Correlation between increase in Ia-bearing macrophages and induction of T cell-dependent antitumor activity by Lactobacillus casei in mice

Summary When Lactobacillus casei YIT 9018 (LC 9018) or Corynebacterium parvum, known to be immunomodulators possessing antitumor activity, were injected i.p. into BALB/c mice, peritoneal exudate macrophage Ia antigen detected by indirect immunofluorescence method was expressed on their cell surface, but it was not expressed following the injection of 10% proteose peptone, an inflammatory agent, or Lactobacillus fermentum YIT 0159 (LF 0159), which have no antitumor activity. The percentage and absolute number of Ia-positive peritoneal macrophages were maximum on the 7th day after the injection of LC 9018. Immunization by injection of Meth A fibrosarcoma cells treated with mitomycin C (MMC-Meth A) 7 days after LC 9018 injection suppressed the growth of Meth A implanted i.p. 14 days after MMC-Meth A injection. A shorter interval between the injections of LC 9018 and MMC-Meth A did not allow suppression of Meth A growth. These results showed that the increase in Ia-positive macrophages in the peritoneal cavity coincided with the effective interval for induction of the antitumor activity by LC 9018. The antitumor activity induced by injections of LC 9018 and MMC-Meth A did not affect the growth of RL l leukemic cells, syngeneic to BALB/c mice. Neutralization (Winn type) tests showed that peritoneal T lymphocytes possessed tumor cytotoxicity and that the antitumor capacity was reduced by in vivo treatment with anti I-A^d monoclonal antibody simultaneously with and 1 day prior to MMC-Meth A injection. These results indicate that LC 9018-induced Ia-positive macrophages, which first encounter a tumor antigen in the peritoneal cavity, play an important role in the in vivo induction of tumor specific T cell-mediated antitumor immunity.     

Prevention of Pathogenic Escherichia coli Infection in Mice and Stimulation of Macrophage Activation in Rats by an Oral Administration of Probiotic Lactobacillus casei I-5

Lactobacillus casei I-5 isolated from an alcohol fermentation broth enhanced immunity and prevented pathogenic infection as a probiotic. Mice fed with I-5 cells for 11 days prior to an intraperitoneal challenge with pathogenic Escherichia coli Juhl exhibited a high survival rate compared with the control group. Rats fed with I-5 cells for 10 days significantly increased the phagocytosis of peritoneal macrophages. In a cell culture system employing peritoneal macrophages from rats, the I-5 administration activated NF-κB stimulated by LPS. It also enhanced LPS-stimulated IL-12 and TNF-α production, but not IL-6 production. These results show that L. casei I-5 effectively prevented infection by pathogenic E. coli possibly through the activation of peritoneal macrophages. The strain would be useful to prevent pathogenic microbial infections in humans and farm animals.     

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.     

Suppressive Effect on Activation of Macrophages by Lactobacillus casei Strain Shirota Genes Determining the Synthesis of Cell Wall-Associated Polysaccharides

Although many Lactobacillus strains used as probiotics are believed to modulate host immune responses, the molecular natures of the components of such probiotic microorganisms directly involved in immune modulation process are largely unknown. We aimed to assess the function of polysaccharide moiety of the cell wall of Lactobacillus casei strain Shirota as a possible immune modulator which regulates cytokine production by macrophages. A gene survey of the genome sequence of L. casei Shirota hunted down a unique cluster of 10 genes, most of whose predicted amino acid sequences had similarities to various extents to known proteins involved in biosynthesis of extracellular or capsular polysaccharides from other lactic acid bacteria. Gene knockout mutants of eight genes from this cluster resulted in the loss of reactivity to L. casei Shirota-specific monoclonal antibody and extreme reduction of high-molecular-mass polysaccharides in the cell wall fraction, indicating that at least these genes are involved in biosynthesis of high-molecular-mass cell wall polysaccharides. By adding heat-killed mutant cells to mouse macrophage cell lines or to mouse spleen cells, the production of tumor necrosis factor alpha, interleukin-12 (IL-12), IL-10, and IL-6 was more stimulated than by wild-type cells. In addition, these mutants additively enhanced lipopolysaccharide-induced IL-6 production by RAW 264.7 mouse macrophage-like cells, while wild-type cells significantly suppressed the IL-6 production of RAW 264.7. Collectively, these results indicate that this cluster of genes of L. casei Shirota, which have been named cps1A, cps1B, cps1C, cps1D, cps1E, cps1F, cps1G, and cps1J, determine the synthesis of the high-molecular-mass polysaccharide moiety of the L. casei Shirota cell wall and that this polysaccharide moiety is the relevant immune modulator which may function to reduce excessive immune reactions during the activation of macrophages by L. casei Shirota.     

