Isolation and identification of bacteriocinogenic strain of Lactobacillus plantarum with potential beneficial properties from donkey milk.

AIMS: The goal of this study was to isolate and characterize a lactic acid bacteria (LAB) from donkey milk with potential beneficial properties. METHODS AND RESULTS: Lactic acid bacteria were isolated from donkey milk and identified based on physiological, biochemical and molecular methods. The isolate that presented highest bacteriocin potential (Lactobacillus plantarum LP08AD) was evaluated for the production of bacteriocin, including stability in the presence of various enzymes, surfactants, salts, pH and temperatures. Bactericidal effect of bacteriocin LP08AD on Listeria monocytogenes, Enterococcus faecium and Lactobacillus curvatus was shown for actively growing and stationary cells. Similar growth and bacteriocin production were observed when strain LP08AD was cultured in MRS broth at 30°C or 37°C. Bacteriocin LP08AD adhered at low levels on the producer cells (200 AU ml(-1) ). The presence of plantaricin W gene on the genomic DNA was recorded based on PCR. Good growth for strain LP08AD was recorded in MRS broth with pH from 5·0 to 9·0 and LP08AD grew well in the absence of oxbile or concentration below 0·8%. Lact. plantarum LP08AD was applied to the small intestinal epithelial polarized monolayers of H4, PSIc1 and CLAB and demonstrated low attachment ability on all cell lines studied, with values with a similar behaviour for cells from human and pig origin. CONCLUSIONS: Bacteriocin-producing Lact. plantarum LP08AD might be useful in the design of novel functional foods with potential probiotic or biopreservation properties. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report on detection and characterization of bacteriocinogenic Lact. plantarum from donkey milk. The strain LP08AD shows to have potential beneficial properties, as demonstrated by the use of noncancerogenic cell lines.

L-arginine intake effect on adenine nucleotide metabolism in rat parenchymal and reproductive tissues.

L-arginine is conditionally essetcial amino acid, required for normal cell growth, protein synthesis, ammonia detoxification, tissue growth and general performance, proposed in the treatment of men sterility and prevention of male impotence. The aim of the present paper was to estimate the activity of the enzymes of adenine nucleotide metabolism: 5'-nucleotidase (5'-NU), adenosine deaminase (ADA), AMP deaminase, and xanthine oxidase (XO), during dietary intake of L-arginine for a period of four weeks of male Wistar rats. Adenosine concentration in tissues is maintained by the relative activities of the adenosine-producing enzyme, 5'-NU and the adenosine-degrading enzyme-ADA adenosine deaminase. Dietary L-arginine intake directed adenine nucleotide metabolism in liver, kidney, and testis tissue toward the activation of adenosine production, by increased 5'-NU activity and decreased ADA activity. Stimulation of adenosine accumulation could be of importance in mediating arginine antiatherosclerotic, vasoactive, immunomodulatory, and antioxidant effects. Assuming that the XO activity reflects the rate of purine catabolism in the cell, while the activity of AMP deaminase is of importance in ATP regeneration, reduced activity of XO, together with the increased AMP-deaminase activity, may suggest that adenine nucleotides are presumably directed to the ATP regenerating process during dietary L-arginine intake.

The effect of depurinized milk draught diet on rat serum uric acid, lipid status and haematological parameters.

Hyperuricaemia and gout are closely related, but hyperuricaemia is an independent risk factor for endothelial damage, autoinflammation and haemodynamic abnormalities. Milk, generally known as a 'purine-free diet', is an essential protein source for patients suffering from hyperuricaemia and gout. As milk still contains different purine ribonucleotides, the new product, depurinized milk, almost free of purine nucleotides and uric acid, was produced. The potential effect of depurinized milk diet on serum uric acid (SUA) level, lipid parameters and blood haematological parameters was explored in rats after 72 h and 15 days, in relation to standard laboratory chow or the untreated milk diet. The beneficial effect on SUA was achieved when depurinized milk draught was given instead of standard chow for 72 h [28.39 ± 4.76 µm; p < 0.001 vs. standard diet (STD) 47.6 ± 6.12, vs. untreated milk diet 31.55 ± 8.50; p < 0.05] or as a supplement for STD for 15 days experiment (35.38 ± 6.40 µm; p < 0.05 vs. STD only 48.05 ± 4.32; vs. untreated milk + STD 46.02 ± 9.48). Depurinized milk diet significantly decreased the low density lipoproteins/high density lipoproteins (LDL/HDL) ratio (p < 0.001), triglycerides (p < 0.05) and leucocyte count (p < 0.001), while both milk draughts enhanced haemoglobin concentration (p < 0.01). In conclusion, considering the detrimental effect of persisting hyperuricaemia, the depurinized milk draught may meet the demand of healthy dairy product for population under hyperuricaemic risk.

Hyperglycemia, oxidative and nitrosative stress affect antiviral, inflammatory and apoptotic signaling of cultured thymocytes.

