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.     

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

Recently, we reported that heat-killed Lactobacillus 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.     

An evaluation of the effect of 2,4-dichlorophenoxyacetic acid derivatives on humoral and cellular immunities

A single oral administration of phenoxy herbicides, viz. 2,4-dichlorophenoxyacetic acid (2,4-D), amino salt, sodium salt, diethylene glycol, iso-octyl and octyl ethers in a dose of LD16, was found to produce a pronounced suppressive effect on humoral immunity on day 5. The 2,4-D derivatives under study, administered in the same dose, stimulated the manifestations of delayed-type hypersensitivity in mice immunized with sheep red blood cells. The effect of the inhibition of antibody formation to thymus-dependent antigen ranged in the order of the increase in the toxicity of substances: amino salt, diethylene glycol ether, 2,4-D, sodium salt, iso-octyl and octyl ethers. On the contrary, the intensity of delayed-type hypersensitivity was more pronounced with regard to substances having greater toxicity.     

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.     

Anti-tumour activity of Lactobacillus casei on lewis lung carcinoma and line-10 hepatoma in syngeneic mice and guinea pigs

Summary The anti-tumour activity of Lactobacillus casei YIT 9018 (LC 9018) on Lewis lung carcinoma (3LL) in C57BL/6 mice and line-10 hepatoma in strain-2 guinea pigs was examined. Intravenous injection of LC 9018 was effective for inhibition of pulmonary metastases in C57BL/6 mice after s.c. inoculation with 3LL tumours. Intralesional (i.l.) injection of LC 9018 was also effective for both prolongation of the survival period and inhibition of pulmonary metastases in 3LL tumour-bearing mice. The combination treatment of i.l. and i.v. injections of LC 9018 before or after surgical excision of the primary tumour remarkably inhibited the pulmonary metastases after inoculation with 3LL tumour. Intralesional injection of LC 9018 was effective for regression of the established tumours of line-10 hepatoma inoculated i.d. and for induction of systemic tumour immunity in strain-2 guinea pigs.     

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 immune interferon in mice treated with a bacterial immunopotentiator,OK-432

Summary When a bacterial immunopotentiator, OK-432, was injected to intact DDI mice, a viral inhibitor with the properties of immune interferon (IF) was induced in the circulation. The maximum titer of antiviral activity (10,240 units/ml) was observed 24 h after intraperitoneal (IP) injection of 50 KE OK-432/kg body weight. The possibility that the induction of immune IF may depend upon the action of thymus-derived (T) lymphocytes and macrophages was inferred from experiments with thymus-defective nude mice and DDI mice treated with either X-rays or immunosuppressive agents.     

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     

Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA.

The chromosomal ccpA gene from Lactobacillus casei ATCC 393 has been cloned and sequenced. It encodes the CcpA protein, a central catabolite regulator belonging to the LacI-GalR family of bacterial repressors, and shows 54% identity with CcpA proteins from Bacillus subtilis and Bacillus megaterium. The L. casei ccpA gene was able to complement a B. subtilis ccpA mutant. An L. casei ccpA mutant showed increased doubling times and a relief of the catabolite repression of some enzymatic activities, such as N-acetylglucosaminidase and phospho-beta-galactosidase. Detailed analysis of CcpA activity was performed by using the promoter region of the L. casei chromosomal lacTEGF operon which is subject to catabolite repression and contains a catabolite responsive element (cre) consensus sequence. Deletion of this cre site or the presence of the ccpA mutation abolished the catabolite repression of a lacp::gusA fusion. These data support the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria.     

Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction

Lactobacillus casei, Lact. paracasei and Lact. rhamnosus form a closely related taxonomic group within the heterofermentative lactobacilli. These three species are difficult to differentiate using traditional fermentation profiles. We have developed polymerase chain reaction primers which are specific for each of these species based on differences in the V1 region of the 16S rRNA gene. Sixty-three Lactobacillus isolates from cheese were identified using these primers. The 12 Lact. rhamnosus and 51 Lact. paracasei identified in this way were also differentiated using a randomly amplified polymorphic DNA (RAPD) primer.     

Radioprotection of mice by a single subcutaneous injection of heat-killed Lactobacillus casei after irradiation

Treatment of whole-body gamma-irradiated mice with a preparation of Lactobacillus casei (LC 9018) immediately after irradiation caused a sustained increase in serum colony-stimulating activity which was followed by an enhanced repopulation of granulocyte-macrophage colony-forming cells in the femoral marrow and spleen. Numbers of blood leukocytes, erythrocytes, and platelets were increased earlier in the treated mice than in the controls, and the survival rate was elevated significantly. The radioprotective effect was dependent on the dose of LC 9018 as well as on the dose of radiation. These results demonstrate the value of LC 9018 for the treatment of myelosuppression after radiotherapy or radiation accidents.     

