Induction of resistance in mice against murine cytomegalovirus by cellular components ofLactobacillus casei

Recently, we reported that heat-killedLactobacillus casei (LC) protected mice from murine cytomegalovirus (MCMV) infection by augmentation of natural killer (NK) cell activity. In the present study, we examined which components of LC cell induce the nonspecific resistance most effectively. Whole cell preparation of original LC, susceptible to bacteriophages SG-T and J1, was more effective than its mutants resistant to either bacteriophage. Although the activity of LC cells decreased upon fractionation, cell wall fractions were more active than cytoplasmic fractions. Glycoprotein (GP), a cell wall constituent, was a potent inducer of the resistance. The relative activity of cellular components to induce the resistance was evaluated by a protection index, a ratio of plaque-forming units (PFU) per 50% lethal dose (LD₅₀) for treated mice to that for untreated mice. The protection indices of LC cells and GP were approximately 80 and 28, respectively. The protective effect of GP was evidenced by a decrease in titers of infectious viruses replicated in the target organs. Not only LC cells but also GP, although to a lesser degree, enhanced NK cell activity both in uninfected mice and MCMV-infected mice. The activity of LC cells and GP to augment NK cell activity correlated with the protection index. GP treatment did not modify interferon (IFN) production during MCMV infection. Thus, GP of LC cells seems to be the active principle to endow mice with resistance to MCMV.     

Host-mediated antiviral activity of Lactobacillus casei against cytomegalovirus infection in mice

The protective effect of heat-killed Lactobacillus casei (LC) against murine cytomegalovirus (MCMV) infection was examined. ICR mice treated once with LC 1 day or 2 days before challenge survived lethal infection, but untreated or Lactobacillus fermentum (LF)-treated mice did not. The protective effect was evidenced by an increase in plaque-forming units (PFU) per 50% lethal dose (LD50) and a decrease in titers of infectious viruses replicated in the target organs. This was further confirmed by severity of histopathological damage to the target organs, especially the liver. LC neither inactivated MCMV nor inhibited its replication in mouse embryonic fibroblasts (MEF). The spleen cells from LC-treated mice inhibited its replication in MEF on co-cultivation. Augmentation by LC of splenic natural killer (NK) cell activity correlated with survival of mice from otherwise lethal MCMV infection. Cytotoxic activity of peritoneal cells and level of serum interferon (IFN) were elevated after MCMV infection, but they were not associated with survival of mice nor with treatment of LC. The protective effect of LC was not clear in NK-deficient beige mutant (bgJ/bgJ) mice, when compared with that in their littermate (bgJ/+) mice. Poor protection of bgJ/bgJ mice by LC treatment correlated with failure to induce NK cell activity by LC treatment in the mutant mice. Thus, it is likely that LC protects mice from MCMV infection by augmentation of NK cell activity.     

Inducible resistance against nisin in Lactobacillus casei

Most strains of Lactobacillus casei tested were found to be nisin-resistant. The addition of nisin to a growing culture of a resistant strain stopped growth for several hours; however, growth then resumed at the previous rate. Nisin induced a resistance mechanism that was lost by one passage in nisin-free medium. During induction with nisin, the cells produced an anionic, phosphate-containing polysaccharide with the subunits rhamnose and galactose. This polysaccharide protected sensitive cells of L. casei against the bactericidal action of nisin. Received: 27 July 1995 / Accepted: 30 October 1995     

Enhancement of host resistance against Listeria infection by Lactobacillus casei: efficacy of cell wall preparation of Lactobacillus casei

Cell wall, cytoplasm, polysaccharide, and peptidoglycan fractions prepared from Lactobacillus casei, L. plantarum, and L. acidophilus were examined for their efficacies to enhance resistance of host mice against Listeria monocytogenes infection. Intraperitoneal injections of those cellular fractions of L. casei led to elicitation of inflammatory cells in the peritoneal cavity and the efficacy was highest in the case of peptidoglycan. Macrophage ratio in the resultant peritoneal exudate cells was also highest in mice given peptidoglycan. Macrophages induced with cell wall fraction of L. casei showed the most potent phorbol myristate acetate (PMA)-triggered respiratory burst (chemiluminescence and O2- production determined on the basis of nitroblue tetrazolium reduction) followed by those elicited with peptidoglycan. All the macrophages induced with cell wall of L. casei (two strains) and L. acidophilus enhanced O2- production in response to PMA but L. plantarum did not enhance O2(-)-producing ability in such a manner. The L. casei-cell wall also enhanced in vitro listericidal activity of mouse peritoneal macrophages, but such an activity was not noted in the case of L. acidophilus-cell wall. When mice were intravenously given the cellular fractions 7 or 13 days before L. monocytogenes infection, cell wall fractions of L. casei caused the most potent protective activity. A weak protective activity was also found in peptidoglycan of L. casei. Therefore, the protective action of L. casei against L. monocytogenes infection in host mice may be attributed to cell wall compounds and partially to the peptidoglycan moiety.     