A high prevalence of various infectious diseases is reported in diabetic patients, which may suggest impaired innate immunity against different pathogen-associated molecular patterns. This study investigated the effects of hyperglycemia, oxidative stress (H(2)O(2)), nitric oxide (NO) and peroxynitrite (ONOO(-)) on the modulation of antiviral (MDA-5, IRF-3 and phospho-IRF-3), inflammatory (NF-kappaB) and pro/anti-apoptotic molecules (Bax and Bcl-2) in BALB/c mice thymocytes. Each of the experimental conditions, except the weakest NO concentration, resulted in down-regulation of MDA-5, IRF-3 and phospho-IRF-3. In contrast, each of the experimental conditions elicited up-regulation of NF-kappaB, Bcl-2 and Bax. These results suggest that hyperglycemia, oxidative and nitrosative stress may contribute to the reduced immunity of the host by altering the MDA-5/IRF-3/phosphoIRF-3 axis, as well as contributing to the mechanisms of inflammatory reaction via increased NF-kappaB, and to augmented turnover rate of thymocyte cells via Bcl2/Bax up-regulation.

Antimicrobial disinfection effect of a laundering procedure for hospital textiles against various indicator bacteria and fungi using different substrates for simulating human excrements.

Recent studies confirm the increase of nosocomial infections and microbial resistance. One of the possible causes is infected textiles due to inappropriate laundering procedures. Most Slovenian laundries use thermal laundering procedures with high energy and water consumption to disinfect hospital textiles. In addition to this fact, there is an increasing number of hospital textiles composed of cotton/polyester blends that cannot endure high temperatures of thermal disinfection. On the other hand, decreasing the temperature of laundering procedures enhances the possibility of pathogenic microorganisms to survive the laundering procedure. In our research, we determined the antimicrobic laundering effect by simulating a common laundering procedure for hospital textiles in the laboratory washing machine at different temperatures by the use of bioindicators. Enterococcus faecium, Staphylococcus aureus, Mycobacterium terrae, Enterobacter aerogenes, and Pseudomonas aeruginosa were used for determining the antibacterial laundering effect. Candida albicans was used for determining the antifungal laundering effect. Swine blood, artificial sweat, and swine fat were used as substrates for simulating human excrements and were inoculated together with the chosen microorganisms onto cotton pieces to simulate real laundering conditions. It was found that E. faecium, S. aureus, E. aerogenes, and P. aeruginosa survived at 60 degrees C, but no microorganisms were found at 75 degrees C.

Implementing hygiene monitoring systems in hospital laundries in order to reduce microbial contamination of hospital textiles.

As textiles sent to hospital laundries contain many types of pathogenic organisms, it is important that laundering not only has an appropriate cleaning effect but also has a satisfactory disinfecting effect. Critical to this process is the maintenance of an appropriate hygiene level in the clean area of laundries in order to prevent recontamination of textiles from manual handling when ironing, folding, packing etc. The aims of this study were to evaluate the hygienic state of a hospital laundry, to introduce continuous sanitary measures, and to introduce a continuous hygiene monitoring system with an infection control programme. Two systems for evaluating hospital laundry hygiene were combined: HACCP principles (hazard analysis and critical control points) and RAL-GZ 992 standards (quality assurance standard for textile care of hospital laundry). Evaluation of the hygienic state of the hospital laundry was carried out by evaluating the number and types of micro-organisms present at the critical control points throughout the whole laundering process, using RODAC agar plates for surface sampling and the pour plate method for investigating water samples. The initial examination showed that the sanitary condition of the laundry did not reach the required hygiene level. Therefore, fundamental sanitation measures were instituted and the examination was repeated. Results were then satisfactory. The most important critical control point was the chemothermal laundering efficiency of the laundering process. To prevent micro-organisms spreading into the entire clean working area, it is important that, in addition to regular sanitary measures such as cleaning/disinfecting all working areas, technical equipment and storage shelves etc., regular education sessions for laundry employees on proper hand hygiene is undertaken and effective separation of the clean and dirty working areas is achieved.

Trophoblastic interferons: do they modulate uterine cellular markers at the time of conceptus attachment in the pig?

Between days 12 and 20 of pregnancy, the trophectoderm of the porcine conceptus secretes two species of interferons (IFN): IFN-gamma (Type II), which is produced in substantial amounts, and IFN-delta (type I), for which secretion peaks at days 15-16 of gestation. The role of these embryonic IFNs is not known. We made the assumption that, in the pig, one possible role of these IFNs may be the remodelling and/or depolarization of the uterine endometrial epithelium, as a prerequisite for implantation and establishment of a functional placenta. A comparative analysis by immunohistochemistry of several cell membrane markers and ECM components of the cyclic and pregnant uterus was performed at day 15 post-oestrus. The markers were those likely to differ between a pregnant and cyclic uterus, or between different stages of pregnancy. A highly specific marker of IFN-gamma activity, namely MHC class II antigens in the uterine mucosa, was also examined. This study provides so far unreported data: in the endometrial epithelium of the pregnant uterus, we observed a partial relocalization of ZO-1, a marker of epithelial tight junctions, thus suggesting significant changes to the endometrial polarity. Heparan-Sulphate Proteoglycan (HSPG) expression did not differ significantly between cyclic and pregnant uteri. In contrast with the accepted rodent model of trophoblast-uterus adhesion, the porcine trophoblast and luminal epithelium were negative for HSPG. Finally, MHC class II antigens were absent from the cyclic uterus, but markedly induced in the day 15 pregnant uterus, particularly in endothelial cells, suggesting that IFN-gamma may indeed cross the maternal epithelium. This hypothesis was supported by the observation of IFN-gamma immunoreactivity associated with clusters of endometrial cells in the pregnant uterus.

