Differential effect of polyphenol-rich dark chocolate on biomarkers of glucose metabolism and cardiovascular risk factors in healthy, overweight and obese subjects: a randomized clinical trial.

The association between excess cortisol and various parameters of metabolic syndrome including hypertension, insulin resistance and dyslipidaemia is increasingly recognised. The present single-blind randomised placebo-controlled cross-over study compared the effect of polyphenol-rich dark chocolate (DC) on biomarkers of glucose metabolism, lipid profile, and blood pressure (BP) in females with BMI ≥ 25 kg m(-2) (n = 21) and females with BMI < 25 kg m(-2) (n = 21). Volunteers consumed 20 g of DC containing 500 mg polyphenols or a placebo DC with negligible polyphenol-content daily for 4 weeks, separated by a 2-week washout period. Systolic BP and diastolic BP decreased after 4 weeks of polyphenol-rich DC. Placebo raised fasting insulin, homeostasis model assessment of insulin resistance (HOMA-IR) and salivary cortisol, an effect that was significantly different from polyphenol-rich DC which had a negligible effect on fasting insulin, HOMA-IR and salivary cortisol. Females with BMI ≥ 25 kg m(-2) responded less favourably to placebo than lean females and consequently had higher fasting insulin and HOMA-IR, in addition to a lower quantitative sensitivity check index (QUICKI) after ingestion of placebo compared to polyphenol-rich DC. No significant changes in lipid profile were observed. This study provides evidence for the metabolic benefits of consuming polyphenol-rich dark chocolate while demonstrating the possibility of adverse effects occurring with polyphenol-poor chocolate placebo.

Differential effectiveness of berry polyphenols as anti-giardial agents.

Following previous work on the anti-giardial effect of blueberry polyphenols, a range of polyphenol-rich extracts from berries and other fruits was screened for their ability to kill Giardia duodenalis, an intestinal parasite of humans. Polyphenol-rich extracts were prepared from berries using solid-phase extraction and applied to trophozoites of Giardia duodenalis grown in vitro. All berry extracts caused inhibition at 166 µg gallic acid equivalents (GAE)/ml phenol content but extracts from strawberry, arctic bramble, blackberry and cloudberry were as effective as the currently used drug, metronidazole, causing complete trophozoite mortality in vitro. Cloudberry extracts were found to be the most effective causing effectively complete trophozoite mortality at 66 µg GAE/ml. The polyphenol composition of the more effective berry extracts suggested that the presence of ellagitannins could be an important factor. However, the potency of cloudberry could be related to high ellagitannin content but also to the presence of substantial amounts of unconjugated p-coumaric acid and benzoic acid. These in vitro effects occur at concentrations easily achievable in the gut after berry ingestion and we discuss the likelihood that berry extracts could be effective anti-giardial agents in vivo.

Effect of nifedipine on capacitive calcium entry in Jurkat T lymphocytes.

The effect of nifedipine-an antagonist of L-type calcium (Ca(2+)) channels-on capacitative Ca(2+) entry (CCE) was studied in Jurkat T lymphocytes. CCE was induced by a variety of treatments each of which depleted intracellular Ca(2+) stores. Cells were treated with thapsigargin, ionomycin, anti-CD3 antibodies, and phytohaemagglutinin, or pre-incubated in a Ca(2+)-free medium. Activity of CCE was evaluated with a Ca(2+)-free/Ca(2+)-readmission protocol, in Fluo-3 pre-loaded cells. Nifedipine inhibited CCE in a dose-dependent manner. CCE inhibition was not due to non-specific effects on K(+) channels. Nifedipine, did not induce any membrane depolarization, as revealed by measurements of the plasma membrane potential with the fluorescent probe bis-oxonol. Moreover, experiments done under depolarizing conditions (i.e. by substituting Na(+) with K(+) ions in the medium) revealed that nifedipine could inhibit capacitative Ca(2+) entry independently of plasma membrane depolarization. We also demonstrated the presence in our Jurkat T-cells of transcripts for Ca(V)1.3 (alpha(1D)) and Ca(V)1.4 (alpha(1F)) L-type Ca(2+) channels. Verapamil and diltiazem, two unrelated blockers of L-type Ca(2+) channels, were less inhibitory on CCE. Possible mechanisms by which nifedipine interferes with Ca(2+) entry in these cells are discussed.

The effect of blueberry extracts on Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts, in vitro.

