A derivative of Lactococcus lactis strain H61 with less interleukin-12 induction has a different cell wall.

Lactococcus lactis H61 can increase the cellular immune responses of aged (14-mo-old) senescence-accelerated mice. The aim of this study was to investigate the factors contributing to IL-12 induction by strain H61 by analyzing strains derived from it. Strain H61 derivative no. 13 was obtained by growing the parent strain at 37°C. This derivative induced significantly lower production of IL-12 from J774.1 macrophage cells than did the parent strain H61. The 2 strains differed in the resistance of their whole cells or cell walls to lysozyme, a cell wall-degrading enzyme. Sodium hydroxide treatment to de-O-acetylate muramic acid in the cell walls of the 2 strains reduced the lysozyme resistance, compared with untreated cell walls: at 3h after adding lysozyme, the lysozyme resistance of untreated and NaOH treated cell wall from strain H61 was 55.4% and 11.7%, respectively. The values of untreated and NaOH-treated cell walls from strain no.13 were 73.7 and 42.8%, respectively. The reduction was higher in strain H61, indicating that the cell walls of strain H61 were highly O-acetylated. Trichloroacetic acid treatment to remove wall-associated polymers such as teichoic acids made the lysozyme resistance of the cell walls of both strains similar. The sugar content of cell walls prepared from strain H61 was significantly higher than that of strain no. 13 cell wall. A derivative with less activity for inducing IL-12 by macrophage cells had less O-acetylation and had lower sugar content in the cell wall than did strain H61. Modifying the cell wall of strain H61 may be a useful way to regulate its ability to induce IL-12. Strain H61 has been used as a starter bacterium in the dairy industry. This study could lead to enhancing the value of dairy products made by strain H61 by characterizing the key factor(s) responsible for its stimulation of immunity.

Interaction between Lactococcus lactis and Lactococcus raffinolactis during growth in milk: development of a new starter culture.

Many milk fermentations use mixed cultures of lactic acid bacteria. To select a new mixed starter culture, 100 acid-producing bacterial strains were isolated from raw cow milk. Of these, 13 strains identified as belonging to the genera Lactococcus, Lactobacillus, Leuconostoc, or Weissella (based on phenotypic and genotypic tests) were assessed for a symbiotic effect between pairs of isolated strains during growth in milk. Among the strains tested, a mixed culture of Lactococcus lactis ssp. lactis strain 54 and Lactococcus raffinolactis strain 37 stimulated greater acid production during fermentation than occurred with pure fermentation. This stimulatory effect was not observed in milk supplemented with yeast extract or glucose or in constituted medium. Addition of a cell-free filtrate from milk fermented by strain 54 increased acid production by strain 37; however, the converse effect was not observed. The increased acid production by this mixed culture was, therefore, due to stimulation of strain 37 by metabolic products of strain 54, suggesting that the interaction between strains 54 and 37 is commensal. Analysis with a taste-sensing system indicated that fermented milk containing the mixed culture was more acidic, had more anionic bitterness, had greater aftertastes of anionic bitterness and astringency, and was less salty and umami than milk containing the individual cultures. This study identifies a new commensal relationship between 2 lactococcal strains that are commonly used for making dairy products.

Lactococcus strains treated with heat and hen-egg-white lysozyme induce abundant interleukin-12 production by J774.1 macrophages and murine spleen cells.

