Spirostanol Saponins from Flowers of Allium Porrum and Related Compounds Indicating Cytotoxic Activity and Affecting Nitric Oxide Production Inhibitory Effect in Peritoneal Macrophages.

Saponins, a diverse group of natural compounds, offer an interesting pool of derivatives with biomedical application. In this study, three structurally related spirostanol saponins were isolated and identified from the leek flowers of Allium porrum L. (garden leek). Two of them were identical with the already known leek plant constituents: aginoside (1) and 6-deoxyaginoside (2). The third one was identified as new component of A. porrum; however, it was found identical with yayoisaponin A (3) obtained earlier from a mutant of elephant garlic Allium ampeloprasun L. It is a derivative of the aginoside (1) with additional glucose in its glycosidic chain, identified by MS and NMR analysis as (2α, 3ß, 6ß, 25R)-2,6-dihydroxyspirostan-3-yl ß-D-glucopyranosyl-(1 → 3)-ß-D-glucopranosyl-(1 → 2)-[ß-D-xylopyranosyl-(1 → 3)]-ß-D-glucopyranosyl]-(1 → 4)-ß-D-galactopyranoside, previously reported also under the name alliporin. The leek native saponins were tested together with other known and structurally related saponins (tomatonin and digitonin) and with their related aglycones (agigenin and diosgenin) for in vitro cytotoxicity and for effects on NO production in mouse peritoneal cells. The highest inhibitory effects were exhibited by 6-deoxyaginoside. The obtained toxicity data, however, closely correlated with the suppression of NO production. Therefore, an unambiguous linking of obtained bioactivities of saponins with their expected immunobiological properties remained uncertain.

Polysubstituted Pyrimidines as Potent Inhibitors of Prostaglandin E2 Production: Increasing Aqueous Solubility.

Water solubility is one of the key features of potential therapeutic agents. In order to enhance the low water solubility of the parent 5-butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-amine, a potent inhibitor of prostaglandin E2 (PGE2 ) production, we synthesized and evaluated a new series of derivatives in which the butyl group at the C5 position of the pyrimidine ring was replaced with a less lipophilic substituent, preferably with a hydrophilic aliphatic moiety. Except for the 5-cyanopyrimidine derivative, all target compounds exhibited increased (2.7-87-fold) water solubility relative to the parent compound. Although nontoxic in mouse peritoneal cells, the prepared compounds were either equipotent or weaker inhibitors of PGE2 production than the parent compound. The most promising compound from the series was found to be the 5-(2,5,8,11-tetraoxadodecyl)pyrimidine derivative (with three polyethylene glycol units at the C5 position), which exhibited 32-fold higher water solubility and only slightly weaker inhibitory activity (22 % of remaining PGE2 production) compared with the parent compound (15 % of remaining PGE2 production).

Polysubstituted Pyrimidines as mPGES-1 Inhibitors: Discovery of Potent Inhibitors of PGE2 Production with Strong Anti-inflammatory Effects in Carrageenan-Induced Rat Paw Edema.

We report an extensive structure-activity relationship optimization of polysubstituted pyrimidines that led to the discovery of 5-butyl-4-(4-benzyloxyphenyl)-6-phenylpyrimidin-2-amine, and its difluorinated analogue. These compounds are sub-micromolar inhibitors of PGE2 production (IC50 as low as 12 nM). In order to identify the molecular target of anti-inflammatory pyrimidines, we performed extensive studies including enzymatic assays, homology modeling and docking. The difluorinated analogue simultaneously inhibits two key enzymes of the arachidonic acid cascade, namely mPGES-1 and COX-2, with mPGES-1 inhibition being the principal mechanism of action. Other pyrimidines studied are potent mPGES-1 inhibitors with no observed inhibition of COX-1/2 enzymes. Moreover, the two most potent compounds proved to be significantly effective in vivo in a model of acute inflammation, suppressing carrageenan-induced rat paw edema by 36 and 46 %. The promising results of this study warrant further preclinical evaluation of selected anti-inflammatory candidates.

