Bile salts and cholesterol induce changes in the lipid cell membrane of Lactobacillus reuteri

AIMS: The objective of this study was to evaluate the effect of bile salts and cholesterol in the lipid profile of Lactobacillus reuteri CRL 1098 and to determine the relationship existing between these changes: the in vitro removal of cholesterol and the tolerance of the cells to acid and cold stress. METHODS AND RESULTS: Lactobacillus reuteri CRL 1098 was grown in the following media: MRS (deMan Rogosa Sharpe; MC, control medium), MB (MC with bile salts), MCH (MC with sterile cholesterol) and MBCH (MC with bile salts and cholesterol). Fatty acids were determined by analytical gas-liquid chromatography, and phospholipids and glycolipids by colorimetric techniques. The cells from different culture media were subjected to cold and acid stress. The MB cultures displayed a decrease in phospholipids and a low ratio of saturated : unsaturated fatty acids. The presence of the unusual C18 : 0,10-OH and C18 : 0,10-oxo fatty acids was the prominent characteristic of the bile salts growing cells. The relative increase in glycolipids and the changes in the fatty acids profiles of the MB cells would be responsible for the cholesterol remotion. The changes induced by bile salts in the lipid profile did not improve the tolerance of L. reuteri CRL 1098 to freezing and acid stress. CONCLUSIONS: The changes in lipid profiles reported in this study would play a key role in the response of Lactobacilli to environmental stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides useful information about the effect of bile salts on the cell membrane of L. reuteri, a probiotic enterolactobacillus. The steady-state response of the cells subjected to bile stress seems to be the appropriate model for evaluating the bacterial behaviour in detergent-containing gastrointestinal tracts, where the bile salts stress would presumably be continuous.     

Karyometry in Barrett's esophagus

OBJECTIVE: To derive a progression curve for lesions in Barrett's esophagus based on karyometric features. STUDY DESIGN: High-resolution imagery of 900 nuclei from normal gastric tissue, Barrett's metaplasia, Barrett's high grade dysplasia and adenocarcinoma of the esophagus was recorded. Karyometric features were computed, and nuclear signatures and lesion signatures for these lesions were derived. A progression curve was defined. RESULTS: Esophageal lesions were distinctly different from the normal gastric fundus tissue, with nuclei from Barrett's metaplasia deviating from normal almost as much as nuclei from high grade dysplasia and adenocarcinoma. There was considerable case-to-case variability and overlap between lesions histologically assigned to different diagnostic categories. CONCLUSION: The karyometric data suggest that Barrett's metaplasia is a more developed lesion than previously assumed.     

Helicobacter pylori in Barrett's esophagus.

Barrett's esophagus is an anatomicoclinical state in which, due to the prolonged action of gastroesophageal reflux, the squamous epithelium is replaced by columnar epithelium. Helicobacter pylori has been implicated in the pathogenesis of various gastrointestinal disorders and has occasionally been observed in Barrett's esophagus. The aim of this study is to determine the incidence of H. pylori in Barrett's esophagus and try to establish its role in the pathogenesis of this disorder. H. pylori was observed in 31 biopsies (44.3%) of the 70 studied, mainly when the epithelium is of the gastric atrophic-fundic type (p less than 0.01). Its presence shows no relation to the degree of inflammatory activity and does not seem, therefore, to play an important role in the pathogenesis of the lesion.     

Bile salts induce resistance to polymyxin in enterohemorrhagic Escherichia coli O157:H7

Many enteric bacteria use bile as an environmental cue to signal resistance and virulence gene expression. Microarray analysis of enterohemorrhagic Escherichia coli O157:H7 (EHEC) treated with bile salts revealed upregulation of genes for an efflux system (acrAB), a two-component signal transduction system (basRS/pmrAB), and lipid A modification (arnBCADTEF and ugd). Bile salt treatment of EHEC produced a basS- and arnT-dependent resistance to polymyxin.     

Failure of capsaicin-containing red pepper sauce suspension to induce esophageal motility response in patients with Barrett's esophagus.

The physiologic importance of afferent sensory pathways in the esophageal motor functions has been recently recognised. Capsaicin-sensitive sensory afferents were shown to play a role in the maintenance of mucosal integrity of the GI tract, and regulation of human esophageal motility. The aim of this study was to investigate the effect of topical application of capsaicin-containing red pepper sauce (Tabasco, 25%v/v, pH:7.0) suspension on the phasic activity of the human esophagus of healthy volunteers and patients with Barrett's esophagus. METHODS: The diagnosis of Barrett's esophagus was based on the findings of esophagoscopy and histology taken from the squamocolumnar junction of the esophagus. Esophageal motility was measured by perfusion manometry before and after application of red pepper sauce. RESULTS: Capsaicin containing red pepper sauce increases the motility response (LES tone, contraction amplitude, propagation velocity) of the human esophagus in healthy volunteers. This response failed in patients with Barrett's esophagus. CONCLUSION: Impaired esophageal sensory motor function may serve as one etiologic role in the development of Barrett's esophagus.     

