8-oxo-7,8-dihydroguanine level - the DNA oxidative stress marker - recognized by fluorescence image analysis in sporadic uterine adenocarcinomas in women.

OBJECTIVES: In the case of carcinogenesis in human endometrium no information exists on tissue concentration of 8-oxo-7,8-dihydroguanine, the DNA oxidative stress marker This was the main reason to undertake the investigation of this DNA modification in human uterine estrogen-dependent tissue cancers. MATERIAL AND METHODS: In order to estimate the level of oxidative damage, 8-oxo-7,8-dihydroguanine was determined directly in cells of tissue microscope slides using OxyDNA Assay Kit, Fluorometric. Cells were investigated under confocal microscope. Images of individual cells were captured by computer-interfaced digital photography and analyzed for fluorescence intensities (continuous inverted 8-bit gray-scale = 0 [black]-255 [white]). Fluorescence scores were calculated for each of 13 normal endometrial samples and 31 uterine adenocarcinoma specimens. Finally the level of the oxidative stress marker was also analyzed according to histological and clinical features of the neoplasms. RESULTS: The obtained data revealed that: 8-oxo-7,8-dihydroguanine levels were higher in uterine adenocarcinomas than in normal endometrial samples (48,32 vs. 38,64; p<0,001); in contrast to normal endometrium there was no correlation between age and DNA oxidative modification content in uterine cancer; highest mean fluorescence intensity was recognized in G2 endometrial adenocarcinomas; level of 8-oxo-7,8-dihydroguanine does not depend on Body Mass Index (BMI) and cancer uterine wall infiltration or tumor FIGO stage. CONCLUSIONS: Our study indicates that accumulation of the oxidized DNA base may contribute to the development of endometrial neoplasia, however oxidative DNA damage does not seem to increase with tumor progression.

Circulating microenvironment of CLL: are nurse-like cells related to tumor-associated macrophages?

B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common hematologic malignancies in Western countries. Accumulation of leukemic lymphocytes in peripheral blood, bone marrow and secondary lymphatic organs of CLL patients is due to decreased apoptosis rather than to increased proliferation. The former is driven by signals from a specific microenvironment, created by stromal cells of mesenchymal origin, follicular dendritic cells, T lymphocytes and others. Nurse-like cells (NLCs) were first described to differentiate from peripheral blood mononuclear cells of CLL patients in vitro, then they have been also found in proliferation centers of their lymphatic tissues. Like tumor-associated macrophages (TAMs) in solid tumors, nurse-like cells promote survival of CLL lymphocytes. NLC gene expression patterns suggest their similarity to TAMs and differ between patients depending on ZAP70 protein expression status. NLC number in vitro corresponds with CD14 expressing cell count and beta-2-microglobulin serum level, and positively correlates with leukemic lymphocyte viability. As NLCs strongly express genes for adhesion molecules and secrete chemokines of antiapoptotic activity, they should be considered as a target for anti-microenvironment therapy of this incurable disease.

Prevention of biofilm formation on urinary catheters: comparison of the sparfloxacin-treated long-term antimicrobial catheters with silver-coated ones.

Urinary catheters are widely used for hospitalized patients and are often associated with high risk of urinary tract infection. The agar and broth diffusion tests, visual TTC (triphenyltetrazolium chloride) method, and confocal scanning laser microscopic (CSLM) observations have shown highly satisfactory antimicrobial and antibiofilm activity of the novel sparfoxacin (SPA)-treated urinary catheters compared with the controversial effectiveness of silver(Ag)-coated catheters against a background of untreated catheters used as controls. SPA-treated catheters were significantly less likely to become colonized (less than 0.01%; inner and outer surfaces against Escherichia coli and Staphylococcus aureus) than both silver-coated (from 0.01% to 39.3 %; outer surface against E. coli and inner surface against S. aureus, resp.) and untreated catheters (from 88.43% to 99.72%; outer and inner surfaces, resp., against S. aureus), and maintained their broad spectrum of antimicrobial and antibiofilm activity during storage for at least 6 months.

The effect of Galleria mellonella apolipophorin III on yeasts and filamentous fungi.

