Immune Response of Healthy Adults to the Ingested Probiotic Lactobacillus casei Shirota.

Daily ingestion of a probiotic drink containing Lactobacillus casei Shirota (LcS; 1.3 × 1010 live cells) by healthy adults for (1) 4-week LcS, (2) 6-week discontinuation of LcS and (3) a final 4 weeks of LcS was investigated. There was a significant increase in expression of the T cell activation marker CD3+ CD69+ in ex vivo unstimulated blood cells at weeks 10 and 14, and there was a significant increase in the NK cell marker CD3+ CD16/56+ in ex vivo unstimulated blood cells at weeks 4, 10 and 14. Expression of the NK cell activation marker CD16/56+ CD69+ in ex vivo unstimulated blood cells was 62% higher at week 10 and 74% higher at week 14. Intracellular staining of IL-4 in ex vivo unstimulated and PMA-/ionomycin-stimulated CD3+ ß7+ integrin blood cells was significantly lower at weeks 10 and 14. Intracellular staining of IL-12 in ex vivo unstimulated and LPS-stimulated CD14+ blood cells was significantly lower at weeks 4, 10 and 14. Intracellular staining of TNF-α in LPS-stimulated CD14+ blood cells was significantly lower at weeks 4, 10 and 14. Mucosal salivary IFN-γ, IgA1 and IgA2 concentrations were significantly higher at week 14, but LcS did not affect systemic circulating influenza A-specific IgA or IgG and tetanus-specific IgG antibody levels. In addition to the decrease in CD3+ ß7+ integrin cell IL-4 and a reduced CD14+ cell pro-inflammatory cytokine profile, at week 14 increased expression of activation markers on circulating T cells and NK cells and higher mucosal salivary IgA1 and IgA2 concentration indicated a secondary boosting effect of LcS.

Selective effects of Lactobacillus casei Shirota on T cell activation, natural killer cell activity and cytokine production.

Modulation of host immunity is an important potential mechanism by which probiotics confer health benefits. This study was designed to investigate the effects of a probiotic strain, Lactobacillus casei Shirota (LcS), on immune function using human peripheral blood mononuclear cells (PBMC) in vitro. In addition, the role of monocytes in LcS-induced immunity was also explored. LcS promoted natural killer (NK) cell activity and preferentially induced expression of CD69 and CD25 on CD8(+) and CD56(+) subsets in the absence of any other stimulus. LcS also induced production of interleukin (IL)-1beta, IL-6, tumour necrosis factor (TNF)-alpha, IL-12 and IL-10 in the absence of lipopolysaccharide (LPS). In the presence of LPS, LcS enhanced IL-1beta production but inhibited LPS-induced IL-10 and IL-6 production, and had no further effect on TNF-alpha and IL-12 production. Monocyte depletion reduced significantly the impact of LcS on lymphocyte activation, cytokine production and natural killer (NK) cell activity. In conclusion, LcS activated cytotoxic lymphocytes preferentially in both the innate and specific immune systems, which suggests that LcS could potentiate the destruction of infected cells in the body. LcS also induced both proinflammatory and anti-inflammatory cytokine production in the absence of LPS, but in some cases inhibited LPS-induced cytokine production. Monocytes play an important role in LcS-induced immunological responses.

Inhibition spectrum studies of microthecin and other anhydrofructose derivatives using selected strains of Gram-positive and -negative bacteria, yeasts and moulds, and investigation of the cytotoxicity of microthecin to malignant blood cell lines.

AIMS: To prepare 1,5-anhydro-d-fructose (AF) derivatives, test their microbial inhibition spectrum, and to further examine the most effective AF derivative against Pseudomonas aeruginosa and malignant blood cell lines. METHODS AND RESULTS: Microthecin and nine other AF derivatives were synthesized from AF. The 10 compounds were tested in vitro against Gram-positive (GP) and Gram-negative (GN) bacteria, yeasts and moulds using a well diffusion method and in a Bioscreen growth analyser. Of the test compounds, microthecin exhibited the most significant antibacterial activity at 100-2000 ppm against both GP and GN bacteria, including Ps. aeruginosa. Further tests with three malignant blood cell lines (Mutu, Ramos, Raji) and one normal cell line indicated that microthecin was a cell toxin, with a cell mortality >85% at 50 ppm. The other nine AF derivatives demonstrated low or no antimicrobial activity. CONCLUSIONS: Microthecin was active 100-2000 ppm against GP and GN bacteria including Ps. aeruginosa, but was inactive against yeasts and moulds. Microthecin was also a cytotoxin to some mammalian cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: Microthecin might have potential for development as a novel drug against Ps. aeruginosa and to target cancer cells. It might also be developed as a food processing aid to control bacterial growth.

Studies on the testicular effects and zinc excretion produced by various isomers of monobutyl-o-phthalate in the rat.

