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Ludmiła Weglarz

Publications and source records attributed to Ludmiła Weglarz.

16 recordsLinked to original sources
PubMed2006

Phytic acid modulates in vitro IL-8 and IL-6 release from colonic epithelial cells stimulated with LPS and IL-1beta.

Phytic acid (PA), a major fiber-associated component of wheat bran and legumes, is physiologically present in the human large gut. The aim of this study was to examine the role of PA in immunologic function of intestinal epithelial cells by analyzing its effect on interleukin (IL)-8 and IL-6 secretion by colonocytes and its role in the response of these cells to bacterial lipopolysaccharides (LPS) and IL-1beta. The human colon cell line Caco-2 was exposed to LPS isolated from two strains of Desulfovibrio desulfuricans, wild intestinal and type soil strains, as well as to LPS from E. coli. Cells were also treated with IL-1beta and with a combination of LPS and IL-1beta. PA had a suppressive effect on IL-8 basal release and it dose dependently reduced IL-8 secretion by colonocytes stimulated with LPS and IL-1beta. On the contrary, PA increased constitutive IL-6 secretion and exhibited differentiated effects on LPS responsiveness of cells depending on its concentration and LPS origin. PA was also an efficient down-regulator of IL-6 secretion stimulated by binary actions of LPS and IL-1beta. The ability of PA to modulate IL-8 and IL-6 release suggests that PA present in the intestinal milieu may exert immunoregulatory effects on colonic epithelium under physiological conditions or during microbe-induced infection/inflammation in order to maintain the colonic mucosa in a noninflammatory state or to counteract infection.

Caco-2 Cells↗
PubMed2006

Quantitative analysis of the level of p53 and p21(WAF1) mRNA in human colon cancer HT-29 cells treated with inositol hexaphosphate.

The aim of this study was to analyze the molecular mechanism of inositol hexaphosphate (InsP(6)) action through which it may inhibit proliferation of colon cancer cells and cell cycle progression. A kinetic study of p53 and p21(WAF1) mRNA increase was performed on human colon cancer HT-29 cells after treatment with 1, 5 and 10 mM InsP(6) for 6, 12, 24 and 48 h. Real-time-QPCR based on TaqMan methodology was applied to analyze quantitatively the transcript levels of these genes. The transcription of beta-actin and GAPDH genes was assessed in parallel to select the control gene with least variability. The 2(-Delta Delta Ct) method was used to analyze the relative changes in gene transcription. InsP(6) stimulated p53 and p21(WAF1) expression at the mRNA level, with the highest increase in p21(WAF1) mRNA occurring at 24 h, i.e., following the highest increase in p53 mRNA observed at 12 h. Based on these studies it may be concluded that the ability of InsP(6) to arrest the cell cycle may be mediated by the transcriptional up-regulation of the p53-responsive p21(WAF1) gene.

Antineoplastic Agents↗
PubMed2006

[Methods of lipopolysaccharide component derivation in evaluating their structures by chromatographic techniques].

The pathogenicity of Gram-negative bacteria is mediated mainly by the outer membrane-associated lipid-polysaccharide structure, called endotoxin or lipopolysaccharide (LPS). This structure consists of three parts, O-antigen, core oligosaccharide, and lipid A, which differ in chemical composition and biological properties. Gas-liquid chromatography coupled with mass spectrometry (GLC/MS) is commonly used to determine LPS structure. The carbohydrate profiles of endotoxins are analyzed after derivation to alditol acetates or partially methylated alditol acetates, which enables one to establish the site of glycosidic linkages. The chromatographic analysis of LPS lipid components requires their modification to ester derivatives, most frequently to methyl esters.

Endotoxins↗
PubMed2005

Butyrate-induced differentiation of colon cancer cells is PKC and JNK dependent.

Butyric acid, a short-chain fatty acid physiologically present in human large gut, is derived from bacterial fermentation of complex carbohydrates. It has been shown to reduce the growth and motility of colon cancer cell lines and to induce cell differentiation and apoptosis. Apoptosis is considered a result of normal colonocyte terminal differentiation in vivo. The aim of this study was to characterize the cellular mechanisms regulating differentiation of colon cancer cells stimulated with sodium butyrate (NaB). The two human colon cancer cell lines Caco-2 and HT-29 were treated with NaB at physiologically relevant concentrations. Alkaline phosphatase (ALP) activity, a marker of colonocyte differentiation, was increased 48 hr after treatment with 1 mM NaB. Higher doses of NaB (5 and 10 mM) induced apoptosis of the cells and failed to stimulate the colonocyte differentiation. Therefore, we assumed that butyrate augments cell differentiation and induces apoptosis, acting via various intracellular mechanisms, and butyrate-mediated programmed cell death cannot be considered a consequence of colonocyte terminal differentiation. The effect of NaB on ALP activity was significantly attenuated in the presence of inhibitors of protein kinase C and JNK. Inhibition of MEK-ERK signal transduction pathways augmented the impact of butyrate on colonocyte differentiation. These results suggest that butyrate could influence the colonocyte differentiation via modulation of the activity of cellular protein kinases and signal transduction.