Influence of the C-5 substitution in polysubstituted pyrimidines on inhibition of prostaglandin E2 production.

As a part of a broader structure-activity relationship study of substituted 2-aminopyrimidines, the influence of the C-5 substitution on inhibition of prostaglandin E2 (PGE2) production was studied. Thirty compounds were prepared starting from the corresponding 2-amino-4,6-dichloropyrimidines using Suzuki cross-coupling. It was shown previously that 2-amino-4,6-dichloropyrimidines with smaller C-5 substituent (hydrogen and methyl) were devoid of significant activity, while 5-butyl derivatives exhibited prominent potency. In this study, on the other hand, both monoaryl- and bisarylpyrimidines were potent inhibitors of PGE2 production regardless the length of the C-5 substituent (hydrogen, methyl, n-butyl). Moreover, the shorter the C-5 substituent the higher potency to inhibit PGE2 production was observed. 2-Amino-4,6-diphenylpyrimidine was the best inhibitor of PGE2 production with IC50 = 3 nM and no cytotoxicity. The most potent inhibitors deserve further preclinical evaluation as potential anti-inflammatory agents.

Polysubstituted 4,6-bis(hetero)arylpyrimidines as dual inhibitors of nitric oxide and prostaglandin E2 production.

As a part of our extensive structure-activity relationship study of anti-inflammatory heterocycles, a novel series of 67 polysubstituted 2-aminopyrimidines was prepared bearing one (at the C-4 position of the pyrimidine ring) or two (in the C-4 and C-6 positions) (hetero)aryl substituents attached directly through the C-C bond. The key synthetic steps involved either Suzuki-Miyaura or Stille cross-coupling reactions carried out on easily available 4,6-dichloropyrimidines. All prepared compounds, except one, were able to inhibit immune-activated production of nitric oxide (NO) significantly. Moreover, several compounds were found to be low micromolar dual inhibitors of NO and prostaglandin E2 (PGE2) production. Although the exact mode of action of the prepared compounds remains to be elucidated, non-toxic dual inhibitors of NO and PGE2 production may have great therapeutic benefit in treatment of various inflammation diseases and deserve further preclinical evaluation.

Dual inhibition of nitric oxide and prostaglandin E2 production by polysubstituted 2-aminopyrimidines.

The present in vitro experiments demonstrate inhibitory effects of polysubstituted 2-aminopyrimidines on high output production of nitric oxide (NO) and prostaglandin E2 (PGE2) stimulated by interferon-γ and lipopolysaccharide (LPS) in peritoneal macrophages of mouse and rat origin. PGE2 production was inhibited also in LPS-activated human peripheral blood mononuclear cells. A tight dependence of the suppressive activities on chemical structure of pyrimidines was observed. Derivatives containing hydroxyl groups at the C-4 and C-6 positions of pyrimidine ring were devoid of any influence on NO and PGE2. Remarkable inhibitory potential was acquired by the replacement of hydroxyl groups with chlorine, the 4,6-dichloro derivatives being more effective than the monochloro analogues. The effects were further intensified by modification of the amino group at the C-2 position, changing it to the (N,N-dimethylamino)methyleneamino or the formamido ones. There was no substantial difference in the expression of NO-inhibitory effects among derivatives containing distinct types of substituents at the C-5 position (hydrogen, methyl, ethyl, propyl, butyl, phenyl, and benzyl). In contrast to NO, larger substituents then methyl were required to inhibit PGE2 production. Overall, no significant correlation between the extent of NO and PGE2 suppression was observed. The IC50s of derivatives with the strongest effects on both NO and PGE2 were within the range of 2-10 µM. Their NO-inhibitory potential of pyrimidines was stronger than that of non-steroidal anti-inflammatory drugs (NSAIDs) aspirin and indomethacin. The PGE2-inhibitory effectiveness of pyrimidines was about the same as that of aspirin, but weaker as compared to indomethacin. The NO- and PGE2-inhibitory activity of tested pyrimidines has been found associated with decreased expression of iNOS mRNA and COX-2 mRNA, respectively, and with post-translation interactions. Selected NO-/PGE2-inhibitory derivatives decreased severity of intestinal inflammation in murine model of ulcerative colitis.