Mono-n-butyl, -iso-butyl, -sec-butyl or -tert-butyl esters of o-phthalic acid were orally administered at 800 mg/kg body wt./day for 6 days, to young male rats. All of the animals except those treated with the mono-tert-ester developed marked testicular atrophy. Additionally, only those isomers producing testicular damage were found to alter zinc metabolism by increasing the urinary excretion of zinc and by depleting the concentration of this element in testicular tissues.

The effect of mono-(2-ethylhexyl) phthalate and other phthalate esters on lactate production by Sertoli cells in vitro.

Sertoli cells produce lactate and pyruvate as energy substrates for the developing germ cells in the testis. Since the Sertoli cells are thought to be the initial target for phthalate esters causing testicular atrophy, the effect of some phthalates on lactate and pyruvate production by primary Sertoli cell-enriched cultures was studied. Mono-(2-ethylhexyl) phthalate (0.1-200 microM) produced a concentration-dependent stimulation of lactate, but not pyruvate production over a 24 h treatment period and an increase in the ratio of lactate/pyruvate concentration in the culture medium. Di-(2-ethylhexyl) phthalate and 2-ethylhexanol (200 microM) had no such effects. Other phthalate monoesters known to cause testicular atrophy also increased Sertoli cell lactate production and the lactate/pyruvate ratio, whereas monoesters not associated with testicular damage in vivo had no such effects. The results suggest that loss of germ cells in phthalate-induced testicular atrophy is not due to inhibition of energy substrate production by the Sertoli cells and that stimulation of lactate production may be a useful in vitro marker for phthalate esters and related compounds that cause testicular injury.

Effect of DI-n-pentyl phthalate treatment on testicular steroidogenic enzymes and cytochrome P-450 in the rat.

Treatment of young male rats with dipentyl phthalate (DPP) produced significant decreases in testicular cytochrome P-450, cytochrome P-450 dependent microsomal steroidogenic enzymes (17 alpha-hydroxylase, 17-20 lyase) and in the maximal binding of a natural substrate (progesterone) to testis microsomes. No effect was demonstrated by this compound on hepatic cytochrome P-450 content. Treatment of animals with a phthalate ester not causing testicular atrophy (diethyl phthalate; DEP) produced no significant changes in any of the parameters measured. This effect on the enzymes responsible for androgen production may be important as a mechanism of action involved in the development of phthalate-induced testicular damage.

Competition between Salmonella and Pseudomonas species growing in and on agar, as affected by pH, sodium chloride concentration and temperature.

pH/sodium chloride (NaCl) gradient plates were used to investigate competition between Pseudomonas and Salmonella species. At 30 degrees C and at particular NaCl/pH conditions the salmonellae inhibited growth of P. fluorescens and not P. putida. At 20 degrees C P. putida and not P. fluorescens inhibited the salmonellae. The growth of the pure and mixed strains in agar plates with the pH/NaCl conditions was compared by viable counts. Competition in pour plates at 30 degrees C was confirmed. At 20 degrees C, sub-surface growth of the salmonellae inhibited the P. putida. With surface growth this did not occur; the salmonellae was slightly inhibited by the P. putida.

Testing multiple variables on the growth of a mixed inoculum of Salmonella strains using gradient plates.

Six Salmonella strains were grown on two-dimensional sodium chloride-pH and temperature-pH gradient plates. Using image analysis the results were expressed in the form of three-dimensional wire frame graphs. On the temperature-pH gradient plates the optimum growth range was 20-30 degrees C and the minimum pH for visible growth was ca. pH 4, except for strain S. typhimurium CRA63 which grew over a narrower temperature and pH range. On the NaCl-pH gradient plates (whose NaCl gradient began at 2.75% (w/v) NaCl) the maximum concentration of salt at which growth was visible varied from 3.9% to 6.0%, and the minimum pH at which growth was observed varied from pH 4.7 to 5.4 according to the strain used. The incorporation of 0.02% (w/v) sodium nitrite reduced the maximum salt concentration and increased the minimum pH at which the strains could grow. The strains were combined and used in a mixed inoculum on NaCl-pH gradient plates containing 6 different concentrations of NaNO2 incubated at 6 different temperatures. Comparison of the data from the mixed inoculum with data from individual strains showed that, apart from one case, the mixed inoculum represented the extremes of the growth domains of the individual strains. The effect of NaNO2 on the ability of the strains to grow at different pH, NaCl concentrations and temperatures, was more clearly shown by subtracting the data of plates containing NaNO2 from the data of plates without NaNO2.

An investigation of the effects of four variables on the growth of Salmonella typhimurium using two types of gradient gel plates.