Apoptosis↗
PubMed2004

Susceptibility of Desulfovibrio desulfuricans intestinal strains to sulfasalazine and its biotransformation products.

BACKGROUND: Desulfovibrio desulfuricans intestinal bacteria may contribute to toxic hydrogen sulfide production in the human gut. Our objective was to examine whether the D. desulfuricans strains isolated from the human body are susceptible to sulfasalazine (SAS) and the products of its biotransformation, i.e. 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP), in order to determine the relationship between the strains' susceptibility to SAS and their ability to reduce the azo bond within this drug. MATERIAL/METHODS: Six wild strains of D. desulfuricans (isolated from feces and biopsy specimens from patients with colitis ulcerosa, Crohn's disease, irritable bowel syndrome, colonic diverticula, primary biliary cirrhosis, or tubular adenomas of the colon) were cultured in the presence of SAS, 5-ASA, and SP. Growth inhibition coefficients were compared with coefficients of inhibition of the azo-bond reduction in SAS. RESULTS: The D. desulfuricans strains present in the human digestive tract were susceptible to a small degree to SAS and to 5-ASA and SP. CONCLUSIONS: The intestinal D. desulfuricans strains differed in their susceptibility to SAS and its biotransformation products. The strains showing higher susceptibility to SAS lost the ability to reduce the azo bond in this drug, which may be attributed to the lower metabolic activity of the bacteria. The presence of D. desulfuricans in the large intestines of patients with ulcerative colitis and the confirmed diversity of the biological activity of the isolated strains demonstrate the need for clinical examination of the role of these bacteria in the development of some inflammatory disorders.

Biotransformation↗
PubMed2004

[Beta2-agonists--what does their chemical structure determine?].

The paper presents the structure and functional relationship of beta2-agonists and the beta2-adrenoceptor. The human beta2-adrenoceptor is a member of the 7-transmembrane family of receptors. Most of the actions of the beta2-receptor are mediated through the Gs protein and the cAMP-dependent PKA system. Alternative cAMP-independent pathways affected following beta2-receptor activation have also been described. Beta2-agonists have been used as bronchodilator agents in the treatment of asthma since the development of inhaled isoprenaline preparations in 1961. Over the years, these agents have been markedly improved through the development of short-acting beta2 selective agents followed by long-acting beta2 selective agents with prolonged duration of action. Efforts directed towards improving the pharmacodynamic and pharmacokinetic properties, including the long-acting and selective beta2-adrenoceptor agonists, included two major pathways of modifying the basic structure of the catecholamines, which are described in this paper. The pharmacological activity usually resides in the (R)-enantiomer. The kinetics of beta2-agonist-stimulated bronchodilation in asthma is determined by the differences in the molecular mechanism of beta2-agonists action. Regulation of the beta2-receptor is influenced by its desensitization following exposure to high concentrations of or repeated challenges with agonists, and up-regulation which can be induced by glucocorticoids and thyroid hormones.

Adrenergic beta-Agonists↗
PubMed2004

[Choice of isolation procedure for endotoxins from the outer membrane of the bacteria Desulfovibrio desulfuricans].

The chemical structure determining properties and biological functions of endotoxins derived from Desulfovibrio desulfuricans species has not been recognized, which considerably hinders the choice of an effective extraction procedure of these lipopolysaccharides (LPS) from the bacterial outer cell membrane. We aimed at selecting the most effective method of LPS isolation from D. desulfuricans in terms of the most efficient extraction solution, the appropriate conditions of isolation and adequate purification technique. For this purpose we tested a few literature-based procedures utilizing various extraction mixtures (phenol-water, phenol-chloroform-petroleum ether and Tri-Reagent, i.e. aqueous solution of guanidinum thiocyanate and phenol). The best yield and purity of the isolated LPS were provided by the application of the extraction with phenol-water according to the modified by Shnyra et.al. (2000) procedure of Westphal et. al. (1952). A satisfactory method of isolation in micro scale appeared to be that based on Tri-Reagent and propagated by Yi and Hackett in 2000. The extraction of LPS from D. desulfuricans with phenol-chloroform-petroleum ether should not be recommended due to its low efficiency.