Immunobiological properties of sesquiterpene lactones obtained by chemically transformed structural modifications of trilobolide.

Our previous research on immunostimulatory properties of trilobolide and its structurally related natural analogues isolated from Laser trilobum (L.) Borkh., encouraged us to investigate structurally related guaianolides belonging to a specific group of sesquiterpene lactones with characteristic glycol moiety attached to the lactone ring. Ever increasing attention has been paid to certain guaianolides such as thapsigargin and trilobolide for their promising anti-inflammatory, anticancer, anti-infectious and SERCA inhibitory activities. However, due to their alkylation capabilities, they might be cytotoxic. Search for compounds with preserved immunobiological properties and decreased cytotoxicity led us to transform some of their structural features, particularly those related to their side chain functionality. For this reason, we prepared a series of over 20 various deacylated, acyl modified, or relactonized derivatives of trilobolide. The immunobiological effects were screened in vitro using the rat peritoneal cells primed with lipopolysaccharide. Secretion of interferon-γ (IFN-γ), interleukins (IL) IL-1ß, IL-6 and tumour necrosis factor-α (TNF-α) were determined by ELISA, and nitric oxide (NO) production by Griess reagent. Relation between the molecular structure and immunobiological activity was investigated. Acetylation at 7-OH and 11-OH positions of the lactone ring, or acyl modification of the guaianolide functionalities (including relactonization) of trilobolide, led to inability to stimulate secretion of cytokines and production of NO. Interestingly, minor structural changes achieved by catalytic hydrogenation or hydrogenolysis retained the original immunoactivity of trilobolide. It can be concluded that several new chemically transformed sesquiterpene lactones resembling the immunobiological properties of trilobolide or thapsigargin were prepared and identified. The implication of the lactone vicinal diol (glycol) moiety, combined with other structure functionality, was confirmed as essential for immune properties of the trilobolide or thapsigargin type of guaianolides.

Acyclic nucleoside phosphonates: a study on cytochrome P450 gene expression.

1. Nucleotide analogues comprise an important class of drugs used in treatment of viral infections but also cancer. These drugs affect the structural integrity of DNA and activate different pathways and processes in the cell and may directly or indirectly influence the drug metabolizing system. Adefovir dipivoxil (AD) and tenofovir disoproxil (TD) are nucleotide analogues approved for the treatment of chronic hepatitis B and/or HIV/AIDS infection. 2. To evaluate the risk of their drug-drug interactions on the level of drug metabolism, an effect of both compounds on cytochromes P450 expression was studied using cDNA microarrays, real-time RT-PCR and immunoblotting. Mice were given intraperitoneally 25 mg/kg of AD or TD, respectively. As a positive control, a combination of prototypic cytochromes P450 (CYP) inducers, phenobarbital and ß-naphthoflavone was chosen. 3. The data obtained showed a significant CYP induction in the positive control group, but no clinically significant induction of CYP genes by AD or TD was observed. Our results support the evidence of safety of AD and TD with respect to drug-drug interactions based on enzyme induction. These findings are important as a plethora of new antivirals of different types are being tested and introduced to clinical practice, mostly to be used in combinations.

Activation of respiratory complex II by interferon-gamma and its inhibition by pyrimidine derivatives.