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.     

Cytotoxic factor production by kupffer cells elicited with Lactobacillus casei and Corynebacterium parvum

Summary The ability of Kupffer cells, spleen macrophages, pulmonary macrophages, and peritoneal macrophages (PM) to produce cytotoxic factor (CTF) was investigated in vitro. The production of CTF by Kupffer cells elicited with Corynebacterium parvum (CP) or Lactobacillus casei YIT9018 (LC9018) was higher than that of spleen, pulmonary macrophages, or PM. In addition, oxygen radical (OR) production by Kupffer cells or PM was measured. The production of OR by Kupffer cells or PM was significantly augmented by i.v. or i.p. injection of LC9018 or CP. No significant correlation was observed between the increase in OR production by Kupffer cells or PM and CTF production by Kupffer cells or PM elicited with either organism. It was suggested that activated Kupffer cells may be one important source of CTF production in serum and that the CTF-producing macrophages may be different from the OR-producing macrophages.     

Suppressive effects of Lactobacillus casei cells,a bacterial immunostimulant,on the incidence of spontaneous thymic lymphoma in AKR mice

 The mean survival age of female AKR/J mice was significantly prolonged, the enlargement of thymus was markedly suppressed, and the proliferation of ecotropic and recombinant murine leukemia viruses (MuLV) was markedly inhibited when 8-week-old female AKR/J mice were injected intraperitoneally (i. p.) with heat-killed Lactobacillus casei cells twice weekly for 8 weeks. In contrast, such actions of heat-killed L. casei cells were not seen in 20-week-old female AKR/J mice. The leukemogenic activity of the cell-free extract of thymus from adult female AKR/J mice in newborn female AKR/J mice was drastically reduced by i. p. treatment with heat-killed L. casei cells. The difference in adjuvant effectiveness of heat-killed L. casei cells on 8- and 20-week-old animals may be dependent on the difference in the enhancing activity of the cell-mediated immune systems between the groups induced by heat-killed L. casei cells, and, as a result, on the difference in the degree of proliferation of ecotropic and recombinant MuLV in thymus, which consequently causes thymic lymphoma. Received: 13 February 1996 / Accepted: 18 April 1996     

Protoplast fusion between Lactobacillus casei and Lactobacillus acidophilus

Summary From the fusion between Lactobacillus casei and Lactobacillus acidophilus, 8 fusants were selected: Four were able to ferment maltose, lactose, galactose and mannose, but two had greater abilities of acid production than parents. Increased values of up to 7.6–8 % in -galactosidase activity were obtained from two when compared to that of L. acidophilus, whereas another 2 had activities of 800 and 548 nmol/mg protein/min comparable to that of L casei giving a value of 400 nmol/mg protein/min in phospho--galactosidase activity.     

Flocculation of Chlorella vulgaris by Lactobacillus casei

Summary The flocculation of Chlorella vulgaris by Lactobacillus casei was studied to determine whether the latter could act as a suitable flocculant for the removal of Chlorella from algal ponds. The flocculating activity of the Lactobacilli was caused by the bacterial cells themselves, and not by diffusible products of bacterial metabolism. Diffusible products of algal metabolism inhibited flocculation. For algae resuspended in water, the best flocculation occurred at pH values less than 3.5 where the charges on the bacterial and algal cells were opposite. For flocculation at least one bacterium was required for every algal cell; in terms of cell concentrations,10 mg/l of bacteria were required to flocculate an algal suspension of 1,000 mg/l. The mechanism of flocculation implied by the results is that positively charged cells of L. casei adsorb to the surface of negatively charged cells of C. vulgaris neutralizing the charge and thus destabilizing the algal suspension. Because of the low pH required and because diffusible products of algal metabolism inhibit the flocculation, it is unlikely that L. casei could be usefully employed as a flocculant of Chlorella from algal ponds.     