Cytotoxic T lymphocytes induce caspase-dependent and -independent cell death in neuroblastomas in a MHC-nonrestricted fashion

The MHC class I- restricted processing and presentation pathway is frequently nonfunctional in tumor cells; therefore, the direct targeting of tumor cells by CTLs may be difficult, if at all possible, to achieve. We used neuroblastoma (NB), which represents a striking example of a tumor with an impaired MHC class I pathway, as a model to study bystander effects of activated T lymphocytes on tumor cells. We found that NB cell lines are susceptible to killing by differentiated CD8(+) CTL clones in a MHC class I-nonrestricted manner that involves two programs of cell death distinguished on the basis of different kinetics, sensitivities to caspase inhibitors, and cytokine-blocking reagents. The "early" death exhibited characteristic features of apoptosis, whereas the "delayed" caspase-independent death exhibited features associated with necrosis and was partially inhibited by TNF-alpha-blocking and prevented by overexpression of Bcl-2 or Bcl-x(L). Our data reveal a previously unappreciated complexity of death pathways induced in tumor cells by immune activation and suggest that redirecting nonspecific effector CTLs to even a small proportion of NB cells or activating CTLs in a tumor's proximity may have therapeutic effects in patients with NB.     

Bile salts mediate hepatocyte apoptosis by increasing cell surface trafficking of Fas

Toxic bile salts induce hepatocyte apoptosis by a Fas-dependent, Fas ligand-independent mechanism. To account for this observation, we formulated the hypothesis that toxic bile salts induce apoptosis by effecting translocation of cytoplasmic Fas to the cell surface, resulting in transduction of Fas death signals. In McNtcp.24 cells the majority of Fas was cytoplasmic, as assessed by cell fractionation and immunofluorescence studies. However, cell surface Fas increased sixfold after treatment with the toxic bile salt glycochenodeoxycholate (GCDC) in the absence of increased Fas protein expression. Moreover, in cells transfected with Fas-green fluorescence protein, cell surface fluorescence also increased in GCDC-treated cells, directly demonstrating Fas translocation to the plasma membrane. Both brefeldin A, a Golgi-disrupting agent, and nocodazole, a microtubule inhibitor, prevented the GCDC-induced increase in cell surface Fas and apoptosis. In conclusion, toxic bile salts appear to induce apoptosis by promoting cytoplasmic transport of Fas to the cell surface by a Golgi- and microtubule-dependent pathway.     

Ciliated cells in papillary adenocarcinomas of Barrett's esophagus.

Eighteen surgical specimens having an adenocarcinoma arising in Barrett's esophagus were reviewed, and special attention was paid to the presence of ciliated cells. The tumors were classified as glandular (9), papillary (4), diffuse (3) and mixed (2) types. Ciliated cells were observed in one specimen, in cystically dilated glands in Barrett's mucosa adjacent to a papillary adenocarcinoma. Ciliated tumor cells were found in three of the four papillary adenocarcinomas. The fourth papillary tumor, 1 mixed papillary-diffuse adenocarcinoma and the remaining 13 adenocarcinomas had no ciliated cells. Thus, the presence of cilia in exfoliated tumor cells from the esophagus should raise the suspicion of a papillary adenocarcinoma arising in Barrett's mucosa.     

SAHA induce hippo pathway in CCA cells without increasing cell proliferation

Background

Cholangiocarcinoma is a malignant tumor originating from bile duct epithelial cells. Since tumor metastasis is associated with poor prognosis and short-term survival of patients, there is an urgent need for alternative therapeutic approaches for CCA. Because of that reason, we aimed to investigate effect of SAHA which is known as HDAC inhibitor on extrahepatic cholangiocarcinoma cell line (TFK-1).

Methods

Cell cycle was measured by Muse Cell Analyzer. YAP, TAZ, TGF-β protein levels were determined by western-blotting method. TEAD (1–3), TIMP2 and TIMP3 genes level were determined by real-time PCR analysis.

Results

We have seen the positive effects of SAHA on the TFK-1 cell line as it reduces cell viability and arresting cells in the G0/G1 phase. We also observed the negative effects of SAHA, as it increases the expression levels of YAP, TAZ, TGF-β protein and TEAD (1–3) gene. We also found that SAHA reduced the expression levels of TIMP2 and TIMP3 in TFK-1 cells, but was not statistically significant.