OBJECTIVES: Formation of formazan is a commonly used measure of cytotoxicity of compounds. It is a product of reduction of tetrazolium salts such as 4-[3- (4-iodophenyl)-2- (4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride. The extent of substrates reduction reflects the activity of enzymes succinate dehydrogenase (SDH; respiratory complex II) and lactate dehydrogenase (LDH), respectively. The aim of present study was a) to investigate formazan formation under the conditions of in vitro stimulation of cells with interferon-γ (IFN-γ) and lipopolysaccharide (LPS), and b) to analyse possible interference of pyrimidine analogues with formazan production. METHODS: Peritoneal cells and splenocytes were obtained from C57BL/6 mice. They were cultured at 37 degrees C, 5% CO2 in humidified incubator. Levels of formazan were determined at the interval of 24 h of culture using the WST-1 and LDH assays. Nitric oxide (NO) was activated by IFN-γ plus LPS and assayed by Griess reagent 24 h afterwards. Pyrimidines were applied concomitantly with immunostimulatory agents. RESULTS: IFN-γ enhanced concentration of SDH-produced formazan by macrophages (not by splenocytes) by approximately 50%. The activity of LDH remained unaffected. LPS was ineffective in both cases. While pyrimidines with NO-inhibitory properties suppressed the IFN-γ-enhanced levels of SDH-produced formazan, they did not change the LDH-dependent formazan production. CONCLUSION: IFN-γ augments the SDH-produced formazan by macrophages. It does not change the LDH-dependent formazan formation. The enhancing effect may have a significant impact upon the appropriate interpretation of cytotoxic properties of drugs investigated under the conditions of immune stimulation of cells.

5-Substituted 2-amino-4,6-dihydroxypyrimidines and 2-amino-4,6-dichloropyrimidines: synthesis and inhibitory effects on immune-activated nitric oxide production.

A series of 5-substituted 2-amino-4,6-dihydroxypyrimidines were prepared by a modified condensation of the corresponding monosubstituted malonic acid diesters with guanidine in an excess of sodium ethoxide. The optimized procedure using Vilsmeier-Haack-Arnold reagent, followed by immediate deprotection of the (dimethylamino)methylene protecting groups, has been developed to convert the 2-amino-4,6-dihydroxypyrimidine analogs to novel 5-substituted 2-amino-4,6-dichloropyrimidines in high yields. Pilot screening for biological properties of the prepared compounds was done in mouse peritoneal cells using the in vitro nitric oxide (NO) assay. Irrespective of the substituent at the 5 position, 2-amino-4,6-dichloropyrimidines inhibited immune-activated NO production. The most effective was 5-fluoro-2-amino-4,6-dichloropyrimidine with an IC 50 of 2 µM (higher activity than the most potent reference compound) while the IC 50s of other derivatives were within the range of 9-36 µM. The 2-amino-4,6-dihydroxypyrimidine counterparts were devoid of any NO-inhibitory activity. The compounds had no suppressive effects on the viability of cells. The Mechanism of action remains to be elucidated.

Two new C-methyl flavanones from the rhizomes and frond bases of Matteuccia struthiopteris.

Two new C-methyl flavanones, (2S)-5,7-dihydroxy-6,8-dimethyl-4'-methoxydihydroflavone-7-O-(6″-O-acetyl)-ß-d-glucopyranoside (1) and (2S)-5,7-dihydroxy-6,8-dimethyldihydroflavone-7-O-(6″-O-acetyl)-ß-d-glucopyranoside (2), together with five known compounds, demethoxymatteucinol-7-O-ß-d-glucopyranoside (3), matteucinol-7-O-ß-d-glucopyranoside (4), 5,7-dihydroxy-6-methyl-4'-methoxydihydroflavone (5), methoxymatteucin (6), and thunberginol C (7), were first isolated from the EtOH extract of the rhizomes and frond bases of Matteuccia struthiopteris. The structures were established by spectral analyses, mainly HR-ESI-MS and 1D and 2D NMR experiments (COSY, HSQC, and HMBC).

Divergent immunomodulatory effects of extracts and phenolic compounds from the fern Osmunda japonica Thunb.