The protozoan parasites Giardia duodenalis and Cryptosporidium parvum are common causes of diarrhoea, worldwide. Effective drug treatment is available for G. duodenalis, but with anecdotal evidence of resistance or reduced compliance. There is no effective specific chemotherapeutic intervention for Cryptosporidium. Recently, there has been renewed interest in the antimicrobial properties of berries and their phenolic compounds but little work has been done on their antiparasitic actions. The effect of various preparations of blueberry (Vaccinium myrtillus) extract on G. duodenalis trophozoites and C. parvum oocysts were investigated. Pressed blueberry extract, a polyphenolic-rich blueberry extract, and a commercially produced blueberry drink (Bouvrage) all demonstrated antigiardial activity. The polyphenol-rich blueberry extract reduced trophozoite viability in a dose dependent manner. At 167 microgml(-1), this extract performed as well as all dilutions of pressed blueberry extract and the Bouvrage beverage (9.6+/-2.8% live trophozoites remaining after 24h incubation). The lowest dilution of blueberry extract tested (12.5% v/v) contained >167 microgml(-1) of polyphenolic compounds suggesting that polyphenols are responsible for the reduced survival of G. duodenalis trophozoites. The pressed blueberry extract, Bouvrage beverage and the polyphenolic-rich blueberry extract increased the spontaneous excystation of C. parvum oocysts at 37 degrees C, compared to controls, but only at a dilution of 50% Bouvrage beverage, equivalent to 213 microgml(-1) gallic acid equivalents in the polyphenolic-rich blueberry extract. Above this level, spontaneous excystation is decreased. We conclude that water soluble extracts of blueberries can kill G. duodenalis trophozoites and modify the morphology of G. duodenalis and C. parvum.

A review of the biological and potential therapeutic actions of Harpagophytum procumbens.

Harpagophytum procumbens (Hp), commonly known as Devil's Claw is a perennial plant which thrives in arid conditions. For centuries, it has been used as a traditional treatment for a variety of illnesses, including fevers, skin complaints, arthritis and diseases of the digestive tract as well as an appetite stimulant. Since its introduction to Europe in the early twentieth century, it has become a popular antiinflammatory and analgesic preparation amongst herbalists for supportive or adjuvant treatment of degenerative joint diseases, tendonitis, headache, backache and menstrual pain. The validity of Hp as an effective antiinflammatory and analgesic preparation, particularly in the relief of arthritic symptoms, has been investigated in numerous animal, clinical and in vitro studies. Although some contradictory evidence exists, the majority of animal studies appear to indicate Hp as an effective antiinflammatory and analgesic preparation in the treatment of acute and subacute inflammation. Clinical trials support Hp as a beneficial treatment for the alleviation of pain and improvement of mobility in a variety of musculoskeletal conditions. Analysis of the in vitro and ex vivo studies that currently exist, indicate that Hp has significant effects on numerous proinflammatory markers. However, the exact mechanism(s) by which Hp may reduce inflammation remain to be elucidated.

Influence of subinhibitory concentrations of plant essential oils on the production of enterotoxins A and B and alpha-toxin by Staphylococcus aureus.

The data presented show the ability of subinhibitory concentrations of plant essential oils to influence the production of enterotoxins A and B and alpha-toxin by Staphylococcus aureus. Subinhibitory concentrations of the oils of bay, clove, cinnamon, nutmeg and thyme had no significant effect on the overall quantity of extracellular protein produced. Haemolysis due to alpha-toxin was significantly reduced after culture with all five plant essential oils. This reduction was greatest with the oils of bay, cinnamon and clove. These three oils also significantly decreased the production of enterotoxin A; the oils of clove and cinnamon also significantly decreased the production of enterotoxin B.

Inhibition of listeriolysin O and phosphatidylcholine-specific production in Listeria monocytogenes by subinhibitory concentrations of plant essential oils.

Successful infection by Listeria monocytogenes is dependent upon a range of bacterial extracellular proteins including a cytolysin termed listeriolysin O and phosphatidylcholine-specific phospholipase C. Five plant essential oils--bay, clove, cinnamon, nutmeg and thyme--significantly reduced the production of listeriolysin O by L. monocytogenes. The greatest change was observed after culture with oil of thyme, which reduced haemolysis to 52.1 haemolytic units (HU)/ml compared with 99.8 HU/ml observed with the control. Oil of clove was the only oil that also significantly reduced phosphatidylcholine-specific phospholipase C activity. These changes were observed despite the oils causing no change to the final bacterial concentration or total extracellular protein concentration.

