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.     

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.     

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.     

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.     

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 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.     

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     

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.     

Effects of Lactobacillus casei on Pseudomonas aeruginosa infection in normal and dexamethasone-treated mice

A single intraperitoneal injection of Lactobacillus casei YIT 0003 into normal or dexamethasone-treated mice led to nonspecific resistance against intraperitoneal challenge with lethal doses of Pseudomonas aeruginosa PAO 3047. The enhanced resistance was retained for 14 days (P less than 0.05) after injection with living L. casei. In contrast, the statistically significant duration of the enhanced resistance in mice treated intraperitoneally with living L. acidophilus YIT 0075 was only 5 days. The in vivo killing activity of peritoneal exudate cells (PECs) against P. aeruginosa 5 and 7 days after intraperitoneal injection of living L. casei was significantly higher than in the case of PECs elicited by L. acidophilus. In the case of intravenous injection of heat-killed L. casei before intraperitoneal challenge with P. aeruginosa, there were no survivors in the late period after administration of L. casei. A high correlation existed between the patterns of in vivo killing of P. aeruginosa by PECs and survival rate of mice injected intravenously with heat-killed L. casei. The reduced in vivo killing activity of PECs from dexamethasone-treated mice against P. aeruginosa infection was also augmented by the intraperitoneal injection of heat-killed L. casei. These results indicate that L. casei possesses a resistance-enhancing capacity against P. aeruginosa infection in vivo. Differences in the duration of the enhanced resistance caused by L. casei and by L. acidophilus may be due to differences in chemical composition and/or physicochemical properties of the cell walls of the two kinds of bacteria.     

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.     

Protective effect of Lactobacillus casei strain Shirota against lethal infection with multi-drug resistant Salmonella enterica serovar Typhimurium DT104 in mice

Aims: The anti‐infectious activity of lactobacilli against multi‐drug resistant Salmonella enterica serovar Typhimurium DT104 (DT104) was examined in a murine model of an opportunistic antibiotic‐induced infection. Methods and Results: Explosive intestinal growth and subsequent lethal extra‐intestinal translocation after oral infection with DT104 during fosfomycin (FOM) administration was significantly inhibited by continuous oral administration of Lactobacillus casei strain Shirota (LcS), which is naturally resistant to FOM, at a dose of 10⁸ colony‐forming units per mouse daily to mice. Comparison of the anti‐Salmonella activity of several Lactobacillus type strains with natural resistance to FOM revealed that Lactobacillus brevis ATCC 14869^T, Lactobacillus plantarum ATCC 14917^T, Lactobacillus reuteri JCM 1112^T, Lactobacillus rhamnosus ATCC 7469^T and Lactobacillus salivarius ATCC 11741^T conferred no activity even when they obtained the high population levels almost similar to those of the effective strains such as LcS, Lact. casei ATCC 334^T and Lactobacillus zeae ATCC 15820^T. The increase in concentration of organic acids and maintenance of the lower pH in the intestine because of Lactobacillus colonization were correlated with the anti‐infectious activity. Moreover, heat‐killed LcS was not protective against the infection, suggesting that the metabolic activity of lactobacilli is important for the anti‐infectious activity. Conclusion: These results suggest that certain lactobacilli in combination with antibiotics may be useful for prophylaxis against opportunistic intestinal infections by multi‐drug resistant pathogens, such as DT104. Significance and Impact of the Study: Antibiotics such as FOM disrupt the metabolic activity of the intestinal microbiota that produce organic acids, and that only probiotic strains that are metabolically active in vivo should be selected to prevent intestinal infection when used clinically in combination with certain antibiotics.     

In vitro antiviral activity of fullerene amino acid derivatives in cytomegalovirus infection]

The in vitro effect of 5 water soluble fullerene C60 amino acid derivatives (FAD) on the development of cytomegalovirus infection was studied in the schemes of the therapeutic, prophylactic and virucidal action. The following compounds as FAD were used: fullerene conjugated with Na salt of gamma-aminobutyric acid (C60-ABA-Na), 2 derivatives based on Na salts of fullerene-gamma-aminobutyric acid and fullerene-omega-caproic acid (C60-ABA-OH-Na and C60-ACA-OH-Na respectively) and 2 derivatives based on methyl ethers of the above mentioned fullerene amino acids (C60-ABA-OH-CH3 and C60-ACA-OH-CH3). All the FAD were able to inhibit the development of the virus cytopathogenic action in the cell culture. However, the compounds had different antiviral properties. C60-ABA-OH-Na, C60-ABA-CH3 and C60-ACA-CH3 showed marked antiviral activity in the prophylactic scheme. 50-Percent inhibition of the virus cytopathogenic action (ID50) was observed when concentrations of the compounds were 0.31, 5 and 25 mcg/ml respectively. C60-ACA-OH-Na inhibited the development of cytomegalovirus infection in the cell culture only in the scheme of the therapeutic action (ID50 4 mcg/ml). C60-ABA-Na had the highest antiviral effect. In a concentration of 0.22 mcg/ml it inhibited the cytomegalovirus plague-forming capacity by 50% in both the prophylactic and the virucidal schemes. The chemotherapeutic index of the compound was within the limits of 2500 to 5450.     