Galleria mellonella apolipophorin III (apoLp-III) has been implicated in the innate immune response against bacterial infections. The protein binds components of bacterial cell wall and inhibits growth of selected Gram-positive and Gram-negative bacteria. Interaction of apoLp-III with fungal ß-1,3-glucan suggests antifungal properties of the protein. In the present study, the effect of apoLp-III on the growth, metabolic activity and cell surface characteristics of selected yeasts and filamentous fungi was investigated using light, confocal and atomic force microscopy. ApoLp-III bound to the cell surface of different yeasts and filamentous fungi as confirmed by immunoblotting with anti-apoLp-III antibodies. Incubation of the fungi in the presence of apoLp-III induced alterations in growth morphology. Candida albicans underwent transition from yeast-like to hyphal growth with formation of true hyphae, whereas Fusarium oxysporum hyphae exhibited decreased metabolic activity, increased vacuolization and appearance of numerous monophialids with microconidia. Atomic force microscopy imaging demonstrated evident alterations in the fungal cell surface after incubation with apoLp-III, suggesting that the protein affected the cell wall components.

The use of morphometric and fractal parameters to assess the effects of 5-fluorouracil, interferon and dexamethasone treatment on colonic anastomosis healing: an experimental study in rats.

Adjuvant chemotherapy and steroid therapy have been demonstrated to interfere with the wound healing process. The aim of this study was to evaluate the effects of 5-fluorouracil, interferon, and dexamethasone, on the healing of colon anastomosis by assessing morphometric and fractal parameters of the colonic wall. An experimental anastomosis of the ascending colon was performed in 60 male Wistar rats, which were then randomly assigned to four groups. On the second to sixth post-operative days, the rats were administered 5-fluorouracil, interferon-α, dexamethasone, or 0.9% NaCl solution as a control. Macroscopic, histomorphometric and microbiological evaluation was performed in order to assess healing of the anastomosis. In three animals from the dexamethasone group, there was leakage of anastomosis; adhesion formation was highest in the interferon group, and significantly higher than in the control and 5-fluorouracil groups. Histomorphometric parameter alterations were most pronounced on the seventh and fourteenth post-operative days in all treatment groups, with submucosal thickness the most affected parameter. Connective tissue fractal dimension was significantly decreased in those animals treated with interferon and dexamethasone. All three pharmaceutical agents impaired healing of anastomosis, and promoted infection in the anastomosis and skin wound sites. As dexamethasone induced both morphometric and macroscopic alterations, it was considered the most detrimental in this study.

Rhizobium leguminosarum bv. trifolii rosR is required for interaction with clover, biofilm formation and adaptation to the environment.

BACKGROUND: Rhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized. RESULTS: In this work, we extend our earlier studies to the characterization of rosR mutants which exhibit pleiotropic phenotypes. The mutants produce three times less exopolysaccharide than the wild type, and the low-molecular-weight fraction in that polymer is greatly reduced. Mutation in rosR also results in quantitative alterations in the polysaccharide constituent of lipopolysaccharide. The rosR mutants are more sensitive to surface-active detergents, antibiotics of the beta-lactam group and some osmolytes, indicating changes in the bacterial membranes. In addition, the rosR mutants exhibit significant decrease in motility and form a biofilm on plastic surfaces, which differs significantly in depth, architecture, and bacterial viability from that of the wild type. The most striking effect of rosR mutation is the considerably decreased attachment and colonization of root hairs, indicating that the mutation affects the first stage of the invasion process. Infection threads initiate at a drastically reduced rate and frequently abort before they reach the base of root hairs. Although these mutants form nodules on clover, they are unable to fix nitrogen and are outcompeted by the wild type in mixed inoculations, demonstrating that functional rosR is important for competitive nodulation. CONCLUSIONS: This report demonstrates the significant role RosR regulatory protein plays in bacterial stress adaptation and in the symbiotic relationship between clover and R. leguminosarum bv. trifolii 24.2.

Glass surface as potential in vitro substratum for Candida famata biofilm.

The biofilm of Candida spp. is a three-dimensional structure consisting of a dense network of yeasts, blastospores and/or filamentous elements (hyphae or pseudohyphae). All species of Candida are able to form biofilm. The aim of this paper is to present data concerning biofilm formation under static conditions by oropharyngeal isolates of C. famata on a glass surface using non-invasive confocal scanning laser microscopy (CSLM ). The changes in five parameters calculated using the CSLM technique, i.e. areal porosity (percent), fractal dimension (D), length of edge line (mm/mm2), length of skeleton line (mm/mm(2)), number of cell clusters/mm(2), describing the biofilm structure of C. famata isolates after 1 h incubation (the adhesion step), 24 h incubation (biofilm formation) and 72 h incubation (mature biofilm), indicate the morphological reorganization of biofilm during maturation. The thickness of biofilm C. famata isolates after 72 h incubation ranged from 35.2 to 81.2 micrometer.