Macrophage activation by Lactobacillus casei in mice

Effects of Lactobacillus casei YIT 9018 (LC 9018), which has antitumor activities against allogeneic and syngeneic murine tumors, on macrophage functions were examined. By intraperitoneal (i.p.) injection of LC 9018, acid phosphatase activity and phagocytic activity of peritoneal macrophages were enhanced significantly compared with those of normal peritoneal macrophages. The phagocytic activities showed peaks 2-3 days after the LC 9018-injection. LC 9018 accelerated the phagocytic function of the reticuloendotherial system in ICR mice tested by the carbon clearance test. The cytostatic activity of peritoneal exudate cells (PEC) induced by i.p. injection of LC 9018 into C57BL/6 mice against EL4 cells was also enhanced. On the other hand, PEC induced by L. fermentum YIT 0159, which has no antitumor activity, did not have cytostatic activity. These observations showed that LC 9018 was able to activate macrophages in mice.     

Host-mediated antiviral activity ofLactobacillus casei against cytomegalovirus infection in mice

The protective effect of heat-killedLactobacillus casei (LC) against murine cytomegalovirus (MCMV) infection was examined. ICR mice treated once with LC 1 day or 2 days before challenge survived lethal infection, but untreated orLactobacillus fermentum (LF)-treated mice did not. The protective effect was evidenced by an increase in plaque-forming units (PFU) per 50% lethal dose (LD₅₀) and a decrease in titers of infectious viruses replicated in the target organs. This was further confirmed by severity of histopathological damage to the target organs, especially the liver. LC neither inactivated MCMV nor inhibited its replication in mouse embryonic fibroblasts (MEF). The spleen cells from LC-treated mice inhibited its replication in MEF on co-cultivation. Augmentation by LC of splenic natural killer (NK) cell activity correlated with survival of mice from otherwise lethal MCMV infection. Cytotoxic activity of peritoneal cells and level of serum interferon (IFN) were elevated after MCMV infection, but they were not associated with survival of mice nor with treatment of LC. The protective effect of LC was not clear in NK-deficient beige mutant (bg^J/bg^J) mice, when compared with that in their littermate (bg^J/+) mice. Poor protection of bg^J/bg^J mice by LC treatment correlated with failure to induce NK cell activity by LC treatment in the mutant mice. Thus, it is likely that LC protects mice from MCMV infection by augmentation of NK cell activity.     

Protective effect of lipoteichoic acid from Lactobacillus casei and Lactobacillus fermentum against Pseudomonas aeruginosa in mice

Lipoteichoic acid (LTA) from Lactobacillus casei YIT 9018 or Lactobacillus fermentum YIT 0159 augmented the resistance of C57BL/6 mice to infection with Pseudomonas aeruginosa, but conferred no resistance to Listeria monocytogenes. It is suggested that LTA was unable to activate macrophages.     

Induction of apoptosis of T cells by infecting mice with murine cytomegalovirus.

Cytomegalovirus (CMV) is associated with several lymphocyte dysfunctions, but the precise mechanisms of the dysfunctions are still unclear. To elucidate the mechanisms, a cell cycle-DNA content analysis was performed on splenic T cells of murine CMV (MCMV)-infected BALB/c mice. T cells from mice infected with 3 x 10(3) PFU of MCMV contained a higher percentage of hypodiploid nuclei after 12 or 24 h of culture than those from naive mice. T cells from infected mice also contained a larger amount of fragmented DNA. Taken together, these results suggested that infection with MCMV induced the apoptotic cell death of T cells. This induction of apoptosis accounted for the dysfunction of lymphocytes, at least partially. Flow cytometric analysis showed that T cells as well as B cells from MCMV-infected mice expressed an augmented level of Fas antigen, an apoptosis-associated cell surface molecule, which might be the cause of the apoptosis of cells. T cells from MCMV-infected C57BL/6-lpr/lpr mice with mutations at the lpr/fas locus, however, also showed a substantial level of apoptosis, which was reproducibly lower than that seen in C57BL/6 mice. Therefore, it was suggested that the Fas-mediated pathway contributed to but was not sufficient for the induction of apoptosis and that mechanisms other than the Fas-associated pathway were also involved in the induction of apoptosis.     