The IL-12-inducing ability of lactic acid bacteria could be a critical index of immunomodulatory activity, especially in promoting T-helper-1 responses and in suppressing T-helper-2-mediated allergic responses. We aimed to develop a simple method for enhancing the IL-12-inducing ability of bacteria. We examined the in vitro effects of strains of lysozyme-modified Lactococcus (ML-LYS), prepared by heat treatment of the Lactococcus strain in the presence of lysozyme, on the ability of mouse macrophage-like J774.1 cells and spleen cells to produce IL-12. An IL-12-inducing ability greater than that of heat-killed bacteria was shown by 41 of 46 ML-LYS strains in J774.1 cells and by all 46 ML-LYS strains in mouse spleen cells. In contrast, bacteria modified by α-lactalbumin, ß-lactoglobulin, or ovalbumin did not enhance IL-12 production in J774.1 cells. Microscopically, ML-LYS showed stronger resistance to lysozyme and macrophage digestion than did heat-killed bacteria or the other modified bacteria. Addition of chitotriose, a lysozyme inhibitor, enhanced IL-12 production by J774.1 cells stimulated with heat-killed bacteria. Therefore, enhancement of resistance to lysozyme may be a key factor in the strong IL-12-inducing ability of ML-LYS. These findings have important implications for the design of dairy products that have an immunomodulatory effect using the modified bacteria.

Effect of fermented liquid diet prepared with Lactobacillus plantarum LQ80 on the immune response in weaning pigs.

Probiotics such as lactic acid bacteria directly influence the host's health and have beneficial effects such as decreasing the number of enteric pathogens, regulating intestinal immune responses and preventing diseases. Among domestic animals, probiotics have been expected to be an alternative to antibiotics added in the diet; and fermented liquid diet (FLD) containing probiotics has great potential as a diet for reducing the use of antibiotics. In this study, we evaluated the immunomodulatory effects of FLD, prepared using Lactobacillus plantarum LQ80 (LQ80), on the immune response of weaning pigs. Ten weaning piglets were divided into two groups and were fed the FLD (n = 5) or a non-fermented liquid diet (NFLD) (n = 5) for 28 days. At the end of the experiment, the total immunoglobulin M (IgM) and immunoglobulin G (IgG) levels in the sera of the FLD-fed piglets were significantly higher than those of the NFLD-fed piglets (P < 0.05). In contrast, the total immunoglobulin A (IgA) levels in the feces and saliva were not significantly affected by FLD feeding. However, the mean fecal IgA levels of FLD-fed piglets at day 28 were higher than those at 14 and 21 days (P < 0.05). Blood cells from the FLD-fed piglets showed a low level of interferon-γ secretion and mitogen-induced proliferation compared to that of the NFLD-fed piglets. Furthermore, the levels of interluekin-8 and tumor necrosis factor-α, which are proinflammatory cytokines, in the blood cells of the FLD-fed piglets were lower than those of the NFLD-fed piglets (P < 0.05). In conclusion, the FLD used in this study could alter the immune responses of weaning piglets by stimulation of the systemic or mucosal antibody response, without unnecessary inflammatory reactions. This indicates, that the FLD feed prepared with the use of LQ80 may be a candidate feed, with regard to enhancing immune responses and preventing diseases in weaning piglets.

Different growth media alter the induction of interleukin 12 by a Lactococcus lactis strain.

Lactococcus lactis subsp. lactis G50 has immunomodulatory activity and is a candidate for use as a probiotic strain. We investigated the factors that affect the immunomodulatory activity of this strain. The macrophage-like cell line J774.1A was exposed to live or dead cells of strain G50 grown in different media, and the interleukin (IL) 12 produced by the cell line was then measured. Live cells grown in M17 supplemented with glucose (GM17 cells) induced IL-12 production by J774.1 cells significantly more than did cells grown in deMan Rogosa Sharpe (MRS) broth (MRS cells; P < 0.05). In the case of dead cells, the opposite results were obtained in these two samples. The sugar content of GM17 cells was significantly higher than that of MRS cells (P < 0.01). The fatty acid compositions of GM17 cells and MRS cells differed. Lysis of GM17 cells by lysozyme, which degrades the cell wall, was greater than in MRS cells. The cell wall fraction prepared from GM17 cells induced significantly more IL-12 production than did the fraction from MRS cells (P < 0.05). These results indicated that alterations in cellular components or in the structure of the cell surface by the growth media affected the immunomodulatory activity of strain G50. Attention should be paid to the selection of growth medium in testing for the immunomodulatory activity of lactic acid bacteria.