Temozolomide, quercetin and cell death in the MOGGCCM astrocytoma cell line.

The aim of the present study was to investigate the effect of Temozolomide (an alkylating chemotherapeutic agent) and quercetin (natural flavonoid) on cell death in the human astrocytoma cell line MOGGCCM (WHO grade III). Our results indicate that Temozolomide induces autophagy, while quercetin promotes severe necrosis in the cell line in a manner dependent on the drug concentration. We demonstrated for the first time that combinations of both drugs were much more effective in programmed cell death induction in glioma cells. At a low (5muM) drug concentration, quercetin potentiated a pro-autophagic effect of Temozolomide, while after treatment with a higher drug concentration (30muM), autophagy switched to apoptosis. Temozolomide attenuated the toxic effect of quercetin. Apoptosis was mediated by the mitochondrial pathway and the activation of caspase 3 and cytochrome C release, but no changes in caspase 8 expression was observed. It was accompanied by decreased mitochondrial membrane potential and inhibition of Hsp27 and Hsp72 expression. Autophagy was correlated with an increased level of LC3II. Temozolomide and quercetin also inhibited migratory phenotype of MOGGCCM cells and changed the nuclei morphology from a circular to an irregular shape. Our results indicate that quercetin acts in synergy with Temozolomide and when used in combination rather than in separate pharmacological application, both drugs are more effective in programmed cell death induction. Temozolomide administered with quercetin seems to be a potent and promising combination which might be useful in glioma therapy.

The importance of release of proinflammatory cytokines, ROS, and NO in different stages of colon carcinoma growth and metastasis after treatment with cytotoxic drugs.

In colorectal cancers, the local cytokine network and the levels of nitric oxide (NO) and reactive oxygen species (ROS) are known to be closely related to cancer progression and metastasis, but the influence of the currently administered therapies on the cancer microenvironment is not completely understood. We analyzed the levels of reactive oxygen species (ROS), nitric oxide (NO), and cachexia-mediated cytokines (IL-1beta, IL-6, TNF-alpha) in cocultures of human colon carcinoma spheroids prepared with cells derived from tumors of different grades with human normal colon epithelial and myofibroblast cells and normal endothelial cells. We also analyzed the influence of standard chemotherapy with 5-fluorouracil (5-FU) and leucovorin (LV) combined with camptothecin (CPT-11) (IFL regimen with drug concentrations adjusted to in vitro conditions) on these parameters. The results indicated that adhesion of colon carcinoma spheroids to colon epithelium and myofibroblast monolayers induced O2- anion production but decreased NO levels compared to the sum of the radicals released by monocultures of the two types of cells. Coculture of colon carcinoma spheroids with endothelium was an exception to this rule, as only HT29 cells decreased NO production. In cocultures, anticancer drugs additionally, though only slightly and insignificantly, increased the production of the radicals compared to a nontreated coculture, but in monocultures, the drugs, and especially CPT-11, were ROS inducers and simultaneously NO production inhibitors. However, the levels of released ROS and NO were dependent on the stage of colon carcinoma that the cells were derived from. LS180 cells (grade B) grown in monocultures produced the lowest ROS levels but were the best producers of NO. Adhesion of tumor spheroids to normal cells influenced the microenvironmental cytokine network compared to monocultures, decreasing IL-1beta and TNF-alpha secretion but significantly enhancing L-6 levels. The addition of the drugs had no effect on IL-1beta levels but increased TNF-alpha production and lowered the amounts of IL-6. In conclusion, cytotoxic drugs may, dependent on the stage of tumor growth or the type of chemotherapy regimen administered, significantly influence the proinflammatory cytokine network and local ROS and NO levels. Moreover, in cocultures of tumor cells with normal epithelial, myofibroblast, and endothelial cells, ROS production seems to be involved in local cell injury, which was detected by confocal microscopy. On the other hand, high level of NO seems to facilitate tumor cell interactions with the endothelium and metastasis as NO production was the highest in a monoculture of HUVEC and remained at high levels in cocultures of colon cancer cells with HUVEC. Among the proinflammatory cytokines, only IL-6 seems to significantly influence colon carcinoma development and metastasis. Attenuation of IL-6 production after chemotherapy can be a useful prognostic factor of its effectiveness.