OBJECTIVE: To study possible immunobiological potential of Osmunda japonica Thunb. METHODS: Immunomodulatory effects of ethanol extracts prepared from rhizomes of O. japonica and phenolic compounds isolated from the extracts were investigated under the in vitro conditions using the rat peritoneal cells (2×10(6)/mL; 24 h culture). Biosynthesis of nitric oxide (NO) was assayed by Griess reagent, production of prostaglandin E2 (PGE2) and secretion of cytokines were determined by enzyme-linked immunoabsorbent assay. RESULTS: The extracts activated dose dependently, with the onset at 2.5-5 µmol/L concentrations, the high output NO production, and secretion of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). Mild enhancement of NO was produced by the aldehyde-type phenolics 4-hydroxybenzaldehyde and 3,4-hydroxybenzaldehyde. In contrasts, the acetone-type phenolics 4-hydroxybenzalacetone and 3,4-hydroxybenzalacetone inhibited production of immune mediators including cytokines (TNF-α, IL-1ß, IL-6), NO, and PGE2. The 3,4-hydroxybenzalacetone was more effective than 4-hydroxybenzaldehyde. The IC50s estimates ranged within the interval of 5-10 µmol/L. No signs of cytotoxicity were observed up to the 50 µmol/L concentration of the compounds. CONCLUSION: Phenolic compounds contained in medicinal herb Osmunda japonica possess distinct immunomodulatory activity.

The effect of probiotic Escherichia coli strain Nissle 1917 lipopolysaccharide on the 5-aminosalicylic acid transepithelial transport across Caco-2 cell monolayers.

The object of this study was to investigate the effect of probiotic Escherichia coli strain Nissle 1917 (EcN) (i) EcN lipopolysaccharide (EcN LPS) and (ii) bacteria-free supernatant of EcN suspension (EcN supernatant) on in vitro transepithelial transport of mesalazine (5-aminosalicylic acid, 5-ASA), the most commonly prescribed anti-inflammatory drug in inflammatory bowel disease (IBD). Effect of co-administered EcN LPS (100 µg/ml) or EcN supernatant (50 µg/ml) on the 5-ASA transport (300 µmol/l) was studied using the Caco-2 monolayer (a human colon carcinoma cell line) as a model of human intestinal absorption. Permeability characteristics for absorptive and secretory transport of parent drug and its intracellularly-formed metabolite were determined. The quantification of 5-ASA and its main metabolite N-acetyl-5-amino-salicylic acid (N-Ac-5-ASA) was performed by high performance liquid chromatography. Obtained results suggest that neither EcN LPS nor EcN supernatant had effect on the total 5-ASA transport (secretory flux greater than absorptive flux) and on the transport of intracellularly formed N-Ac-5-ASA (preferentially transported in the secretory direction). The percent cumulative transport of the total 5-ASA alone or in combination with EcN LPS or EcN supernatant did not exceed 1%.

Effects of sesquiterpene, flavonoid and coumarin types of compounds from Artemisia annua L. on production of mediators of angiogenesis.

BACKGROUND: In addition to recognized antimalarial effects, Artemisia annua L. (Qinghao) possesses anticancer properties. The underlying mechanisms of this activity are unknown. The aim of our experiments was to investigate the effects of distinct types of compounds isolated from A. annua on the immune-activated production of major mediators of angiogenesis playing a crucial role in growth of tumors and formation of metastasis. METHODS: Included in the study were the sesquiterpene lactones artemisinin and its biogenetic precursors arteannuin B and artemisinic acid. The semi-synthetic analogue dihydroartemisinin was used for comparative purposes. The flavonoids were represented by casticin and chrysosplenol D, the coumarin type of compounds by 4-methylesculetin. Their effects on the lipopolysaccharide (LPS)-induced in vitro production of nitric oxide (NO) were analyzed in rat peritoneal cells using Griess reagent. The LPS-activated production of prostaglandin E2 (PGE2) and cytokines (VEGF, IL-1ß, IL-6 and TNF-α) was determined in both rat peritoneal cells and human peripheral blood mononuclear cells using ELISA. RESULTS: All sesquiterpenes (artemisinin, dihydroartemisinin, artemisinic acid, arteannuin B) significantly reduced production of PGE2. Arteannuin B also inhibited production of NO and secretion of cytokines. All NO, PGE2 and cytokines were suppressed by flavonoids casticin and chrysosplenol D. The coumarin derivative, 4-methylesculetin, was ineffective to change the production of any of these factors. CONCLUSIONS: The inhibition of immune mediators of angiogenesis by sesquiterpene lactones and flavonoids may be one of the mechanisms of anticancer activity of Artemisia annua L.