Effects of lactoferrin on collagen gel contractile activity and myosin light chain phosphorylation in human fibroblasts.

When fibroblasts are plated on a type I collagen gel they reduce the size of the gel and the extent of collagen gel contraction reflects the motile activity of the fibroblasts. We found that both bovine and human lactoferrin (Lf) enhanced the collagen gel contractile activity of WI-38 human fibroblasts. Rho inhibitor (exoenzyme C3), Rho kinase inhibitor (Y-27632), myosin light chain kinase inhibitor (ML-7), MEK inhibitor (PD98059) and Src family tyrosine kinase inhibitor inhibited the Lf-enhanced collagen gel contraction. Treatment of fibroblasts with Lf induced the phosphorylation of myosin light chain (MLC) within 30 min. Lf-enhanced MLC phosphorylation was inhibited by Y-27632 and ML-7. These results suggest that Lf promotes the motility of fibroblasts by regulating MLC phosphorylation.

Interleukin-10-secreting Peyer's patch cells are responsible for active suppression in low-dose oral tolerance.

We demonstrate the induction of antigen-specific interleukin-10 (IL-10)-secreting cells in murine Peyer's patches (PPs) after low-dose beta-lactoglobulin (BLG) feeding. In addition, we show that PP cells can inhibit the T-cell proliferative response in vitro as well as T-cell-mediated inflammation in vivo. The active suppression mediated by these regulatory cells was seen only within a narrow range of antigen dosage (feeding), with the most prominent effect at 5 x 1 mg BLG. On either side of this range, T-helper 1-like cytokine responses were observed when PP cells were stimulated with antigen in vitro. This result correlated with reduced production of regulatory cytokines as well as reduced activity of bystander suppression. We found that changes in IL-10 production correlated inversely with changes in interferon-gamma production. Inhibitory effects mediated by CD4(+) PP cells were partially neutralized by antibodies to IL-10 and transforming growth factor-beta. Interestingly, the generation of such regulatory cells after low-dose BLG feeding exhibited organ dependence. Among spleen, lymph node and PP cells derived from orally tolerized mice, PP cells were the most effective in promoting bystander suppression in the presence of BLG, indicating the significance of PPs as an inductive site for antigen-specific regulatory cells upon induction of low-dose oral tolerance. Moreover, PP cells from mice fed 5 x 1 mg BLG were shown to suppress a BLG-specific delayed-type hypersensitivity response induced in footpads, suggesting that IL-10-secreting PP cells regulate systemic inflammation.

Factors in bovine colostrum that enhance the migration of human fibroblasts in type I collagen gels.

Bovine colostrum has an activity that increases the migration of WI38 fibroblasts. We evaluated the motility of fibroblasts by their ability to contract collagen gels. Part of the activity was absorbed by anion-exchange chromatography at pH 6.4, and eluted by 0.2-0.3 M sodium chloride. The activity was separated into many fractions corresponding to 20-150 kDa by gel filtration chromatography under acidic conditions. The major peak of the activity coincided with 50-70 kDa.

Localization of sequential antigenic determinants on bovine beta-casein with synthetic peptides and antisera from mouse, rabbit, and goat.

The antigenic determinants of bovine beta-casein (beta-CN) were localized by using twenty overlapping peptides encompassing the entire sequence of beta-CN and anti-beta-CN antisera from outbred mouse, rabbit and goat. The profile of the reactions was characteristic to the species, the dominant antigenic regions being 80-95, 143-158 and 195-209 in mouse, 1-16 in rabbit and 100-115 in goat. Regions 1-16, 100-115, 121-136 and 143-158 were antigenic in all three species. The number of antigenic regions recognized by goat was much fewer than that by mouse and rabbit, possibly because of the homology between bovine and goat beta-CN. A mixture of the twenty peptides could absorb about 50-60% of beta-CN specific antibodies from each species. Furthermore, the mouse and rabbit anti-beta-CN antibodies were also specific to the phosphorylated regions. We therefore conclude that the major antigenic determinants on beta-CN would be largely sequential and include the phosphorylated sites.