Anti-tumour effect of humoral and cellular immunities mediated by a bacterial immunopotentiator,Lactobacillus casei,in mice

Summary Administration of a mixture containing Lactobacillus casei YIT 9018 (LC9018) and methylcholanthrene-induced fibrosarcoma (Meth A) cells into the peritoneum of syngeneic BALB/c mice suppressed the tumour growth and protected the mice from tumour death. With the appearance of the anti-tumour activity, serum complement-dependent tumour cytotoxic (CDC) antibody was induced on the 5th day after the administration as a result of the adjuvant effect. The cytotoxic antibody was not found in serum on the 5th day after inoculation of Meth A cells alone, but it was induced before the mice died of the tumours. Adjuvant induction of the cytotoxic serum antibody at an early time was also observed using Kirsten murine sarcoma virus-transformed tumour (K234) cells. Both of these cytotoxic antibodies in sera from Meth A-suppressed and the tumour-bearing mice were specific for the tumour cells and were IgM class, since they were absorbed with rabbit anti-mouse IgM antibody. However, the cytotoxic antibody was not found in the peritoneal cavity which was the tumour inoculation site, but binding antibody against the tumour cells was faintly detected in the region using an enzyme-linked immunoabsorbent assay (ELISA). In neutralization tests, the cytotoxic antibody did not exert anti-tumour activity in recipient mice when it was administered to the mice along with the tumour cells or when it was administered i. v. at the time of tumour inoculation. Moreover, the cytotoxic antibody was not available for the antibody-dependent cell-mediated cytotoxicity (ADCC). These results suggest that the cytotoxic antibody did not exert anti-tumour activity in the tumour-suppressed mice. In contrast, peritoneal exudate cells (PEC) on the 5th day, and PEC and spleen cells on the 15th day after i. p. administration of the mixture exerted strong anti-tumour activity as measured by the Winn test.In conclusion, the adjuvant effect of LC9018 induced tumour-specific humoral and cellular immunities but the anti-tumour activity was dependent only on the cellular effectors of the host. The possible use of LC9018 in tumour immunotherapy is discussed.     

Enhancement of Host Resistance against Listeria Infection by Lactobacillus casei: Activation of Liver Macrophages and Peritoneal Macrophages by Lactobacillus casei

The functions of liver macrophages and peritoneal macrophages obtained after injection of Lactobacillus casei were examined. Listericidal activity in vivo was enhanced in liver macrophages 13 days after L. casei injection but was somewhat suppressed in the macrophages 2 days after the injection. The listericidal activity in vitro was enhanced in peritoneal macrophages obtained 13 days after L. casei injection but was suppressed in cells obtained 2 days later. The PMA-triggered respiratory burst in the liver macrophages elicited by L. casei was higher than that of resident macrophages. Alkaline phosphodiesterase activity in the liver macrophages was decreased by L. casei injection, as was also the case with peritoneal macrophages. These observations indicate that L. casei augmented cellular functions of both liver and peritoneal macrophages.     

On the benefit of whey-cultured Lactobacillus casei in murine colitis

The objective of this study was to examine the prophylactic and therapeutic effect of whey-cultured Lactobacillus casei (L. casei) in a murine model of colitis. Colitis was induced by intracolonic administration of a mixture of 2,4,6-trinitrobenzenesulphonic acid (TNBS)/absolute ethanol in male Wistar rats. Animals were divided into 5 groups including sham (normal group), control (vehicle-treated), positive control (dexamethasone 1 mg/kg/day, orally), prevention (10(8) cfu L. casei/day, orally, 14 days before induction of colitis), and treatment (10(8) cfu L. casei/day, orally, 14 days after induction of colitis). After 14-days treatment, the animals were sacrificed on the day 15. Distal colons were removed for examining histological and biochemical assays. Biomarkers including TNF-α, myeloperoxidase (MPO), and lipid peroxidation (LPO) were measured in the homogenate of colon. Results indicated an apparent improvement in colon histopathology scores, TNF-α, MPO, and LPO in the treatment group, whereas prevention group did not demonstrate positive efficacy in prevention of colonic damage. It is concluded that L. casei grown in whey culture is very effective in ameliorating both biochemical and histopathological markers of colitis if used post induction of colitis but not if used before induction of colitis. The difference between effects of L. casei when used pre-colitis and post-colitis confirms its mechanism of action as an anti toxic stress agent. Further studies should be made in IBD patients.     