Trilobolide and related sesquiterpene lactones from Laser trilobum possessing immunobiological properties.

Three new and five known sesquiterpene lactones were isolated from the roots of Laser trilobum (L.) Borkh. Chemical identity of the known compounds and structural analysis of the new ones were determined by HR MS and NMR spectroscopy. The two new sesquiterpene lactones: 2-acetoxytrilobolide and 2-hydroxy-10-deacetyltrilobolide belong to the guaianolide type, and the third one, eudeslaserolide, to the biogenetically related eudesmanolide type. Both types, together with their biogenetic precursor of germacranolide type (laserolide) are present in L. trilobum, as well as in the related Laserpitium species. Purposefully selected set of these native sesquiterpene lactones was tested for specific immunobiological properties. The obtained results demonstrate that trilobolide and its acetoxy analog are strong activators of cytokine secretion. On the contrary, the other L. trilobum and Laserpitium siler constituents are only very mild activators, or even inhibitors of the cytokine and nitric oxide production.

Microfiltration method of removal of bacterial contaminants and their monitoring by nitric oxide and Limulus assays.

Similar to lipopolysaccharide (LPS), a product of Gram-negative bacteria, the signal macromolecules of Gram-positive bacteria lipoteichoic acid (LTA) and peptidoglycan (PGN) possess multiple biological activities. They may be a source of misinterpretation of experimental findings. We have found that not only LPS but also LTA and PGN can be detected by the Limulus amebocyte lysate (LAL) assay. All of them stimulate the high output in vitro nitric oxide (NO) production of in rat peritoneal cells. The onset of the NO enhancement was observed with 25-100pg/ml of LPS and 25-100ng/ml of PGN and LTA. Polymyxin B (PMX), if applied at concentration 10,000-fold higher than that of LPS, can completely inhibit the NO and LAL binding responses of LPS. The NO-stimulatory and LAL-binding properties of LTA and PGN are not eliminated by PMX. Handling of LPS contamination with PMX may be associated with serious problems because it possesses intrinsic biological activity and becomes cytotoxic at concentration >25µg/ml. The present findings suggest a convenient alternative avoiding these issues. As monitored by the NO and LAL assays, even high amounts of LPS as well as PGN and LTA can be removed by molecular mass cutoff microfiltration. All types of the filters (3kDa to 100kDa) are equally effective. It is suggested that the microfiltration procedure may be considered as a preferable, general and easy method of sample decontamination.

Stimulation of nitric oxide, cytokine and prostaglandin production by low-molecular weight fractions of probiotic Lactobacillus casei lysate.

OBJECTIVES: Major medical indications of probiotic bacteria are conditions associated with the gastrointestinal tract. They exhibit not only the local but also systemic effects, the molecular mechanisms of which are poorly understood. We hypothesized that the action at remote sites of the body could be at least partially attributed to substances of the low molecular mass released from digested bacteria and able to cross the intestinal barrier. The aim of the study was the analysis of immunobiological properties of bacterial lysates and characterization of chemical constituents participating on this mode of action. METHODS: Lactobacillus casei probiotic strain DN-114001 was employed. Lysates were prepared by passing bacteria through a French press (1500 psi) followed by lyophilisation. The fractions were prepared by the microfiltration of the crude lysate using the 3-, 10-, 30-, 50-, and 100-kDa cutoff filters (Amicon® Ultra 0.5 ml, Millipore Corp.). This procedure completely removes biologically active bacterial macromolecules such as peptidoglycan (PGN), lipoteichoic acid (LTA) and lipopolysaccharide (LPS). Effects of microfiltrates on the in vitro production of nitric oxide (NO), cytokines, and prostaglandin E2 (PGE2) were investigated in rat peritoneal cells. RESULTS: The original crude lysate (≤10 µg/ml) activated the biosynthesis of NO, PGE2, and secretion of cytokines. The amount of the lysate needed for the preparation of microfiltered fractions exhibiting immunostimulatory effects was 10-fold higher (100 µg/ml). The molecules with the molecular mass ≤3 kDa were responsible for approximately 45% and 83% of the NO- and PGE2-enhancing activities of the crude lysate, respectively. The microfiltered fractions of the lysate also enhanced secretion of interleukin-6 and tumor necrosis factor-α but not that of interleukin-10 and interferon-γ. CONCLUSION: The Lactobacillus casei probiotic strain DN-114001 contains low molecular mass (≤3 kDa) molecules possessing immunostimulatory properties. Their chemical nature remains to be identified.