Enhanced immune response to pneumococcal infection in malnourished mice nasally treated with heat-killed Lactobacillus casei

The present study analyzed whether nasal administration of viable and non-viable Lactobacillus casei CRL 431 to immunocompromised mice was capable of increasing resistance against Streptococcus pneumoniae. Weaned mice were malnourished after consuming a PFD for 21 days. Malnourished mice were fed a BCD for 7 days or BCD for 7 days with viable or non-viable L. casei nasal treatments on day 6 and day 7 (BCD+LcV and BCD+LcN, respectively). The MNC group received PFD whereas the WNC mice consumed BCD. MNC mice showed greater lung colonization, more severe lung injuries, impaired leukocyte recruitment and reduced antibodies and cytokine production when compared with WNC mice. Administration of L. casei increased the resistance of malnourished mice to the infection. Both BCD+LcV and BCD+LcN treatments prevented the dissemination of the pathogen to the blood and induced its lung clearance. BCD+LcV or BCD+LcN groups showed improved production of TNF-α and activity of phagocytes in the respiratory tract, an effect that was not observed in the BCD control group. In addition, IL-4 and IL-10 were significantly increased in BCD+LcV and BCD+LcN groups, which correlated with the increase in the levels of specific respiratory IgA. The nasal treatments with L. casei were also effective at stimulating the production of specific IgG at both the systemic and the respiratory levels. The comparative study between the viable and the non-viable bacteria demonstrated that viability would be an important factor to achieve maximum protective effects. However, the results from this study suggest that heat-killed lactic acid bacteria are also effective in the immunomodulation of the systemic and respiratory immune system.     

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.     

Thymidine kinase not required for antiviral activity of acyclovir against mouse cytomegalovirus.

Previous studies of herpesvirus infections have indicated that a virus-specified thymidine kinase is required for the initial phosphorylation of acyclovir [acycloguanosine or 9-(2-hydroxyethoxymethyl)guanine] in the formation of acycloguanosine triphosphate. The latter compound accumulates in infected cells and competitively inhibits the viral DNA polymerase. We found that mouse cytomegalovirus, which does not express a thymidine kinase, was sensitive to the antiviral effects of acyclovir at a 50% inhibitory dose of approximately 0.23 microM. Acyclovir was equally effective against mouse cytomegalovirus in normal 3T3 cells and in 3T3 cells deficient in cellular thymidine kinase. Furthermore, the activity of acyclovir could not be reversed by excess thymidine, which easily reversed the antiviral activity of acyclovir against herpes simplex virus. Using a high-pressure liquid chromatography technique that easily detected acycloguanosine triphosphate in cells infected with herpes simplex virus, we could not detect acycloguanosine triphosphate in mouse cytomegalovirus-infected cells. These experiments demonstrated that the activity of acyclovir against mouse cytomegalovirus is not dependent on a thymidine phosphorylation pathway. Additional experiments are underway to determine whether acycloguanosine triphosphate is produced by another pathway in concentrations sufficient to inhibit mouse cytomegalovirus DNA polymerase.     

Effect of acute rat cytomegalovirus infection on the antiviral activity of peritoneal macrophages

The effect of rat cytomegalovirus (RCMV) infection on the extrinsic antiviral activity of peritoneal rat macrophages was investigated. The presence of a non-interferon-like antiviral substance in the supernatant of the culture of peritoneal macrophages of normal rats was shown. This antiviral activity was altered during an RCMV infection in vitro resulting in the absence of activity in the supernatant of macrophages harvested at 4 days post RCMV infection. The antiviral activity was also present in vivo, namely in the peritoneal cavity of normal rats. RCMV infection altered this antiviral effect resulting in an enhancement at days 2 to 5 post infection.     

Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection.

The aim of this study was to compare the antagonistic properties of Lactobacillus casei GG exerted in vitro against Salmonella typhimurium C5 in a cellular model, cultured enterocyte-like Caco-2 cells, to those exerted in vivo in an animal model, C3H/He/Oujco mice. Our results show that a 1-h contact between the invading strain C5 and either the culture or the supernatant of L. casei GG impeded the invasion by the Salmonella strain in Caco-2 cells, without modifying the viability of the strain. After neutralization at pH 7, no inhibition of the invasion by C5 was observed. The antagonistic activity of L. casei GG was examined in C3H/He/Oujco mice orally infected with C5 as follows: (i) L. casei GG was given daily to conventional animals as a probiotic, and (ii) it was given once to germ-free animals in order to study the effect of the population of L. casei GG established in the different segments of the gut. In vivo experiments show that after a single challenge with C5, this strain survives and persists at a higher level in the feces of the untreated conventional mice than in those of the treated group. In L. casei GG germ-free mice, establishment of L. casei GG in the gut significantly delayed the occurrence of 100% mortality of the animals (15 days after C5 challenge versus 9 days in germ-free mice [P < 0.01]). Cecal colonization level and translocation rate of C5 to the mesenteric lymph nodes, spleen, and liver were significantly reduced during the first 2 days post-C5 challenge, although the L. casei GG population level in the gut dramatically decreased in these animals.