Thermal and chemical resistance of Lactobacillus casei and Lactobacillus paracasei bacteriophages

AIMS: The survival of two collection Lactobacillus casei and L. paracasei bacteriophages when subjected to thermal and chemical treatments was investigated. METHODS AND RESULTS: Thermal resistance was evaluated by heating phage suspensions at 63, 72 and 90 degrees C in three different media [Tris-magnesium gelatin (TMG) buffer: 10 mmol l(-1) Tris-Cl, 10 mmol l(-1) MgSO(4) and 0.1% w/v gelatin; Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)]. A marked heat sensitivity was evident in both phages, as 15 min at 72 degrees C was enough to completely inactivate (6 log(10) reduction) them. No clear influence was demonstrated by the suspension media. The phages also showed similar resistance to biocides. Peracetic acid and sodium hypochlorite (800 ppm) were the most effective ones, destroying the phages within 5 min. Concentrations of 75 and 100% ethanol were not suitable to inactivate phage particles even after 45 min. Isopropanol did not show an effect on phage viability. CONCLUSIONS: The data obtained in this work are important to design more effective control procedures in order to inactivate phages in dairy plants and laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will contribute to enhance the background knowledge about phages of probiotic bacteria.     

Immunomodulatory and protective effect of probiotic Lactobacillus casei against Candida albicans infection in malnourished mice

The effect of Lactobacillus casei CRL 431 (Lc), when administered as a supplement to a repletion diet, on the resistance of malnourished mice to Candida albicans infection was studied. Weaned mice were malnourished by being given a protein-free diet (PFD) for 21 days. The malnourished mice were then fed a balanced conventional diet (BCD) for 7 days or BCD for 7 days with supplemental Lc on days 6 and 7 (BCD+Lc). Malnourished (MNC) and well-nourished (WNC) mice were used as controls. At the end of the treatments the mice were infected intraperitoneally with C. albicans. Animals that had received probiotics had improved survival and resistance against this infection compared to those in the BCD and MNC groups. The number and fungicidal activity of phagocytes, and the concentrations of tumor necrosis factor-α, interferon-γ and interleukin-6 (IL-6), increased in blood and infected tissues in all experimental groups, but MNC mice showed lower concentrations than those in the WNC group. BCD and BCD+Lc mice showed higher concentrations of these variables than those in the MNC group, but only the BCD+Lc group presented values similar to the WNC mice. Malnutrition also impaired the production of IL-17 and IL-10 in response to infection. Both repletion treatments normalized IL-17 concentrations, but IL-10 in the BCD+Lc group was significantly higher than in WNC mice. The addition of L. casei to the repletion diet normalized the immune response against C. albicans, allowing efficient recruitment and activation of phagocytes, as well as effective release of pro-inflammatory cytokines. In addition, probiotic treatment induced an increase in IL-10 concentrations, which would have helped to prevent damage caused by the inflammatory response.     

Protective and therapeutic efficacy of Lactobacillus casei against experimental murine infections due to Mycobacterium fortuitum complex

A bacterial immunopotentiator, LC 9018 (heat-killed Lactobacillus casei), was studied for its protective and therapeutic efficacies against Mycobacterium fortuitum and M. chelonae infections in mice. This agent reduced the incidence of spinning disease and gross renal lesions and enhanced the elimination of organisms at the site of infection in the host mice, when administered intramuscularly six times a week (0.1 mg dry weight per injection, one injection on each day of treatment) from 1 week before to 2 weeks after infection. The LC 9018 injections in this protocol caused a marked increase in the phagocytic function, O2- -producing ability and chemiluminescence of host peritoneal macrophages. Moreover, LC 9018 injections using the same schedule resulted in an enhancement of interleukin-1-producing function of the macrophages, particularly in the infected mice. These findings indicate that LC 9018 administration with the present protocol can activate macrophage functions, in particular those related to microbicidal activity. This would partly explain the protective and therapeutic efficacy of LC 9018 against infection due to M. fortuitum complex.     

Antitumor effect of intrapleural administration of Lactobacillus casei in mice

Summary The antitumor effect of intrapleural (i.pl.) administration of Lactobacillus casei YIT 9018 (LC 9018) on Meth A sarcoma in BALB/c mice was examined. Inoculation of Meth A cells into the thoracic cavity of BALB/c mice caused growth of the cells and the mice died from the tumor with an increased amount of pleural fluid. LC 9018 was given i.pl. to BALB/c mice before or after i.pl. inoculation of Meth A cells and the survival of the mice was determined. The i.pl. administration of LC 9018 was effective in prolonging the survival of the mice after i.pl. inoculation of Meth A tumor, and pretreatment with LC 9018 i.pl. also prolonged survival. Moreover, i.pl. administration of LC 9018 not only increased the number of thoracic exudate cells (TEC) but also augmented both cytolytic activity of thoracic macrophages and natural killer cell activity of TEC. Furthermore, phagocytic activity of thoracic macrophages against sheep red blood cells was enhanced and Ia antigen-positive cells in TEC were increased by the i.pl. treatment with LC 9018. These results showed that TEC induced by i.pl. administration of LC 9018 had antitumor activity against Meth A tumor inoculated i.pl. into BALB/c mice.