Effect of bifidobacteria feeding on fecal flora and production of immunoglobulins in lactating mouse.

To elucidate the effects of probiotics on the stimulation of immunoglobulin production during lactation, feeding trials of bifidobacteria in lactating mice were conducted. Bifidobacteria appeared in feces at 9.67+/-0.17 log 10 number per gram levels. All bifidobacteria found in the feces were the administered strain. Mice fed bifidobacteria for 12 days showed significantly high levels of fecal total IgA compared to that of the control group (P < 0.05). The levels of anti-beta-lactoglobulin IgA in milk as well as in fecal extracts were significantly higher in the bifidobacteria-fed group than that of the control group (P < 0.05). These results suggest that the intake of bifidobacteria can enhance local production of IgA in milk and the intestine, which may help to protect both pups and dams from exposure to food antigens.

Effect of whey hydrolysate formula on the transfer of beta-lactoglobulin into serum and milk in mice.

This study was performed to elucidate the effect of whey hydrolysate formula on the transfer of an antigen into serum and milk. The concentrations of beta-lactoglobulin in serum and milk were positively correlated (p < 0.01). The concentration of beta-lactoglobulin in serum tended to become low in the mouse and to be accompanied with a high level of fecal anti-beta-lactoglobulin IgA concentration. The fecal anti-beta-lactoglobulin IgA of mice fed hydrolysate formula for 12 weeks was significantly higher than that of the control formula-fed mice (p < 0.05). These results suggest that a high level of intestinal IgA elicited by the feeding of hydrolysate formula may reduce the transfer of beta-lactoglobulin into serum and milk.

[Effects of exogenous adenosine on myocardial recovery after acute hemorrhage].

This study was designed to investigate whether reperfusion with adenosine had an effect on myocardial high energy phosphate levels and cardiac function in hearts extracted from acutely hemorrhaged rats. Rats were bled to a mean arterial pressure of 0 mmHg for 5, 10 or 15 minutes and hearts were removed and assigned to one of three groups: 1) Hearts freeze clamped for measuring high energy phosphates; 2) Hearts perfused by Langendorff method at a constant perfusion pressure of 90 mmHg for 30 minutes followed by freeze clamping and determining high energy phosphates and; 3) Hearts perfused with adenosine (20 microM) and treated in the same way as in group 2. In group 1 myocardial ATP was significantly reduced as compared with control. When reperfusion started within 10 min, ATP contents recovered to the levels of control, and there were no significant changes between groups 2 and 3. LVP and LV dp/dt in group 3 were significantly higher than those in group 2. When reperfusion started after 15 min, ATP remained at a low level and few hearts could be resuscitated. These findings suggest that early resuscitation with adenosine might facilitate cardiac recovery following acute hemorrhage.

Localization of T-cell determinants on bovine beta-lactoglobulin.

T-cell determinants of bovine beta-lactoglobulin (beta-LG) in BALB/c(H-2d), C57BL/6(H-2b) and C3H/He(H-2k) mice were identified using a set of overlapping synthetic peptides encompassing the entire primary structure of the protein. Lymph node cells from mice immunized with beta-LG were subjected to cell proliferation assay in the presence of these peptides and uptake of 3H-labeled thymidine was measured. Determinant regions were indicated to lie in residues 42-56, 62-76 and 139-153 in BALB/c mice, residues 11-26, 72-86, 100-113 and 119-133 in C57BL/6 mice, residues 72-86, 91-104, 129-143 and 139-153 in C3H/He mice. Some of these fragments included the antigenic motifs predicted by hypotheses according to amphipathicity and sequential patterns of peptides. We reported elsewhere that residues 42-56 and 72-86 represent one of the B-cell antigenic determinants in BALB/c and C3H/He, respectively. These peptides serve as good models of colinear T- and B-cell determinants as they contain both of T- and B-cell determinants.