Up-regulation of polymeric immunoglobulin receptor mRNA in mammary epithelial cells by IFN-gamma.

As shown in previous in vivo experiment, the amount of polymeric immunoglobulin receptor (pIgR), which mediates the transcytosis of pIgA across epithelial cells, is regulated by lactogenic hormones (PRL and cortisol) during the development of the mammary gland. In the present in vitro study, it appeared that these hormones were insufficient to induce the strong expression of the gene that we observed in vivo. Several papers have shown that IFN-gamma is a strong stimulator of pIgR gene expression in different models. In contrast, nothing is known of the effects of IFN-gamma on pIgR gene expression in the mammary gland. We report here that IFN-gamma strongly increased pIgR mRNA levels through a direct effect on mammary epithelial cells. We show that IFN-gamma activated not only Stat1 but also Stat5 and that expression of the pIgR and IRF-1 genes was strongly correlated following IFN-gamma stimulation in mammary epithelial cells. In conclusion, these experiments enabled the analysis of different types of regulation of pIgR gene expression in the mammary gland and suggest possible co-operation between circulating hormones and locally produced cytokines, leading to pIgR gene expression in the mammary gland.

Tetracycline-controlled expression of glycosylated porcine interferon-gamma in mammalian cells.

Tetracycline-controlled expression plasmids that allow inducible expression of proteins in mammalian cells (Gossen & Bujard, 1992), have been used to express porcine interferon-gamma in the RK-13 rabbit kidney cell line. Following neomycin selection, stable clones produced recombinant, glycosylated porcine interferon-gamma (rGPoIFN-gamma) only after removal of tetracycline (Tc). Southern blot analysis of one clone showed that approximately 50 copies of IFN-gamma cDNA were present in the cell genome. In the absence of Tc, stable clones secreted large amounts of rGPoIFN-gamma (up to 16 microg/ml) into the medium supplemented with 10% FCS and high glucose concentration. Molecular weight comparison of 35S-Methionine, labelled rGPoIFN-gamma with natural leukocytic IFN-gamma after immunoprecipitation, revealed 4 major glycoforms with apparent Mr of 27,000; 25,000; 20,000 and 18,500, that are almost identical in both IFN-gamma species. In both cases, all 4 glycoforms resolved into 2 polypeptide monomers with apparent Mr of 16,500 and 14,500 upon deglycosylation with N-glycosydase F. The biological activity of rGPoIFN-gamma was in the same range as that of natural leukocytic PoIFN-gamma (2 x 10(6) U/mg). Eventually, this recombinant mammalian IFN-gamma should constitute a very useful substitute for leukocyte PoIFN-gamma in in vitro or in vivo experiments.

Porcine blood cell separation by porous cellulose acetate membranes.

Leukocytes were separated from whole porcine blood using laboratory prepared polymeric asymmetric porous membranes from cellulose acetate (CA) and by applying standard blood cell separation methods: centrifugation in a Ficoll solution gradient and in sucrose solution concentration gradient. Leukocytes, obtained by different separation methods were characterised by their quantity, type, viability and growth ability. Membranes prepared by a wet phase inversion process from different cellulose acetate/acetone/water and magnesium chlorate VII systems, were characterised according to: permeability to deionised water, surface morphology and by the determination of the flux of the permeate during the whole porcine blood separation. Cellulose acetate membranes prepared from 300 µm thick cast solution (14.8 wt% of cellulose acetate, 19.9 wt% of water, 2.3 wt% of Magnesium perchlorate, and 63.0 wt% of acetone), have separation characteristics comparable with the standard separation methods; in the dead-end mode filtration, 21.3% of leukocytes from porcine whole blood are separated. The leukocyte number in peripheral blood before separation was 450,000 ml(-1); the number passed through after was 95,000±6620. The main interest of the study was to introduce the CA membrane filters for the continus technological separation of the leukocyte/lymphocytes from animal (= porcine, bovine, horse..) blood.

Porcine interferon-gamma inhibits the growth of Legionella pneumophila in WiREF cells in vitro.

The intracellular growth of Legionella pneumophila in WiREF (Wistar rat embryonal fibroblast) cells was inhibited by porcine interferon-gamma. The effect was compared with that of different human interferons (alpha and gamma). The growth inhibition was dose-dependent and required the pretreatment of WiREF cells with interferon. The development of an antibacterial state of the cells was observed. When interferon was added together with bacteria or 1 d after the infection there was no inhibition. Also, there was no direct antibacterial effect of the interferon. In addition, cell pretreatment with a combination of interferon and antibiotics failed to show a synergistic effect.