High-fat diet (HFD) consumption leads to metabolic disorders, gastrointestinal dysfunction and intestinal dysbiosis. Antibiotics also disrupt the composition of intestinal microbiota. The aim of the present study was to investigate the impact of a short-term feeding with HFD on oxidative status, enteric microbiota, intestinal motility and the effects of antibiotics and/or melatonin treatments on diet-induced hepato-intestinal dysfunction and inflammation. Male Sprague–Dawley rats were pair-fed with either standard chow or HFD (45 % fat) and were given tap water or melatonin (4 mg/kg per d) or melatonin plus antibiotics (ABX; neomycin, ampicillin, metronidazole; each 1 g/l) in drinking water for 2 weeks. On the 14th day, colonic motility was measured and the next day intestinal transit was assessed using charcoal propagation. Trunk blood, liver and intestine samples were removed for biochemical and histopathological evaluations, and faeces were collected for microbiota analysis. A 2-week HFD feeding increased blood glucose level and perirenal fat weight, induced low-level hepatic and intestinal inflammation, delayed intestinal transit, led to deterioration of epithelial tight junctions and overgrowth of colonic bacteria. Melatonin intake in HFD-fed rats reduced ileal inflammation, colonic motility and perirenal fat accumulation. ABX abolished increases in fat accumulation and blood glucose, reduced ileal oxidative damage, suppressed HFD-induced overgrowth in colonic bacteria, and reversed HFD-induced delay in intestinal transit; however, hepatic neutrophil accumulation, hepatic injury and dysfunction were further enhanced. In conclusion, the results demonstrate that even a short-term HFD ingestion results in hepato-intestinal inflammatory state and alterations in bacterial populations, which may be worsened with antibiotic intake, but alleviated by melatonin.

Trypanosoma musculi are readily killed when phagocytosed by mononuclear phagocytes but the nature of the mediators of this cytotoxicity is unclear. Among the most potent mediators are oxygen-derived species. The generation of chemilumine-scence (CL) by peritoneal macrophages from 12 day T. musculi-infected mice, which phagocytose and kill parasites when opsonizing antibodies are present, was recorded in the presence of antibody-coated trypanosomes. Taurine, a specific quencher of hypochlorous acid (HOCl) inhibited CL production by peritoneal macrophages, showing that HOCl is produced during phagocytosis of T. musculi. In vitro, HOCl alone exerted a powerful trypanocidal activity which was inhibited in the presence of specific quenchers. The role of HOCl generated by phagocytes in trypanosome killing was studied using granulocytes which produce more oxygen-derived species than macrophages when stimulated. Phorbol myristate acetate-triggered granulocytes can destroy T. musculi and trypanosome killing is inhibited in the presence of taurine. These data demonstrate that HOC1 produced by phagocytes can effectively destroy T. musculi.

The cDNA probe, Lmet2, was labelled with digoxigenin and used in a chemiluminescent system to detect fewer than 100 membrane-immobilized Leishmania parasites. The probe was found to hybridize primarily with members of the L. donovani complex but a slight cross-reaction was also observed with greater than 5 x 104L. major. This cross-reaction was reduced by hybridizations in 50% formamide at 37 °C. Formamide also significantly reduced non-specific binding of the digoxigenin-labelled probe to the membrane support which, in hybridizations without formamide, masked the specific hybridization signal. This background was not observed with the corresponding radio-isotope labelled probe. With hybridizations in formamide the sensitivity achieved by the chemiluminescent system after exposure to film for 3 h was greater than that achieved by the isotopic system even after autoradiography for 24 h.

Using an ultrafiltration membrane (molecular cut-off, 3000), low molecular weight compounds in bovine milk were collected (YM-3 filtrate). A hydrogen peroxide (H2O2)-like substance was generated in the YM-3 filtrate. This substance was undetected at 0 h, but increased in a time-dependent manner, peaking after 2 h of incubation at 38°C. After incubating the YM-3 filtrate with catalase and lactoperoxidase, the signal showing the presence of this substance disappeared. The substance was quantified using one chemiluminescence and three colorimetric H2O2 detection systems. In all systems, their estimates were within the same range. The amount of substance, as estimated by the chemiluminescence H2O2 detection system, was correlated with that estimated by the other three colorimetric systems (r=0·98, 0·95 and 0·87). The substance was eluted at the same position as H2O2 by gel filtration on Superdex 30. Thus, the substance had the same characteristics as H2O2. An H2O2-generating substance in either the YM-3 filtrate or whey had a molecular mass of about 600. In this study, we clarify that bovine milk is capable of generating H2O2 by utilizing a low molecular weight compound. Thus, we present a new type of H2O2-supplying system in bovine milk.

Udder defence mechanisms are not completely explained by current mastitis research. The anatomical construction of the udder implies that infection of one udder quarter does not influence the immune status of neighbouring quarters. To test this hypothesis, we compared the immune reactions of individual udder quarters in response to microbial attacks. In the course of immune reactions, polymorphonuclear leucocytes (PMN) release oxygen radicals, which can be determined by chemiluminescence (CL). Milk from 140 udder quarters of 36 cows was analysed for somatic cell count (SCC), differential cell count, viability and CL activity. Quarters with an SCC <100000 cells/ml and free of pathogens were defined as uninfected, all other quarters were categorized as infected. Three groups of cows were classified cytologically: group A (healthy, 11 animals, SCC limit <100000 cells/ml); group B (moderate mastitis, 8 cows, SCC [ges ]100000 and <400000 cells/ml in at least one quarter); and group C (severe mastitis, 17 cows, SCC [ges ]400000 cells/ml in at least one quarter). Infected and uninfected quarters in groups B and C were analysed separately. Viability of PMN leucocytes was significantly (P=0·0012) lower in group A (72·6%) than in healthy quarters of group C (84·0%). Lowering the SCC limit of healthy quarters to <50000 cells/ml (group A: all quarters within the udder) revealed striking differences between samples of groups B and C: in addition to varying differential cell counts and viabilities, CL activity of group B<50 (2929 CL units/million PMN) was markedly lower than that of the other groups (5616 in group A<50 and 6445 CL units/million PMN in group C<50). These results allow the conclusion that the infection of one udder quarter influences the cell activity of neighbouring quarters. When the SCC threshold for healthy quarters was reduced to 50000 cells/ml, greater differences in cell activities were detected between healthy udders and healthy quarters of infected udders.