Gut bacterium of Dendrobaena veneta (Annelida: Oligochaeta) possesses antimycobacterial activity.

The new bacterial strain with antimycobacterial activity has been isolated from the midgut of Dendrobaena veneta (Annelida). Biochemical and molecular characterization of isolates from 18 individuals identified all as Raoultella ornithinolytica genus with 99% similarity. The bacterium is a possible symbiont of the earthworm D. veneta. The isolated microorganism has shown the activity against four strains of fast-growing mycobacteria: Mycobacterium butiricum, Mycobacterium jucho, Mycobacterium smegmatis and Mycobacterium phlei. The multiplication of the gut bacterium on plates with Sauton medium containing mycobacteria has caused a lytic effect. After the incubation of the cell free extract prepared from the gut bacterium with four strains of mycobacteria in liquid Sauton medium, the cells of all tested strains were deformed and divided to small oval forms and sometimes created long filaments. The effect was observed by the use of light, transmission and scanning microscopy. Viability of all examined species of mycobacteria was significantly decreased. The antimycobacterial effect was probably the result of the antibiotic action produced by the gut bacterium of the earthworm. The application of ultrafiltration procedure allowed to demonstrate that antimicrobial substance with strong antimycobacterial activity from bacterial culture supernatant, is a protein with the molecular mass above 100 kDa.

Behavioural and histological effects of preconditioning with lipopolysaccharide in epileptic rats.

Sublethal stress stimuli such as systemic endotoxin treatment can induce tolerance of the brain to subsequent ischemic stress, which results in a decreased infarct size. Based on this evidence, we hypothesized that lipopolysaccharide (LPS)-induced preconditioning could protect hippocampal neurons in epileptic rats. To test this hypothesis, the anticonvulsant effect of a low dose of LPS against seizures elicited by pilocarpine hydrochloride was measured. Using the pilocarpine model of temporal lobe epilepsy and LPS-preconditioning, we also investigated hippocampal pathology in the rat brain. Based on the behavioural observations conducted, it can be assumed that the preconditioning procedure used may decrease seizure excitability in epileptic rats. However, determination of the seizure excitability threshold needs to be elaborated. Qualitative and quantitative analyses of histological brain sections in the LPS-preconditioned rats showed markedly decreased intensity of neurodegenerative changes in the CA1, CA3 and DG hippocampal fields. The tendency was observed in all the periods of the pilocarpine model of epilepsy. We suggest that preconditioning with LPS may have neuroprotective effects in the CA1, CA3 and DG hippocampal sectors; however, it has no influence on the course of the seizures in rats in the pilocarpine model of epilepsy.

A comparison of cytokine production in 2-dimensional and 3-dimensional cultures of bone marrow stromal cells of multiple myeloma patients in response to RPMI8226 myeloma cells.

We examined cytokine production by bone marrow stromal cells (BMSCs) of patients with multiple myeloma (MM) in response to contact with myeloma RPMI8226 cells in standard 2-dimensional (2D) cultures and in 3-dimensional (3D) cultures on a gelatine sponge scaffold. It was detected that BMSCs in the 3D cultures produced more IL-11 and HGF and less IL-10 than in the 2D cultures. Moreover, RPMI8226 cells after contact with BMSCs in 3D cultures produced more sIL-6R than in the classic 2D cultures. We concluded that 3D cultures of BMSCs with myeloma cells offered a promising model for in vitro examination of interactions between myeloma cells and the bone marrow stroma and for examination of potent antimyeloma agents.

Different sensitivity of neurons and neuroblastoma cells to quercetin treatment.

Quercetin is a natural flavonoid with pro-apoptotic and antiproliferative properties. In this study, we determined the sensitivity of neurons and neuroblastoma cells on apoptosis and necrosis induction upon quercetin treatment. No expression of Hsp72 was observed in neurons, which were more sensitive to cell death upon quercetin treatment than neuroblastoma cells, where Hsp72 expression was observed. Reduction of Hsp72 gene expression in neuroblastoma cells by antisense oligonucleotides made them more sensitive to pro-apoptotic action of quercetin. Moreover, the flavonoid decreased Hsp27, procaspase-3, MRP and PKB expression in neuroblastoma cells and in neurons. Nuclear localization of mainly cytoplasmic Hsp27 was observed in neuroblastoma cells after treatment with high quercetin concentrations, while in neurons, the protein was present in nuclei both in control and quercetin treated cells. Our results suggest that quercetin induce apoptosis more effectively in cells with low level of Hsp 72 expression. Higher sensitivity of neurons for cell death after treatment with high quercetin concentrations in comparison to neuroblastoma cell line should also be taken into consideration in further studies on using studied flavonoid as therapeutic agent.