Establishment of CD4+ T cell clones specific to bovine beta-lactoglobulin and analysis of their specificity.

Bovine beta-lactoglobulin (beta-LG) specific T cell clones, H1.1, 5G, 2.11G, and 6.11G were established from BALB/c mice and characterized. Surface phenotypes of clones were Thy1+, CD3+, CD4+, and CD8-. All clones recognize beta-LG in a I-Ad restricted manner. The T cell determinant region for H1.1 and 5G was identified as residues 42-56 of beta-LG by proliferation assay with overlapping synthetic peptides covering the entire sequence of beta-LG. The T cell determinant region for H1.1 was further delimited to residues 43-52 by using analog peptides. Previously we analyzed the T cell determinant regions of beta-LG in BALB/c, C3H/He, and C57BL/6 mouse by polyclonal T cell proliferation assay and reported that residues 42-56 of beta-LG constitute one of the major T cell determinants in BALB/c mice. Specificities of 2.11G and 6.11G were not identified by these synthetic peptides. Although reduction of disulfide bonds and CNBr cleavage of beta-LG did not affect the antigen recognition by 2.11G and 6.11G, tryptic degradation of the protein caused a clearer decrease of response in 6.11G than in 2.11G.

[Alterations in number of rabbit myocardial beta-adrenergic receptors in endotoxic shock: down regulation in hyperdynamic sepsis model and effects of cytokines administration].

We investigated alterations in myocardial beta- and beta 1-adrenergic receptor (BAR and B1AR) number during hyperdynamic state induced by endotoxin or cytokines. [METHODS] Twenty-nine Japanese White rabbits were divided into 2 groups. Hearts were removed 18 h after intraperitoneal administration of sterile saline (SAL) or E. coli endotoxin (LPS; 50 micrograms/kg) (Group E, n = 12), or 3 h after intravenous injection of SAL or cytokines (interleukin 1-beta; 5 micrograms/kg followed by 25 ng/kg/min for 2 h, or tumor necrosis factor; 5 micrograms/kg) (Group C, n = 17). BAR and B1AR numbers were determined in myocardial membranes from rabbit left ventricles with techniques of radioactive ligand binding study. We used [3H] dihydroalprenolol (3H-DHA) as radioactive ligand, and specific 3H-DHA binding to BARs was defined as the difference between the presence and the absence of 10 microM propranolol. B1AR number was assessed through the specific binding of 3H-DHA in the presence of ICI 118, 551 (5 x 10(-8) M), a highly selective beta 2-adrenergic receptor antagonist. In Group E, mean arterial blood pressure (MAP), heart rate (HR), and cardiac output (CO) (by thermodilution) were measured under pentobarbital sodium anesthesia before excision of hearts. [RESULTS] In Group E, CO was significantly (p less than 0.05) increased in rabbits injected with LPS (E-LPS) as compared with that in rabbits injected with SAL (E-SAL) (E-LPS; 0.75 +/- 0.02 l.min-1, E-SAL; 0.61 +/- 0.05 l.min-1, mean +/- SEM). MAP and HR were slightly decreased in E-LPS but not significantly. Maximum binding (Bmax) of 3H-DHA to BARs was significantly (p less than 0.05) decreased by 18% in myocardial membranes from E-LPS compared to E-SAL (E-LPS; 48.2 +/- 4.3 fmol/mg protein, E-SAL; 58.9 +/- 2.9 fmol/mg protein, mean +/- SEM). Similarly, Bmax of 3H-DHA to B1ARs was decreased by 18% in E-LPS, although no statistical significance was detected. In Group C, both BAR and B1 AR number was slightly, but not significantly decreased 3 h after administration of cytokines. [CONCLUSION] These data suggest that down regulation of cardiac BARs may occur during hyperdynamic stage of endotoxic shock.

Decreased beta-adrenergic receptor density in rat myocardium during hemorrhagic shock.