Conclusions

Although observing its antiproliferative effects, these negative effects may be related to the cells being resistant to the drug or the remaining cells having a more aggressive phenotype. Therefore, we think that caution should be exercised in the use of this drug for CCA treatment.

     

Platelets induce human umbilical vein endothelial cell proliferation through P-selectin

We studied whether platelets could participate in the endothelial cell monolayer regeneration in the case of a vessel damage. Incorporation of [3H]-thymidine into the DNA of human umbilical vein endothelial cells (HUVECs) was measured after 48 h of co-incubation with platelets. The effect of platelets was compared to that of platelet-free supernatants from thrombin-activated platelets that had secreted their active granule constituents. Platelets dose-dependently induced HUVEC proliferation. Platelets preactivated by thrombin induced similar proliferation as did unactivated platelets (proliferation factor = 7 - 8), indicating that preactivation of platelets was not required. Platelets fixed with paraformaldehyde had no effect, suggesting that the platelet mitogenic effect required a mobile, alive membrane. Ketanserine and suramin reduced by at most 30 % the platelet-induced proliferation; supernatants of thrombin-activated platelets caused only minor proliferation (proliferation factor = 2), suggesting that secreted 5-hydroxytryptamine and growth factors poorly contributed to the proliferative effect. When the co-incubation was performed in the presence of an anti P-selectin antibody, the platelet-induced HUVEC proliferation was inhibited. The results suggest that platelet adhesion participate in the control of the endothelial regeneration and that platelet P-selectin is a molecular determinant of the proliferative signal.     

Human T-helper clones induce IgG production in a subclass-specific fashion.

The mechanisms governing the induction of IgG subclasses by T-helper cells in humans were investigated. As preliminary bulk-culture experiments had indicated that a direct B cell contact with viable T cells was an essential requirement for optimal IgG subclass production, 256 CD4+ human T cell clones were preactivated with PHA and cultured in direct contact with autologous B cells. These clones induced IgG production in a strikingly subclass-specific fashion. Moreover, the distribution of subclass-specific helper clones was very similar to the IgG subclass profile observed in serum and peripheral lymphoid tissue plasma cells (IgG1 approximately 60%, IgG2 approximately 30%, IgG3 approximately 5-10%, IgG4 less than or equal to 5%) and unlike that observed in resting B cells (which is IgG1 approximately 40% and IgG2 approximately 50%). It would, therefore, seem that a predominance of T cells capable of delivering IgG1-specific, as opposed to IgG2-specific, help is an essential factor for the preferential induction of IgG1 antibodies during B cell proliferation and differentiation. There was no relationship between IL2, IL4, IL6, and IFN-gamma secreted by the T-helper clones and their IgG subclass induction patterns. In addition, only a few supernatants were able to reproduce the helper effects of the clones themselves. Therefore, direct contact of B cells with helper clones is crucial for IgG-subclass production in humans.     

Low concentrations of ouabain induce vascular smooth muscle cell proliferation.

Ouabain is a well known inhibitor of the Na+ pump in all mammalian cells. We have demonstrated that ouabain at concentrations below those which inhibit the pump, i.e. 0.1 nM and 1.0 nM, induce proliferation of saphenous vein smooth muscle cells as measured by bromodeoxyuridine (BrdU) uptake. Ouabain at these low concentrations also activated MAPK. Proliferating concentrations of the drug did not increase levels of Ca(i)2+, suggesting no effect of this ion in the process. In addition, incubation of the cells in low levels of K+, which has been shown to inhibit the pump, had no effect on proliferation. These data show that low concentrations of ouabain that do not inhibit the Na+ pump can activate proliferation of vascular smooth muscle cells, suggesting that the pump complex may act as a transducing receptor.     

Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts

A colorimetric method to assay cell proliferation of microorganisms in 96-well microtiter plates using water-soluble tetrazolium salts and electron mediators was developed. Combinations of 6 kinds of water-soluble tetrazolium salts and 27 kinds of electron mediators that considered the metabolic efficiency of microorganisms and the influence with medium components were investigated. 2-Methyl-1,4-naphthoquinone (NQ) was reduced most effectively by various species of microorganisms, and a combination of WST-8 as a water-soluble tetrazolium salt with 2-methyl-1,4-NQ repressed the increase in background due to medium components. In the presence of 2-methyl-1,4-NQ, WST-8 was reduced by microbial cells to formazan, which exhibited maximum absorbance at 460 nm. The proposed tetrazolium method could be applied to measure proliferations of various microbial cells including 3 kinds of yeast, 9 kinds of Gram-positive bacteria, and 10 kinds of Gram-negative bacteria. Linear relationships between the absorbance and viable microbial cell density were obtained in all microorganisms, suggesting that the absorbance change reflected the microbial cell proliferation.     