Stimulation of macrophages by immunobiotic Lactobacillus strains: influence beyond the intestinal tract

Lactobacillus rhamnosus CRL1505 (Lr1505), L. rhamnosus CRL1506 (Lr1506) and L. casei CRL431 (Lc431) are able to stimulate intestinal immunity, but only Lr1505 and Lc431 are able to stimulate immunity in the respiratory tract. With the aim of advancing the understanding of the immunological mechanisms involved in stimulation of distant mucosal sites, this study evaluated the effects of orally administered probiotics on the functions of alveolar and peritoneal macrophages. Compared to a control group, these three lactobacilli were able to significantly increase phagocytic and microbicidal activities of peritoneal macrophages. After intraperitoneal challenge with pathogenic Candida albicans, mice treated with immunobiotics had significantly lower pathogen counts in infected organs. Moreover, lactobacilli‐treated mice had a stronger immune response against C. albicans. On the other hand, only Lc1505 and Lc431 were able to improve activity of and cytokine production by alveolar macrophages. Only in these two groups was there better resistance to respiratory challenge with C. albicans, which correlated with improved respiratory immune response. The results of this study suggest that consumption of some probiotic strains could be useful for improving resistance to infections in sites distant from the gut by increasing the activity of macrophages at those sites.     

Expression of the Lactobacillus casei lactate dehydrogenase gene in Bacillus subtilis

Summary A gene for allosteric lactate dehydrogenase (LDH) of Lactobacillus casei ATCC393 was transferred into Bacillus subtilis. The LDH was produced in a growth-associated type, and comprised up to 40 % of the total cellular protein. The maximum specific activity in the transformant was 208 U/mg protein which was approximately 16 times higher than in L. casei or in the previously constructed Escherichia coli transformant.     

Augmentation of Mouse Natural Killer Cell Activity by Lactobacillus casei and Its Surface Antigens

Lactobacillus casei YIT 9018 (LC 9018) augmented the natural killer (NK) cell activity of spleen cells from inbred BALB/c mice injected intravenously with LC 9018 or intraperitoneally with polyinosinate-polycytidylate. Augmentation of this activity by LC 9018 was also observed in male C3H/He, CBA/N, and C57BL/6 mice. The spleen cells exhibited no cytolytic activity against P815, a cell line insensitive to NK cells. The cytolytic activity of the spleen cells increased 2 days after the injection of 250 μg of LC 9018/mouse, peaked on day 3, and gradually declined thereafter. The increase caused by LC 9018 was also observed in normal and Meth A-bearing mice. In vitro treatment with anti-asialo GM1 antibody plus complement completely-abrogated the LC 9018-augmented murine NK cell activity. The NK activity on the 3rd day after LC 9018 injection was reduced by in vitro treatment with anti-Thy 1.2 monoclonal antibody plus complement to half of that observed when treatment was with complement alone. This suggests that there were two populations of NK cells in the spleen cell suspension derived from LC 9018-treated mice. One population was asialo GM1-positive and Thy 1-negative, the other was asialo GM1-positive and Thy 1-positive.     

Phage Receptor Material in Lactobacillus casei Cell Wall

A trichloroacetic acid (TCA)-soluble fraction, extracted using cold TCA, was derived from the cell wall of Lactobacillus casei strain S-1. It not only inhibited the adsorption of phage J-l but also desorbed these phages, in their active form, which had previously been adsorbed onto the cell walls. l-Rhamnose, one of the components of this TCA-soluble fraction, had an identical activity to this TCA-soluble fraction, on phage adsorption. This suggested that l-rhamnose is a part of phage receptor material in the cell wall of L. casei strain S-l; and the binding of the phage to the cell wall is reversible, even at 37 C.     

