthetic analogues of 1α,25‐dihydroxyvitamin D with reduced effect on calcium metabolism. The aim of this study was to analyse the effects of tacalcitol and calcipotriol on cell viability, proliferation and migration in the human glioblastoma cell line T98G. Glioblastoma is the most lethal type of primary tumours in the CNS. Both analogues decreased cell viability and/or growth, dose‐dependently, in concentrations between 1 nM and 10 μM. Manual counting indicated suppressive effects by the vitamin D analogues on proliferation. Treatment with tacalcitol strongly suppressed thymidine incorporation, indicating that the vitamin D analogues mainly inhibit proliferation. Also, effects on cell migration were measured with wound‐healing assay. Both calcipotriol and tacalcitol reduced the migration rate of T98G cells compared to vehicle‐treated cells. However, they had no effect on caspase‐3 and ‐7 activities, suggesting that their mechanism of action does not involve induction of apoptosis. The current results indicate that the vitamin D analogues tacalcitol and calcipotriol strongly reduce proliferation and migration of human glioblastoma T98G cells, suggesting a potential role for this type of compounds in treatment of brain cancer.     

Evidence for a differential translocation of actinides across human lung epithelial cell monolayer in vitro according to their physicochemical properties and the presence of a chelating agent

The epithelial cell plays a key role in the transfer of radionuclides from lungs to blood following pulmonary exposure. The present study was designed to evaluate the transfer across human lung epithelial cells of various actinides (plutonium, americium and uranium), the influence of the physicochemical properties of plutonium compounds and of the chelating agent diethylene triamine pentaacetic acid (DTPA). To address this question, Calu-3 cells grown in a bicameral culture system were used. The integrity of the epithelial barrier was evaluated by measuring transepithelial electrical resistance (TEER) and the passage of a fluorescent marker, lucifer yellow. Activity measurement in basal compartment following periodic collection of culture medium was made from 2 h to seven days. To facilitate data handling and analysis, the statistical tool STATBIODIS was used. The results indicate differences in transfer for the different elements, and according to Pu physicochemical properties. Though to various extents, the chelating agent DTPA always increased the transfer of Pu and Am across the epithelial cells, without altering the integrity of the epithelial barrier.This in vitro cell culture model, by mimicking translocation of actinides from lungs to blood, can represent a valuable tool to further understand the underlying mechanisms and properties controlling this process.