Bacteriological Techniques↗
PubMed2004

Evaluation of biotransformation of sulphasalazine in the colon epithelial Caco-2 cells.

Sulphasalazine (salicyl-azo-sulphapiridine, SAS) is a drug commonly used in the treatment of non-specific inflammatory bowel diseases, such as ulcerative colitis and Crohn disease. Chronic inflammatory states of the colon can lead to neoplastic changes of the intestinal mucosa. There are some suggestions in the literature that the intestinal bacterial azo-reductases are involved in biotransformation of SAS into 5-aminosalicylic acid (5-ASA) and sulphapyridine (SP). For this reason, it seemed worth of investigating whether transformation of SAS could be performed by the colon epithelial cells themselves. No enzymatic systems presumably exist in Caco-2, which could be responsible for SAS metabolism, because after 72 h-incubation of cell cultures with 1 mM SAS, its metabolites i.e., 5-ASA and SP were not detected in cells, neither in culture media. SAS metabolism, therefore, seems to depend on the presence of intestinal bacterial enzymatic systems. It was confirmed that 5-ASA is converted by Caco-2 cells to N-acetyl-5-aminosalicylic acid (Ac-5-ASA), which migrates to the culture medium. The other metabolite of SAS i.e., SP, was not transformed in the human colon cancer cells at all.

Anti-Inflammatory Agents↗
PubMed2004

Application of HPLC for determination of phytic acid in the colonic epithelial Caco-2 cells.

Phytic acid (myo-inositol hexaphosphate, IP6) is currently receiving a considerable interest because of its anticancer (preventive and therapeutic) potential against colon tumors and the need for methods of its determination. The aim of this study was to analyze the uptake of IP6 by human colon adenocarcinoma cells (Caco-2 cell line) and to evaluate the method of its intracellular quantification with the use of high performance liquid chromatography (HPLC) technique. Chromatographic analysis revealed a rapid uptake of IP6 by cells. The intracellular accumulation of IP6 was saturable at 0.5 h and it did not change with the prolongation of incubation up to 72 h.

Caco-2 Cells↗
PubMed2003

Evaluation of hydralazine and procainamide effects on fibroblast membrane fluidity.

In this study the membrane fluidity of fibroblasts under different pharmacological treatment was investigated. Two drugs, hydralazine and procainamide, were used to treat the immortalized mouse NIH 3T3 and hamster B14 fibroblasts. Membrane lipid dynamics was measured by fluorescence spectroscopy and electron spin resonance techniques. Two kinds of fluorescent probes (TMA-DPH and 12-(9-anthroyloxy)-stearic acid (12-AS)) and two spin labels (5-doxylstearic acid (5-DS) and 12-doxylstearic acid (12-DS)) were used to monitor fluidity in the upper polar and in the hydrophobic core regions of the lipid bilayer. The drugs influenced the membrane hydrophobic core, of which hydralazine induced fluidization and procainamide increased the rigidity. The membrane fluidity at the surface of the lipid bilayer was not modified by the drugs which indicates that both drugs intercalated mainly into the inner core of the cell membrane.

Animals↗
PubMed2003

Evaluation of arbitrarily primed PCR for typing of Desulfovibrio desulfuricans strains.

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of 15 (soil and intestinal) Desulfovibrio desulfuricans strains. The primer M 13, which is a core sequence of phage M 13, was found to be appropriate for the differentiation of isolates of this species. The analysis revealed characteristic band patterns for all of the examined strains of which two soil strains (DV-7 and DV-8) showed identical DNA fingerprints. According to Jaccard's coefficient, the soil bacterial group as well as intestinal bacterial group formed two different clusters. Furthermore, the soil strains showed greater variability than the intestinal isolates. Based on the AP-PCR fingerprints D. desulfuricans strains were differentiated depending on their origin. This study demonstrates that the typing method AP-PCR can be useful in epidemiologic investigations as a rapid and valuable tool for differentiation of the strains of D. desulfuricans species.

Bacterial Typing Techniques↗
PubMed2003

[Polymorphism of beta 2-adrenergic receptors].