The Preventive Effects of Lactobacillus casei on Acute Lung Injury Induced by Lipopolysaccharide

Lactobacillus has been reported to inhibit acute lung injury (ALI). However, the molecular mechanism of Lactobacillus casei (L. casei) in preventing ALI has not been identified, so we investigated whether L. casei pretreatment could inhibit the activation of TLR4/MyD88/NF-κB signaling pathway following ALI. ALI model was established by intraperitoneal injection of 2 mg/kg lipopolysaccharide (LPS) to female BALB/c mice. In L. casei LC2W group, mice were intragastrically administrated L. casei LC2W for a week, before the ALI modeling. The serum of normal BALB/c mice after intragastric administration of L. casei LC2W was used for in vitro cell assays. The serum was pre-incubated with mouse macrophage cell line (RAW264.7) and human lung cell line (HLF-A), then LPS was added to co-incubate. Compared with ALI model group, L. casei LC2W pretreatment significantly reduced lung pathological damage, the number of neutrophils and total cells in bronchoalveolar lavage fluid. Besides, L. casei LC2W pretreatment could significantly reverse the abnormal expression of ICAM-1, IL-6, TNF-α and IL-10 in lung tissue and serum, plus, L. casei LC2W significantly reduced the phosphorylation levels of IRAK-1 and NF-κB p65. In vitro, the serum decreased the up-regulation of IL-6 and TNF-α in cell lines induced by LPS. In conclusion, L. casei LC2W intragastric administration pretreatment could significantly improve LPS-induced ALI in mice, probably through circulation to reach the lungs so as to inhibit the inflammatory response induced by activation of TLR4/MyD88/NF-κB signaling pathway.     

Augmentation of resistance of mice to bacterial infection by a polysaccharide-peptidoglycan complex (PSPG) extracted fromLactobacillus casei

A water-soluble polysaccharide-peptidoglycan complex (PSPG) was prepared from heat-killedLactobacillus casei by digesting the bacteria with N-acetylmuramidase. The molecular weight of PSPG was over 30,000, and the polysaccharide portion of PSPG, its main component was composed of rhamnose, glucose, galactose, glucosamine and galactosamine. Mice pretreated intraperitoneally with PSPG survived after a lethal infection withListeria monocytogenes orPseudomonas aeruginosa. The growth of infecting bacteria (L. monocytogenes, P. aeruginosa, Salmonella typhimurium, Escherichia coli) in both the peritoneal cavity and the liver was inhibited markedly in the mice that had been treated with PSPG. It was suggested that macrophages may be the main effector for the anti-infectious effect of PSPG since treatment of mice with carrageenan, a selective macrophage blocker, markedly reduced the anti-infectious effect of PSPG.     

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.     

Genomic Adaptation of the Lactobacillus casei Group

Lactobacillus casei, L. paracasei, and L. rhamnosus form a closely related taxonomic group (Lactobacillus casei group) within the facultatively heterofermentative lactobacilli. Here, we report the complete genome sequences of L. paracasei JCM 8130 and L. casei ATCC 393, and the draft genome sequence of L. paracasei COM0101, all of which were isolated from daily products. Furthermore, we re-annotated the genome of L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG), which we have previously reported. We confirmed that ATCC 393 is distinct from other strains previously described as L. paracasei. The core genome of 10 completely sequenced strains of the L. casei group comprised 1,682 protein-coding genes. Although extensive genome-wide synteny was found among the L. casei group, the genomes of ATCC 53103, JCM 8130, and ATCC 393 contained genomic islands compared with L. paracasei ATCC 334. Several genomic islands, including carbohydrate utilization gene clusters, were found at the same loci in the chromosomes of the L. casei group. The spaCBA pilus gene cluster, which was first identified in GG, was also found in other strains of the L. casei group, but several L. paracasei strains including COM0101 contained truncated spaC gene. ATCC 53103 encoded a higher number of proteins involved in carbohydrate utilization compared with intestinal lactobacilli, and extracellular adhesion proteins, several of which are absent in other strains of the L. casei group. In addition to previously fully sequenced L. rhamnosus and L. paracasei strains, the complete genome sequences of L. casei will provide valuable insights into the evolution of the L. casei group.     