Bile salt export pump inhibitors are associated with bile acid-dependent drug-induced toxicity in sandwich-cultured hepatocytes

Drug-induced liver injury (DILI) is a major reason for the dropout of candidate compounds from drug testing and the withdrawal of pharmaceuticals from clinical use. Among the various mechanisms of liver injury, the accumulation of bile acids (BAs) within hepatocytes is thought to be a primary mechanism for the development of DILI. Although bile salt export pump (BSEP) dysfunction is considered a susceptibility factor for DILI, little is known about the relationship between drug-induced BSEP dysfunction and BA-dependent hepatotoxicity. Furthermore, few methods are at hand for the systematic and quantitative evaluation of BA-dependent DILI. This study aimed to construct a model of DILI by employing sandwich-cultured hepatocytes (SCHs). SCHs can be used to assess functions of canalicular transporters such as BSEP and the activity of metabolic enzymes. Here, the impact of 26 test compounds (ritonavir, troglitazone, etc.) was investigated on BA-dependent cytotoxicity in SCHs. SCHs were exposed to each compound for 24h with or without BAs (glycochenodeoxycholic acid, deoxycholic acid, etc.). As a result, BA-dependent toxicity was observed for 11 test compounds in SCHs treated in the presence of BAs, while no signs of toxicity were observed for SCHs treated in the absence of BAs. Of the 11 compounds, nine were known BSEP inhibitors. Moreover, for some compounds, an increase in the severity of BA-dependent toxicity was observed in SCHs that were co-treated with 1-aminobenzotriazole, a non-selective inhibitor of cytochrome P450 (CYP450)-mediated drug metabolism. These results indicate that the SCH-based model is likely to prove useful for the evaluation of BA-dependent DILI, including the effects of drug metabolism and BSEP inhibition on liver injury.     

重组人乳铁蛋白对食管癌细胞体外增殖的影响

目的:研究重组人乳铁蛋白(rhLF)对体外培养的食管癌细胞的生长特性的影响.方法:MTT法评定rhLF对人食管癌细胞Eca-109、人肝脏细胞L0-2、CHO中国昌鼠细胞系的生长抑制作用.结果:重组人乳铁蛋白对人食管癌细胞系Eca-109的增殖有抑制作用并呈现剂量依赖性,对正常细胞无抑制作用.结论:重组人乳铁蛋白作为新的抗肿瘤化学治疗荆,可以抑制食管癌细胞Eca-109的增殖.     

Aluminum ions induce DNA synthesis but not cell proliferation in human fibroblasts in vitro

The effect in vitro of aluminum (Al) ions on DNA synthesis and human dermal fibroblast proliferation using [Al] concentrations from 1.85 to 74 μM and incubation periods of 1, 2, 3, 4, and 5 d was assessed. The lowest concentration of Al that exerted a slight positive, although not significant, effect on DNA synthesis was 1.85 μM, after d 3 or 5 of incubation. The stimulating action of Al was more evident and statistically significant from concentrations of 3.7 μM and 2 d exposure onward. This Al-induced effect on [³H] thymidine incorporation into DNA increased in a time-dependent manner as [Al] in the culture medium rose, provoking increments of up to 322% above the control at [Al] 74 μM and 5 d incubation. In contrast, Al salts moderately increased fibroblast division in a continuous manner only from 7.4 to 74 μM after 3 d of incubation. Although significant overall, the minimal and inconstant mitogenic activity of Al differs greatly from and is not parallel to DNA synthesis, which is not clearly related to exposure times or Al concentrations. Abnormalities in Al-induced cellular metabolic processes described herein and their influence on the cell cycle may constitute a toxicity mechanism for human tissues, leading to disease development. Further studies are required to determine whether these findings can be extrapolated to in vivo situations.     