Differential effects of acyclic nucleoside phosphonates on nitric oxide and cytokines in rat hepatocytes and macrophages.

Acyclic nucleoside phosphonates (ANP) are virostatics effective against viruses like hepatitis B virus and human immunodeficiency virus. Our previous reports indicated immunomodulatory activities of ANP in mouse and human innate immune cells. Recently, evidence has increased that hepatocytes may play an active role in immune regulation of the liver homeostasis or injury. In this study we investigated possible immunomodulatory effects of ANP on rat hepatocytes and macrophages. Nitric oxide (NO) production and secretion of cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-13, IL-18, IFN-γ, TNF-α and GM-CSF) were analyzed under in vitro conditions. Test compounds included: 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; adefovir); 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP); (R)- and (S)-enantiomers of 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir] and [(S)-PMPA]; 9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine [(R)-PMPDAP] and [(S)-PMPDAP]. The group of test compounds also included their N(6)-substituted derivatives. Some of ANP which are able to induce NO production and cytokine secretion in cultured macrophages possess the same immunobiological activity in isolated hepatocytes. The extent of responses is in range of LPS/IFN-γ stimulation in both types of cells. The effects of active ANP on NO expression and cytokine secretion are dose- and time-dependent. Interestingly, the spectrum of detected cytokines induced by ANP is broader in hepatocytes. The results also confirm immunomodulatory effects of some ANP on rodent macrophages. Moreover, we demonstrate for the first time immunobiological reactivity of primary rat hepatocytes induced by exogenous ANP compounds. The potential of hepatocytes to synthesize cytokines can contribute to better understanding of liver immune function and can serve for pharmacological intervention in liver diseases.

Effects of Lactobacillus casei on the expression and the activity of cytochromes P450 and on the CYP mRNA level in the intestine and the liver of male rats.

OBJECTIVES: The aim of the study was to find whether probiotic Lactobacillus casei influences the expression or the activity of cytochromes P450 (CYP) and whether it has an influence on the level of CYP mRNA in male rats. DESIGN: Live bacterial suspension of L. casei was administered orally (gavage) to healthy male Wistar rats daily for 7 days. Control group of rats was treated with the saline solution. Sections of the duodenum, jejunum, ileum, caecum and colon were dissected from each experimental animal. In all individual samples, the expression of selected CYPs was determined by Western blotting. The levels of expression of CYPs were also evaluated by mRNA using the real-time PCR method. RESULTS: There were changes observed in the expression of CYP enzymes and in the CYP mRNA levels along the intestine after application of L. casei. The expression of CYP1A1 enzyme was found to be decreased in the proximal part of the jejunum and colon, CYP1A1 mRNA level was decreased in the distal part of the jejunum, ileum and caecum. Thus, the changes in CYP1A1 protein or mRNA were observed along the intestine of male rats. Similarly, a decreased expression of the caecal CYP2E1 mRNA and of the duodenal CYP3A9 mRNA after treatment of rats with L. casei was found. CONCLUSION: Probiotic L. casei might be able to contribute to prevention against colorectal cancer by decreasing levels of certain forms of xenobiotic-metabolizing enzymes; moreover, in general, there is a possibility of interactions with concomitantly taken pharmacotherapeutic agents.