Phenotypic changes in the lipopolysaccharide of Pseudomonas aeruginosa and Escherichia coli grown in milk-based enteral nutrition solutions.

Previous studies have shown enteral nutritional solutions (ENS) contaminated with large numbers of microorganisms from the environment or gastrointestinal (GI) tract of patients have caused respiratory infections, acute and chronic enteritis, and septicemia. The introduction of "closed" enteral feeding systems has been used to prevent contaminating organisms from entering enteral feeding systems in large numbers. However, there is some discussion as to whether this has been an effective measure in reducing ENS-related infections because there is anecdotal evidence to suggest that disease processes resulting from enteral feeding are still commonplace in the hospital and home. This is because there is very little information about the growth of microorganisms in ENS and whether growth in ENS may affect the virulence and pathogenicity of microorganisms. This study shows that Escherichia coli and Pseudomonas aeruginosa may grow at 25 degrees C from either high or low initial numbers to up to 9.2 log colony-forming units per mL in a range of milk-based ENS. However, these organisms did not grow in the fruit-based ENS. The effect on the lipopolysaccharide (LPS) of culturing E. coli and P. aeruginosa in milk-based ENS as opposed to standard laboratory media was examined using polyacrylamide gel electrophoresis. We found that there were significant qualitative changes in the phenotype of O-polysaccharide side chains of the LPS from these organisms. O-polysaccharide is known to mediate in the complement, antibiotic and bile resistance, and affect adherence. Therefore, changes in the virulence and pathogenicity of these microorganisms when cultured in ENS may be indicated. Thus, the study provides further evidence for reevaluating the microbiologic and immunologic effects of enteral feeding, especially on the microbial flora of the GI tract.

Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens.

The antimicrobial properties of 21 plant essential oils and two essences were investigated against five important food-borne pathogens, Campylobacter jejuni, Salmonella enteritidis, Escherichia coli, Staphylococcus aureus and Listeria monocytogenes. The oils of bay, cinnamon, clove and thyme were the most inhibitory, each having a bacteriostatic concentration of 0.075% or less against all five pathogens. In general, Gram-positive bacteria were more sensitive to inhibition by plant essential oils than the Gram-negative bacteria. Campylobacter jejuni was the most resistant of the bacteria investigated to plant essential oils, with only the oils of bay and thyme having a bacteriocidal concentration of less than 1%. At 35 degrees C, L. monocytogenes was extremely sensitive to the oil of nutmeg. A concentration of less than 0.01% was bacteriostatic and 0.05% was bacteriocidal, but when the temperature was reduced to 4 degrees, the bacteriostatic concentration was increased to 0.5% and the bacteriocidal concentration to greater than 1%.

Inhibition of Listeria monocytogenes and Salmonella enteriditis by combinations of plant oils and derivatives of benzoic acid: the development of synergistic antimicrobial combinations.

This study describes inhibitory properties of combinations of oil of fennel, oil of anise or oil of basil with either benzoic acid or methyl-paraben against Listeria monocytogenes and Salmonella enteriditis. Micro-organisms were cultured at 37 degrees C in broth and viable counts measured over a 48-h period. S. enteriditis was particularly sensitive to inhibition by a combination of oil of anise, fennel or basil with methyl-paraben where there was < 10 CFU/ml after 1 h. L. monocytogenes was less sensitive to inhibition by each combination however there was a significant reduction in growth of 4-8 log by combinations of all oils and methyl-paraben at 8, 24 and 48 h. Synergistic inhibition by one or more combinations was evident against each micro-organism.

Murine HIV-1 p24 specific T lymphocyte activation by different antigen presenting cells: B lymphocytes from immunized mice present core protein to T cells.

Mice were injected with three doses of baculovirus-produced recombinant HIV-1 p24 core protein in alum adjuvant. CD4 positive T lymphocytes from immunized animals proliferated in vitro in the presence of antigen and peritoneal macrophages (Mps) or splenic dendritic cells (DCs) from non-immunized mice as antigen presenting cells (APCs). DCs were approximately three times more efficient than Mps on a cell for cell basis. No synergy was observed between Mps and DCs in this system. B lymphocytes from immunized animals also presented p24 antigen to the specific T cells. Mps did synergize with B cells to enhance the level of T lymphocyte proliferation. This may have implications for the induction of specific immune responses to pathogens after administration of single protein vaccines.