Picogram quantities of anti-Ig antibodies coupled to dextran induce B cell proliferation

To investigate the properties which enable type 2 Ag, as exemplified by dextran and Ficoll, to stimulate high levels of antibody responses in the relative absence of T cells, we conjugated anti-IgD and anti-IgM mAb to both dextran and Ficoll and examined their B cell-activating properties. Such conjugated anti-Ig antibodies stimulated both early and later stages of B cell activation at picogram concentrations, which are at least 1000-fold lower than that required for B cell stimulation by unconjugated anti-Ig antibodies, and the level of proliferation they stimulated was on average 10-fold greater. Furthermore, concentrations of anti-Ig dextran (100 pg/ml) which modulated little sIgD from the B cell surface were strong inducers of enhanced B cell expression of MHC class II molecules. Conjugation of Fab fragments of anti-IgD or nonmitogenic anti-IgM mAb to dextran rendered them as mitogenic as dextran conjugated to strongly stimulatory anti-IgD or anti-IgM antibodies. The ability of dextran and Ficoll to serve as effective carrier molecules for anti-IgD was not related solely to their large m.w., because anti-IgD coupled to polymerized BSA (m.w. 1.5 X 10(6), was only 10- to 50-fold more potent than unconjugated anti-IgD antibodies at stimulating B cell DNA synthesis. These results suggest, therefore, that the unique ability of picogram concentrations of haptenated type 2 Ag to stimulate Ig secretion in the absence of T cells may be a function of their ability to promote effective cross-linking without resulting in the modulation of sIg. This would enable such Ag to mediate repetitive B cell signaling, a situation that cannot be achieved by unconjugated anti-Ig antibodies which result in modulation of sIg at their mitogenic concentrations. These compounds therefore may be employed to study B cell activation stimulated by sIg cross-linking at concentrations that may more closely reflect those which are achieved under physiologic conditions by type 2 Ag.     

Toll-like receptor agonists synergize with CD40L to induce either proliferation or plasma cell differentiation of mouse B cells

In a classical dogma, pathogens are sensed (via recognition of Pathogen Associated Molecular Patterns (PAMPs)) by innate immune cells that in turn activate adaptive immune cells. However, recent data showed that TLRs (Toll Like Receptors), the most characterized class of Pattern Recognition Receptors, are also expressed by adaptive immune B cells. B cells play an important role in protective immunity essentially by differentiating into antibody-secreting cells (ASC). This differentiation requires at least two signals: the recognition of an antigen by the B cell specific receptor (BCR) and a T cell co-stimulatory signal provided mainly by CD154/CD40L acting on CD40. In order to better understand interactions of innate and adaptive B cell stimulatory signals, we evaluated the outcome of combinations of TLRs, BCR and/or CD40 stimulation. For this purpose, mouse spleen B cells were activated with synthetic TLR agonists, recombinant mouse CD40L and agonist anti-BCR antibodies. As expected, TLR agonists induced mouse B cell proliferation and activation or differentiation into ASC. Interestingly, addition of CD40 signal to TLR agonists stimulated either B cell proliferation and activation (TLR3, TLR4, and TLR9) or differentiation into ASC (TLR1/2, TLR2/6, TLR4 and TLR7). Addition of a BCR signal to CD40L and either TLR3 or TLR9 agonists did not induce differentiation into ASC, which could be interpreted as an entrance into the memory pathway. In conclusion, our results suggest that PAMPs synergize with signals from adaptive immunity to regulate B lymphocyte fate during humoral immune response.     

Gain of allelic gene expression for IGF-II occurs frequently in Barrett's esophagus

The IGF-II gene normally exhibits genomic imprinting, a DNA modification that allows the expression of only one of the two inherited alleles. With loss of imprinting, there is a gain of allelic gene expression (GOAGE) due to IGF-II being expressed by both alleles. GOAGE for IGF-II has been demonstrated in a number of malignancies and in normal epithelia surrounding malignancies, but not in epithelia without associated neoplasia. We hypothesized that nonneoplastic Barrett's epithelium might have GOAGE for IGF-II that could facilitate its progression to neoplasia. Endoscopic biopsies were obtained from metaplastic esophageal, normal gastric, and normal duodenal epithelia from 43 patients with Barrett's esophagus. Genomic DNA were analyzed using PCR followed by ApaI restriction enzyme digestion or allele-specific PCR to identify an ApaI polymorphism of IGF-II. cDNA from patients with the ApaI polymorphism were analyzed for IGF-II GOAGE using exon connection PCR, followed by a secondary nested PCR and ApaI restriction enzyme digestion. We found that 13 (30%) of 43 samples of Barrett's metaplasia contained the ApaI polymorphism and were thus informative for IGF-II, and sufficient material was available for GOAGE analysis in 9 of those 13 cases. GOAGE for IGF-II was demonstrated in five (56%) of those nine cases. All patients with GOAGE in Barrett's metaplasia also demonstrated GOAGE in the gastric and duodenal epithelia. In contrast, patients without GOAGE in Barrett's metaplasia also had no GOAGE in their gastric and duodenal epithelia. We conclude that in patients with Barrett's esophagus, GOAGE for IGF-II is found frequently in the metaplastic esophageal epithelium as well as in normal gastric and duodenal epithelia.