The effect of four variables (pH, temperature, sodium chloride concentration and sodium nitrite concentration) on the growth of Salmonella typhimurium CRA663 was investigated using a two-dimensional gradient gel technique. Two methods were used. In the first method the gradients comprised NaCl and pH and in the second method a temperature gradient incubator was used to produce a temperature-pH gradient. Using image analysis, the growth on the plates was depicted as three-dimensional wire frame graphs. At neutral pH and in conditions of low salt, growth was observed over the temperature gradient range of 14-41 degrees C. The optimum growth range was reduced to 21-29 degrees C in conditions of acid pH and/or increased NaCl concentration. The growth on the temperature-pH gradient plates had an irregular surface appearance suggesting that changes in growth rate were occurring at different points of temperature and pH. The effects of increased salt concentration together with acidic pH increased the inhibitory effect of the sodium nitrite. The gradient gel plate technique may be a means of rapidly screening the effect of multiple variables on the growth on microorganisms that may be found in food.

Effect of three preservatives on the growth of Bacillus cereus, Vero cytotoxigenic Escherichia coli and Staphylococcus aureus, on plates with gradients of pH and sodium chloride concentration.

The effect of temperature, pH, sodium chloride concentration and a preservative (sodium benzoate, sodium nitrite or potassium sorbate) on the growth of three foodborne bacterial pathogens (Bacillus cereus, Vero cytotoxigenic Escherichia coli and Staphylococcus aureus) was studied using gradient gel plates. Growth, expressed in optical density units, was recorded using image analysis techniques, and was expressed as three-dimensional grids. These gave a visual indication of the effects of any three of the environmental factors on bacterial proliferation. Sorbate was completely effective against E. coli at all temperature/pH/NaCl combinations, and was the most effective preservative tested against B. cereus. Increase in the acidity and/or the NaCl concentration improved the effect of all the preservatives, except nitrite when used against St. aureus. Nitrite was the least effective preservative, particularly against St. aureus. At < 25 degrees C, sorbate was more effective than benzoate against St. aureus when used with higher concentrations of NaCl. At 35 degrees C benzoate was the most effective preservative against St. aureus, especially when used at pH < 6.

Studies on the metabolism of deoxynivalenol in the rat.

The metabolism and tissue distribution of [14C]deoxynivalenol have been studied in male PVG rats. Following administration of a single oral 10-mg/kg dose, radioactivity excreted in the urine and faeces accounted, respectively, for 25 and 64% of the administered dose within 96 hr. Less than 0.15% of the dose was detected in the respired air. Very little radioactivity appeared to be retained in any of the tissues examined after 96 hr. HPLC separation of several urinary and faecal metabolites was achieved on a reversed-phase column, using two different elution systems, one at neutral pH and one acidified. Two of the major non-polar HPLC peaks were identified by gas chromatography-mass spectrometry as unchanged deoxynivalenol and 3 alpha,7 alpha,15-trihydroxytrichothec-9,12-dien-8-one.

Effective use of nisin to control lactic acid bacterial spoilage in vacuum-packed bologna-type sausage.

Lactic acid bacteria (LAB) commonly cause spoilage in minimal heat-treated vacuum-packed cured delicatessen meats. Predominant species are Lactobacillus sake and L. curvatus. LAB strains isolated from spoiled products of this type (liver sausage, ham and bologna sausage) were found to be sensitive to low nisin concentrations (maximum of 1.25 microg g(-1)). Addition of 25 microg g(-1) nisin (as Nisaplin) inhibited the growth of LAB spoilage organisms inoculated into vacuum-packed pasteurized bologna-type sausages stored at 8 degrees C. Control sausages became spoiled (>10(8) LAB CFU g(-1)) by day 7, whereas sausages containing nisin remained unspoiled for >50 days. The effect of three types of phosphates (used as emulsifiers) on nisin activity in the sausages was compared. LAB growth rate was fastest in samples containing orthophosphate, and slowest in sausages containing diphosphate. The shelf life was also greatly extended in the latter. Fat content also affected nisin activity. Nisin activity (as indicated by LAB inhibition) was greatest in samples containing 15% > 25% > 37% (wt/wt) fat. In a sausage formulation containing 37% fat and incorporating diphosphate as emulsifier, levels of nisin as low as 2.5 microg g(-1) showed antibacterial effects. A nisin level of 6.25 microg g(-1) totally inhibited LAB growth for over 4 weeks and 25 microg g(-1) for 5 weeks. Spoilage control was achieved in the same sausage formulation but with 25% (wt/wt) fat; 12.5 microg g(-1) nisin prevented LAB growth for 5 weeks.

Investigation of the effect of combined variations in temperature, pH, and NaCl concentration on nisin inhibition of Listeria monocytogenes and Staphylococcus aureus.