Aged garlic extract and allicin improve performance and gastrointestinal tract development of piglets reared in artificial sow.

This study was performed to investigate whether postnatal administration with aged garlic extract (AGE) and allicin influences performance and systemic development of piglets exposed to early weaning. Twenty-four male piglets were weaned from sows at the age of two days of life, divided into 4 weight-matched groups and kept under conditions of artificial sow for 6 days. The first group consisted of control animals, while piglets that were given AGE daily per os at the dosages of 1 ml and 2 ml/kg body weight, respectively belonged to the second and third group. The fourth group consisted of piglets administered orally with allicin at the dosage of 1.0 mg/kg body weight/day. At the age of 8 days of life all animals were sacrificed. Next to body weight gain and morphological properties of the gastrointestinal tract, the haematological examination was performed, and activity of lysozyme and ceruloplasmin as well as level of gamma-globulins were determined. The obtained results showed that AGE and allicin improved final body weight, morphological properties of intestine villi and non-specific defence mechanisms of pigs. All these results indicate that AGE and allicin induced beneficial effects on health status, performance and systemic development of piglets.

Dietary alpha-ketoglutarate reduces gastrectomy-evoked loss of calvaria and trabecular bone in female rats.

OBJECTIVE: Surgical removal of the stomach (gastrectomy, Gx) leads to osteopenia in animals and in humans. In the rat, Gx adversely affects calvaria and trabecular bone. alpha-Ketoglutarate (AKG) is a precursor of hydroxyproline--the most abundant amino acid in bone collagen. The purpose of this study was to investigate the effects of dietary AKG on Gx-induced osteopenia. MATERIAL AND METHODS: Twenty female Sprague-Dawley rats were subjected to Gx and divided between two groups: Gx+AKG in the drinking water and Gx+Vehicle (i.e. drinking water without AKG). Another 20 rats were sham-operated and divided between two groups: Sham+AKG and Sham+Vehicle. The daily dose of AKG was 0.43 g per 100 g rat. All the rats were killed 8 weeks later and the calvariae, femora and tibiae were collected. The integrity of the calvariae was analysed planimetrically, following transillumination and photography. The bone mineral content (BMC) and bone mineral density (BMD) were measured in the right femorae and tibiae (bone densitometry), leaving the left femorae and tibiae to be analysed histomorphometrically (measurement of trabecular bone volume and trabecular fractal dimension). RESULTS: Gx caused calvarial bone degradation, reduced trabecular bone (femur and tibia) and impaired trabecular architecture. In addition, Gx lowered the femoral/tibial BMC and BMD (mainly cortical bone). Dietary AKG counteracted the Gx-evoked impairment of calvaria and trabecular bone but failed to affect the BMC and the BMD in either sham- operated or Gx rats. CONCLUSIONS: Gx resulted in loss of calvarial, trabecular and cortical bone in the rat. AKG counteracted the effect of Gx on calvaria and trabecular bone but not on cortical bone.

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group.

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.

Quercetin suppresses heat shock-induced nuclear translocation of Hsp72.

The effect of quercetin and heat shock on the Hsp72 level and distribution in HeLa cells was studied by Western blotting, indirect immunofluorescence and immunogold electron microscopy. In control cells and after quercetin treatment, Hsp72 was located both in the cytoplasm and in the nucleus in comparable amounts. After hyperthermia, the level of nuclear Hsp72 raised dramatically. Expression of Hsp72 in cytoplasm was also higher but not to such extent as that observed in the nucleus. Preincubation of heated cells with quercetin inhibited strong Hsp72 expression observed after hyperthermia and changed the intracellular Hsp72 distribution. The cytoplasmic level of protein exceeded the nuclear one, especially around the nucleus, where the coat of Hsp72 was noticed. Observations indicating that quercetin was present around and in the nuclear envelope suggested an involvement of this drug in the inhibition of nuclear translocation. Our results indicate that pro-apoptotic activity of quercetin may be correlated not only with the inhibition of Hsp72 expression but also with suppression of its migration to the nucleus.