The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores of Myxobolus cerebralis. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested with Xho I restriction enzyme, cDNA fragments less than 400 bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packaged in vitro using Gigapack III gold packaging extract. The primary cDNA library titre contained 0·5×106 clones, with 97% recombinant and only 3% non-recombinant clones. The cDNA library was then screened using the anti-triactinomyxon antibodies. Positive clones were selected and re-screened twice more to give a final selection of 526 clones. One clone (46-5) was selected randomly and subjected to in vivo excision of the pBK-CMV phagemid from the ZAP express vector. The sequence of the entire clone was obtained using rapid amplification of the cDNA ends. A search of the clone sequence against GenBank revealed that it related to ribosomal protein L23 and it had a high percentage similarity to this protein from different species. A conserved domain for ribosomal protein L23 was also identified in the clone sequence.

The effect of regular intake of low doses of an effervescent multivitamin preparation on the free-radical-producing activity of murine peritoneal macrophages under conditions resembling a possible infection was studied in vitro. Initially, several groups of mice were fed a basal diet and given, for 2 weeks, water without or with supplementation of either α -tocopherol, ascorbic acid, riboflavin or a multivitamin preparation. The supplementation period was followed by a 2-week wash-out time interval during which control and multivitamin groups received deionized water. Macrophage stimulation tests using chemiluminescent spectroscopy were performed at the end of the supplementation and wash-out periods to determine cell counts and their capacity to produce free radicals. Multivitamin supplementation increased the number, and the reactive oxygen species-producing activity, of macrophages. This effect persisted for 2 weeks after higher doses of supplementation were stopped. Multivitamin supplementation lowered the steady-state free radical concentrations of liver and spleen as measured by electron paramagnetic resonance spectroscopy. It also increased the antioxidant reactivity of the same organs, while there was no effect on the free radical concentration and antioxidant capacity of the kidney and brain. When taken regularly, low doses of multivitamin supplementation may have a beneficial effect on the defence mechanisms of the organism.

Background and objective To investigate the ability of lidocaine to inhibit reactive oxygen and/or nitrogen species generation by either human leukocytes or cell-free systems via luminol- and lucigenin-enhanced chemiluminescence.

Methods Venous blood was obtained from healthy volunteers and leukocytes were isolated, from which chemiluminescence was generated. Also, chemiluminescence, induced by H2O2, HOCl, peroxynitrite or ferrous iron, was generated in cell-free systems.

Results Lidocaine produced a concentration-dependent inhibition in chemiluminescence generated by leukocytes (92 ± 1%, 1 mM). In cell-free experiments, lidocaine (1 mM) markedly inhibited chemiluminescence of xanthine-xanthine oxidase (24 ± 3%), while it slightly suppressed hypochlorous acid-induced chemiluminescence (9 ± 2%). Peroxynitrite-induced luminol- and lucigenin-enhanced chemiluminescence were also inhibited by lidocaine at 1 mM (19 ± 3% and 48 ± 8%, respectively). Lidocaine did not affect chemiluminescence generated by FeSO4. However, lidocaine produced a biphasic effect on H2O2-induced chemiluminescence (37 ± 5% inhibition at 0.01 mM and 61 ± 17% activation at 1 mM).

Conclusions Lidocaine can elicit direct scavenging activity at high concentrations that might be important at or near the site of injection in local anaesthetic use.

The non-stimulated and phorbol 12-myristate 13-acetate (PMA)-stimulated luminol-augmented cellular chemiluminescence (CL) response and viability of milk and blood polymorphonuclear leukocytes (PMN) were determined in lactating dairy cows during different stages of lactation. In the first study, ten healthy cows each in early, mid and late lactation were compared. In a second study, the same measurements as in the first study were evaluated longitudinally in 12 cows during 1 month following parturition. The CL activity and myeloperoxidase (MPO) content of milk PMN and macrophages (M) were also compared. Milk M did not possess MPO activity and were devoid of any luminol-enhanced CL. The CL activity of milk and blood PMN was significantly lower in early lactation than in mid and late lactation (P < 0·001). Whereas little changes were observed in viability of blood PMN, the viability of milk PMN was lower in early lactation than in mid and late lactation (P < 0·001). The percentage of PMN in isolated milk cells was also lower during early lactation than during mid and late lactation (P < 0·001). The CL activity in response to PMA during early, mid and late lactation increased 13, 59 and 42-fold in blood PMN and 1·7, 2·6 and 2·4-fold in milk PMN, respectively, in comparison with non-stimulated PMN. The CL activity, both in milk and blood PMN, the milk PMN viability and the percentage of milk PMN were lowest between 3 d and 11 d post partum. These observed changes immediately after calving could contribute to a higher susceptibility to mastitis in that period.