We investigated alterations in the number and affinity of cardiac beta-adrenergic receptors during hemorrhagic shock. Forty male Wistar rats were divided into two groups: (1) a shock group (n = 20), in which mean arterial blood pressure was decreased to 40-50 mmHg by bleeding and kept constant for 6 h; and (2) a control group (n = 20), which underwent a sham operation. We used (-)[(3)H]dihydroalprenolol for the determination of the number and affinity of beta-adrenergic receptors in myocardial membranes. An additional 25 rats were used for determination of plasma epinephrine and norepinephrine concentrations. Scatchard analysis showed a 20% reduction ( P < 0.05) in beta-adrenergic receptor density in the shock group (70.3 +/- 3.5 fmol.mg(-1) protein) compared to the control group (90.0 +/- 4.8 fmol.mg(-1) protein) but no significant change in the affinity (2.52 +/- 0.06 vs. 2.31 +/- 0.09 nmol. l(-1), control vs. shock). Plasma catecholamine concentrations were increased significantly at 1, 2, 4 and 6 h after the start of hypotension. These data suggest that increased levels of plasma catecholamines in hemorrhagic shock may be correlated a significant loss of beta-adrenergic receptors in rat myocardium.

[Changes in myocardial energy charge following cardiac arrest and subsequent resuscitation].

Cardiac resuscitation becomes more difficult as time of arrest is prolonged. This study was designed to investigate changes in energy charge (EC) during cardiac arrest and subsequent resuscitation periods. The experiment was divided into 2 parts. In the first part, 36 rats were divided into 6 groups. Control rats were anesthetized only with pentobarbital and the heart was extirpated. In other rats, an acute exsanguination was carried out until mean arterial pressure (MAP) fell to 0, and then the rats were divided into 5 groups according to the time of extirpation following the cardiac arrest: 1, 5, 10, 15 and 20 minutes, respectively. The extirpated hearts were rapidly frozen with liquid nitrogen. ATP, ADP and AMP were enzymatically measured and EC was calculated. ECG was monitored to ascertain complete electrical arrest. Disappearance of ECG activity was observed about 9 minutes after MAP reaching zero. In the second part; 36 rats were also divided into 6 groups. After the same manipulation as in the first part, the extirpated heart was perfused for 30 minutes using Langendorff method and both cardiac function (LVP, LV dp/dt and HR) and EC were investigated. Total adenine nucleotide (TAN) was already diminished at 1 minute and decreased progressively. On the contrary, EC was well maintained around control level until ECG activity disappeared completely, and an abrupt decrease in EC occurred after electrical arrest. When the heart was perfused about 30 minutes, EC recovered almost to the control level in the groups in which perfusion was started within 10 minutes, but its recovery was incomplete in the groups in which perfusion was begun after 10 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of shock-related cardiotoxic peptide.

The aim of the present work is to confirm the presence of MDF (myocardial depressant factor), which has long been postulated to be one of the cardiotoxic substances in the shock state. Twenty male mongrel dogs were divided into two groups, a hemorrhagic shock group (n = 10) and an endotoxic shock group (n = 10). Blood samples were obtained from each animal at 1, 2, 4, and 7 h after hypotensive events occurred. Inotropic properties of the plasma samples were evaluated by the isometric contraction of a cat papillary muscle preparation, and chromatographic analysis was performed on the peptides in the plasma. Developed tension of the muscle was increased significantly by changing the bathing medium from Krebs-Henseleit solution to plasma obtained 1 to 4 h after the onset of hemorrhagic and endotoxin induced hypotension. The positive inotropic change was associated with a significant increase in plasma epinephrine concentration. None of these plasma samples possessed a negative inotropic effect (i.e., the property of MDF activity). The elution profile by gel column chromatography of samples obtained from shocked animals was almost identical to that recognized as MDF. However, the presence of MDF was not confirmed by column parameters and color development by the ninhidrin reaction. In conclusion, we found no evidence to support the presence of cardiotoxic peptide in plasma of shocked animals.