Gradient plates were used to investigate the effects of varying temperature, pH, and sodium chloride (NaCl) concentration on nisin inhibition of Staphylococcus aureus and Listeria monocytogenes, Nisin was incorporated into the plates of 0, 50, 100, 250, and 500 IU ml -1. Gradients of pH (3.7 to 7.92) at right angles to NaCl concentration (2.1 to 7% [wt/vol]) were used for the plates, which were incubated at 20, 25, 30 and 35 degrees C. Growth on the plates were recorded by eye and by image analysis. The presence of viable but nongrowing cells was revealed by transfer to nongradient plates. Lower temperatures and greater NaCl concentrations increased the nisin inhibition of S. aureus synergistically. Increasing the NaCl concentration potentiated the nisin action against L. monocytogenes; the effect of temperature difference was not so apparent. Between pH 7.92 and ca. pH 5, a fall pH appeared to increase nisin's effectiveness against both organisms. At more acid pH values (ca. pH 4.5 to 5), the organisms showed resistance to both nisin and NaCl at 20 and 25 degrees C. Similar results were obtained with one-dimensional liquid cultures.

Method for Investigation of Competition between Bacteria as a Function of Three Environmental Factors Varied Simultaneously.

Competition between microorganisms as affected by temperature, pH, and the sodium chloride (NaCl) concentration was investigated by selective replication from gradient plates. Salmonella typhimurium was inhibited by Pseudomonas putida at 20 and 23 degrees C but not 30 and 35 degrees C. P. putida no longer grew at the extremes of pH and NaCl concentration, particularly at 30 and 35 degrees C.

The occurrence of colonisation factors (CFA/I, CFA/II and E8775) in enterotoxigenic Escherichia coli from various countries in South East Asia.

Four hundred and fifty-eight enterotoxigenic strains of Escherichia coli (ETEC) were examined for the presence of colonisation factor antigens (CFA) I and II, and the putative colonisation factor, E8775, using an immunodiffusion technique with specific antisera. The ETEC strains had been isolated in Thailand, Bangladesh and from travellers returning to Japan from abroad. Approximately 14% of the ETEC strains possessed CFA/I and a further 13% of the strains possessed CFA/II. The E8775 antigen was found on 5% of the strains. CFA/I was found on strains of the serogroups 04, 015, 063, 078, 090, 0110, 0126, 0128, 0153, and 0? CFA/II was found on strains of the serogroups 06, 08, 09, 078, 0115, 0139, 0? and 0 rough. The E8775 antigen was found on strains of the serogroups 025, 0115, and 0167. The results of this study emphasise the need to continue the search for other mechanisms of adhesion used by ETEC strains, and in particular strains of the serogroups 027, 034, 0148 and 0166.

Enterotoxigenic strains of Escherichia coli O128 are not restricted to subgroup ac but also belong to subgroups ab and abc.

Ninety strains of Escherichia coli O128 isolated in many different countries were examined. The majority (77 strains) belonged to the antigenic subgroup O128ab, and 41 of these strains produced heat-stable enterotoxin (ST), heat-labile enterotoxin (LT), or both. Eight strains were of the antigenic subgroup O128ac; six produced ST only, and two were nontoxigenic. Five strains were of the antigenic subgroup O128abc; two produced ST and LT, and three were nontoxigenic. Ten of the strains studied produced Vero cytotoxin, and all belonged to the antigenic subgroup O128ab. It was concluded that, contrary to the report of Guth et al. (B. E. C. Guth, M. L. M. Silva, I. C. A. Scaletsky, M. R. F. Toledo, and L. R. Trabulsi, Infect. Immun. 47:338-340, 1985), enterotoxigenic strains of E. coli O128 are not restricted to subgroup ac but also belong to the most common subgroup, ab.

In strains of Escherichia coli O167 a single plasmid encodes for the coli surface antigens CS5 and CS6 of putative colonization factor PCF8775, heat-stable enterotoxin, and colicin Ia.

Twenty Escherichia coli strains of serogroup O167 were examined. They all produced the two surface antigens CS5 and CS6 of the putative colonization factor PCF8775, together with heat-stable enterotoxin and colicin Ia. A plasmid coding for CS5, CS6, heat-stable enterotoxin, and colicin Ia was demonstrated in each strain.

Enterotoxigenic Escherichia coli strains belonging to a new serogroup, Escherichia coli O166.

Thirty-two strains of Escherichia coli belonging to a new O group, O166, were examined. Twenty-one strains had the flagella antigen H27, five had the H15 antigen, five had the H7 antigen, and one was nonmotile. All the H27 strains and the nonmotile strain produced heat-stable enterotoxin but not heat-labile enterotoxin. All the H7 strains produced heat-labile enterotoxin but not heat-stable enterotoxin. The remaining strains were nonenterotoxigenic. None of the strains possessed colonization factor antigens CFA/I, CFA/II, or PCF8775.