Human papillomavirus type 16 E6-specific antitumor immunity is induced by oral administration of HPV16 E6-expressing Lactobacillus casei in C57BL/6 mice

Given that local cell-mediated immunity (CMI) against the human papillomavirus type 16 E6 (HPV16 E6) protein is important for eradication of HPV16 E6-expressing cancer cells in the cervical mucosa, the HPV16 E6 protein may be a target for the mucosal immunotherapy of cervical cancer. Here, we expressed the HPV16 E6 antigen on Lactobacillus casei (L. casei) and investigated E6-specific CMI following oral administration of the L. casei-PgsA-E6 to mice. Surface expression of HPV16 E6 antigens was confirmed and mice were orally inoculated with the L. casei-PgsA or the L. casei-PgsA-E6. Compared to the L. casei-PgsA-treated mice, significantly higher levels of serum IgG and mucosal IgA were observed in L. casei-PgsA-E6-immunized mice; these differences were significantly enhanced after boost. Consistent with this, systemic and local CMI were significantly increased after the boost, as shown by increased counts of IFN-γ-secreting cells in splenocytes, mesenteric lymph nodes (MLN), and vaginal samples. Furthermore, in the TC-1 tumor model, animals receiving the orally administered L. casei-PgsA-E6 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei-PgsA) immunization. We also found that L. casei-PgsA-E6-induced antitumor effect was decreased by in vivo depletion of CD4⁺ or CD8⁺ T cells. Collectively, these results indicate that the oral administration of lactobacilli bearing the surface-displayed E6 protein induces T cell-mediated cellular immunity and antitumor effects in mice.     

Production of a Heterologous Nonheme Catalase by Lactobacillus casei: an Efficient Tool for Removal of H2O2 and Protection of Lactobacillus bulgaricus from Oxidative Stress in Milk

Lactic acid bacteria (LAB) are generally sensitive to H₂O₂, a compound that they can paradoxically produce themselves, as is the case for Lactobacillus bulgaricus. Lactobacillus plantarum ATCC 14431 is one of the very few LAB strains able to degrade H₂O₂ through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species: L. bulgaricus ATCC 11842, Lactobacillus casei BL23, and Lactococcus lactis MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in L. casei. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in L. plantarum and L. casei but at least 10- and 100-fold lower in Lactococcus lactis and L. bulgaricus, respectively. The expression of the MnKat gene in L. casei conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H₂O₂, and improved long-term survival in aerated cultures. In mixtures of L. casei producing MnKat and L. bulgaricus, L. casei can eliminate H₂O₂ from the culture medium, thereby protecting both L. casei and L. bulgaricus from its deleterious effects.     

Lactobacillus casei Is Able To Survive and Initiate Protein Synthesis during Its Transit in the Digestive Tract of Human Flora-Associated Mice

Live Lactobacillus casei is present in fermented dairy products and has beneficial properties for human health. In the human digestive tract, the resident flora generally prevents the establishment of ingested lactic acid bacteria, the presence of which is therefore transient. The aim of this work was to determine if L. casei DN-114 001 survives during transit and how this bacterium behaves in the digestive environment. We used the human flora-associated (HFA) mouse model. L. casei DN-114 001 was genetically modified by the introduction of erm and lux genes, encoding erythromycin resistance and luciferase, respectively. For this modified strain (DN-240 041), light emission related to luciferase expression could easily be detected in the contents of the digestive tract. When inoculated into the digestive tract of HFA mice, L. casei (DN-240 041) survives but is eliminated with the same kinetics as an inert transit marker, indicating that it does not establish itself. In pure culture of L. casei, luciferase activities were high in the exponential and early stationary growth phases but decreased to become undetectable 1 day after inoculation. Viability was only slightly reduced even after more than 5 days. After transit in HFA mice, luciferase activity was detected even when 5-day-old L. casei cultures were given to the mice. In culture, the luciferase activity could be restored after 0.5 to 7 h of incubation in fresh medium or milk containing glucose, unless protein synthesis was inhibited by the addition of chloramphenicol or rifampin. These results suggest that in HFA mice L. casei DN-240 041, and thus probably L. casei DN-114 001, is able to initiate new protein synthesis during its transit with the diet. The beneficial properties of L. casei-fermented milk for human health might be related to this protein synthesis in the digestive tract.