Murine immune response to HIV-1 p24 core protein following subcutaneous, intraperitoneal and intravenous immunization.

The murine immune response to baculovirus-produced human immunodeficiency virus type-1 (HIV-1)p24 was examined after injection by three different routes: subcutaneously (s.c.), intraperitoneally (i.p.) and intravenously (i.v.). Both antigen-specific T-cell proliferation and serum antibody were induced by i.p. injection. In contrast, s.c. and i.v. injection of antigen resulted in specific antibody generation alone. Lympho-proliferative responses seen after i.p. injection were confined to splenocytes, and were greater after a low dose of antigen than after a high dose. p24-specific proliferation was not detected in lymph node cells. CD4:CD8 ratios were normal in lymph nodes and spleen at all times, irrespective of the dose or route of administration. p24-specific serum IgG antibodies were detected in all animals after the second injection of antigen. After s.c. and i.v. administration of high doses of antigen, the median antibody titres continued to rise after a third injection, but plateaued in animals injected by the i.p. route. In contrast, low doses of antigen given i.p. increased the median titre during and after the course of four injections. A low antigen dose given s.c. resulted in a plateau of median titre between the third and fourth injections. In i.v.-injected animals the median titre decreased between the third and fourth injections. IgG1 p24-specific antibodies were detected in all immunized mice, whereas IgM antibodies were detectable only following i.p. injection of antigen.

Identification of major common extracellular proteins secreted by Aeromonas salmonicida strains isolated from diseased fish.

Ten different strains of Aeromonas salmonicida that were isolated from diseased fish were grown under identical conditions (24 h at 25 degree C) in 3% (wt/vol) tryptone soya broth medium supplemented with vitamins and inorganic ions. In each case the extracellular proteins that were formed were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was found that there were two significant common components, one with a molecular weight of 70,000 and the other with a weight of 56,000. Application of enzyme purification techniques to the supernatant fraction proteins of a culture of one of the strains resulted in the isolation of a 70-kilodalton (kDa) component, which was found to be a serine protease, and a 56-kDa component, which was hemolytic to trout erythrocytes. Rocket immunoelectrophoresis with rabbit antibodies to the isolated protease and hemolysin showed the same antigenic components in the supernatant fractions of all the cultures. These activities were assayed, and protease activity was found to vary by a factor of three, from 59 to 195 U/ml, while the range of hemolytic activity was over a narrow band, from 28 to 43 U/ml. There was an inconsistency between the immunoelectrophoretic and direct assay data in only one case. This indicated the presence of additional hemolytic activity, in addition to the 56-kDa component. The detection of large amounts of the same protease and hemolysin, two potent degradative activities, in a random series of strains of A. salmonicida suggests that they may be obligatory virulence factors in the development of furunculosis.

A comparative study of the formation of extracellular proteins by Aeromonas salmonicida at two different temperatures.

Aeromonas salmonicida was grown in a supplemented 3% (w/v) tryptone soya broth medium at 10 degrees C, a temperature at the lower end of the range over which furunculosis has been observed to occur in the field, and 25 degrees C, the optimum temperature for growth. Similar bacterial densities in the range 2.35 +/- 0.05 mg dry wt/ml were achieved in the two cultures at the beginning of the stationary phase of the growth cycle, after 125 h at 10 degrees C and 18 h at 25 degrees C. At this point, at the higher temperature 1.5 times more exoprotein was formed, 80 +/- 2.8 micrograms/ml compared with 54 +/- 1.7 micrograms/ml. Exoprotein contained the same proportion of haemolysin at both temperatures and twice as much protease at the higher temperature. The most marked difference was in an unidentified 100 kD protein which was formed in a 10-fold greater amount at 10 degrees C.

A comparison of the distribution of extracellular proteins produced by the protease-secreting organism Aeromonas salmonicida during aerobic and anaerobic growth.

Aeromonas salmonicida was grown aerobically and anaerobically in supplemented 3% (w/v) tryptone soya broth medium for 24 h at 25 degrees C. Although the bacterial density achieved was 4.9 times higher in the aerobic culture, the exoprotein produced per unit of bacterial dry weight was only 1.9 times higher than in the anaerobic culture. However, the protease activity of the exoprotein showed a marked reduction anaerobically, being only one-tenth of that of the exoprotein produced aerobically. This finding was consistent with the differing SDS-PAGE patterns of the extracellular proteins from the two cultures, which also showed marked loss and reinforcement of other, as yet unidentified extracellular products.