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.     

Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus zeae isolates identified by sequence signature and immunoblot phenotype

Species taxonomy within the Lactobacillus casei group of bacteria has been unsettled. With the goal of helping clarify the taxonomy of these bacteria, we investigated the first 3 variable regions of the 16S rRNA gene, the 16S-23S rRNA interspacer region, and one third of the chaperonin 60 gene for Lactobacillus isolates originally designated as L. casei, L. paracasei, L. rhamnosus, and L. zeae. For each genetic region, a phylogenetic tree was created and signature sequence analysis was done. As well, phenotypic analysis of the various strains was performed by immunoblotting. Both sequence signature analysis and immunoblotting gave immediate identification of L. casei, L. rhamnosus, and L. zeae isolates. These results corroborate and extend previous findings concerning these lactobacilli; therefore, we strongly endorse recent proposals for revised nomenclature. Specifically, isolate ATCC 393 is appropriately rejected as the L. casei type strain because of grouping with isolates identified as L. zeae. As well, because all other L. casei isolates, including the proposed neotype isolate ATCC 334, grouped together with isolates designated L. paracasei, we support the use of the single species L. casei and rejection of the name L. paracasei.     

Lactobacillus casei administration reduces lung injuries in a Streptococcus pneumoniae infection in mice

The effect of the oral administration of Lactobacillus casei on the prevention of a Streptococcus pneumoniae lung infection in a mouse experimental model was studied, analyzing the innate and specific immune response. Adult Swiss albino mice were treated with L. casei (10(9)CFU/day) for 2, 5 and 7 d. Mice were infected intranasally with S. pneumoniae (10(6)CFU/mouse) after each treatment and the microbiological, histopathological and host responses were determined for 15 d after infection. Feeding L. casei for 2 d induced a faster clearance of S. pneumoniae, with a lower number of pneumococci in lung and a shorter period of septicemia than in the control group. L. casei administration induced activation of phagocytes as evidenced by the strong myeloperoxidase activity and the nitro blue tetrazolium assay in lung. Mice given L. casei for 2 d showed higher levels of anti-pneumococcal serum IgG and bronchoalveolar lavage IgA than the control mice. The group fed L. casei for 2 d could beneficially regulate the balance between tumor necrosis factor alpha and interleukin 10, allowing a more effective immune response against infection and modulating the inflammatory response, with less damage to the lung.     

Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains.

Hydrophilic and electrostatic cell surface properties of eight Lactobacillus strains were characterized by using the microbial adhesion to solvents method and microelectrophoresis, respectively. All strains appeared relatively hydrophilic. The strong microbial adhesion to chloroform, an acidic solvent, in comparison with microbial adhesion to hexadecane, an apolar n-alkane, demonstrated the particularity of lactobacilli to have an important electron donor and basic character and consequently their potential ability to generate Lewis acid-base interactions with a support. Regardless of their electrophoretic mobility (EM), strains were in general slightly negatively charged at alkaline pH. A pH-dependent behavior concerning cell surface charges was observed. The EM decreased progressively with more acidic pHs for the L. casei subsp. casei and L. paracasei subsp. paracasei strains until the isoelectric point (IEP), i.e., the pH value for which the EM is zero. On the other hand, the EM for the L. rhamnosus strains was stable from pH 8 to pH 3 to 4, at which point there was a shift near the IEP. Both L. casei subsp. casei and L. paracasei subsp. paracasei strains were characterized by an IEP of around 4, whereas L. rhamnosus strains possessed a markedly lower IEP of 2. The present study showed that the cell surface physicochemical properties of lactobacilli seem to be, at least in part and under certain experimental conditions, particular to the bacterial species. Such differences detected between species are likely to be accompanied by some particular changes in cell wall chemical composition.     

Release of aminopeptidase from Lactobacillus casei sp. casei by cell disruption in a Microfluidizer

Cell disruption in a Microfluidizer was a function of both operating pressure and number of passes. The operating pressure had greater effect on the disruption than did the number of passes as indicated by the magnitude of the constants from the cell disruption equation. Protein release correlated with aminopeptidase release by Lactobacillus casei sp. casei. The optimum operating pressure for enzyme extraction was 76 MPa with loss of enzyme activity about 15 to 20%.