The human beta 2-adrenoceptor is a member of the seven-transmembrane family of receptors. It is expressed in many cell types such as airway smooth muscle cells, neutrophils, eosinophiles, alveolar macrophages and airway epithelial cells. The beta 2-adrenoceptor agonists are the most important group of bronchodilator drugs used in the treatment of asthma. They are classified by their selectivity, affinity for the receptor, potency and pharmacological efficacy. The gene encoding the beta 2-adrenergic receptor is highly polymorphic in the human population. Polymorphism affecting amino acids 16, 27 and 164 are the most common and they have been shown to correlate with some clinical features of asthma, including airways reactivity. They can modulate the behaviour of the beta 2-receptor, altering ligand binding and the characteristics of down-regulation following agonist exposure. The homozygous glycine-16 (Arg-->Gly) variant of the beta 2-adrenoceptor is known to predispose to agonist-induced down-regulation and desensitization, and may play a role in the pathogenesis of asthma severity. The polymorphism at position 27 (Gln-->Glu) is associated with decreased airway responsiveness. The polymorphic variant 164 (Thr-->Ile) shows impaired agonist binding and decreased adenylyl cyclase activity. No convincing evidence has been presented demonstrating a linking of asthma per se with this receptor polymorphism.

Adrenergic beta-Agonists↗
PubMed2003

Desulfovibrio desulfuricans lipopolysaccharides induce endothelial cell IL-6 and IL-8 secretion and E-selectin and VCAM-1 expression.

The aim of this study was to determine whether Desulfovibrio desulfuricans-derived LPS stimulate the release of IL-6 and IL-8 from ECs and the expression of their adhesion molecules at the transcriptional level. Confluent monolayers of HUVEC were incubated in the absence or presence of 20 microg/ml and 60 microg/ml LPSs derived from the DdT and DdA bacterial strains. Also, the simultaneous stimulation of cells with LPSs and IL-1beta was evaluated. The levels of cytokines released were measured using ELISA. LPS-activated HUVEC increased the secretion of both IL-6 and IL-8, which was not LPS dose dependent. The expression of E-selectin and VCAM-1 was assessed by TR-PCR. The transcripts were detectable at all the concentrations (20, 40, 60 microg/ml) of LPSs used. These results suggest that D. desulfuricans LPS may activate immune functions in endothelial cells and influence the inflammatory response during bacteremia caused by these bacteria.

Cells, Cultured↗
PubMedDate not supplied

Quantification of p21 gene expression in Caco-2 cells treated with sodium butyrate using real-time reverse transcription-PCR (RT-PCR) assay.

Butyric acid, a short chain fatty-acid derived from bacterial fermentation of complex carbohydrates in the large intestine has been shown to be a growth inhibitory in many colon cancer cell lines. Butyrate induced inhibition of cellular proliferation is considered to result from the induction of P21 gene expression through the activation of this gene transcription. P21 is an inhibitor of cyclin-dependent protein kinases that are required for the cells to enter the DNA synthesis phase. In the present study the kinetics of the changes of the P21 transcription in Caco-2 colon adenocarcinoma cells treated with various concentrations of sodium butyrate was determined using a novel real-time quantitative RT-PCR (TaqMan) technique. Beta-actin mRNA and GAPDH mRNA levels were used as the endogenous references. Colonocytes were incubated with sodium butyrate at concentrations of 5 mM, 10 mM and 20 mM for 3, 6, 12, 24 and 48 h. The results of this study indicated that butyrate strongly induced P21 gene expression as early as 3 h after treatment. Characteristic patterns of time-dependent changes of the target gene expression were observed. The increases in P21 mRNA level were generally more pronounced at higher butyrate concentrations. Because Caco-2 cells are lacking the wild allele of the P53 gene, the present results support the hypothesis that butyrate induces P21 gene expression by P53-independent mechanism.

Butyrates↗
PubMedDate not supplied

The effect of sulphasalazine and its metabolites on the colonic epithelial Caco-2 cells.

Sulphaselazine (SAS) is a drug commonly used to treat patients suffering from chronic inflammatory states such as inflammatory bowel diseases. It was shown that besides bacteriostatic, antiinflammatory and immunosuppressive activity of this drug, the risk of neoplastic changes in the colon and rectum was substantially diminished during ulcerative colitis therapy with SAS. In the present study the effects of SAS and its main metabolites--sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) on colon adenocarcinoma Caco-2 cells viability and proliferation was evaluated. Significant inhibitory impact of SAS was observed already at 1 mM concentration whereas 5-ASA and SP impaired cellular growth when used at 5 mM concentration. 5 mM SAS exerted a strong cytotoxic effect on Caco-2 cells resulting in their necrotic death. The inhibition of cellular proliferation and the cytotoxic effects of SAS and its metabolites (5-ASA and SP) on the colonic carcinoma cells (Caco-2) confirm the suggestions that these compounds at appropriate concentrations may reduce the risk of neoplastic changes frequently initiated by prolonged inflammatory states.

Caco-2 Cells↗