Role of macrophages in serum colony-stimulating factor induction by Lactobacillus casei in mice.

Heat-killed Lactobacillus casei YIT9018 (LC9018), when injected intravenously into mice at a dose of 4 to 40 mg/kg, induced the production of serum colony-stimulating factor (CSF). Since this induction was observed in both C3H/HeJ and C3H/HeN mice, LC9018 was considered to act differently from lipopolysaccharide. The amount of serum CSF induced by LC9018 in nude mice and whole-body-X-ray-irradiated mice was similar to that in control mice, but the induction of serum CSF was suppressed by the previous administration of carrageenan, indicating that macrophages, but not T cells, were responsible for serum CSF induction by LC9018. To determine whether macrophages themselves produce CSF or help other cells produce CSF in response to LC9018, we prepared adherent cells from the peritoneal cavity of normal mice and examined CSF activity in their conditioned media. Peritoneal adherent cells did not produce CSF without LC9018, but when cultivated with 1 mg of LC9018 per ml, they produced CSF at the same time that serum CSF was induced after the intravenous administration of LC9018. Additionally, in vitro-induced CSF formed macrophage, granulocyte, and mixed colonies, as serum CSF did. CSF production by peritoneal adherent cells was completely inhibited by cycloheximide (50 micrograms/ml), and neither the elimination of T cells from the peritoneal adherent cells by treating them with anti-Thy-1.2 antibody plus complement nor the addition of T cells affected CSF production. These results suggest that heat-killed LC9018 induces serum CSF in mice via direct stimulation of macrophages to produce CSF de novo.     

Effects on antitumor activity and cytokine production in the thoracic cavity by intrapleural administration of Lactobacillus casei in tumor-bearing mice

The effects Lactobacillus casei YIT9108 (LC 9018) on antitumor activity and cytokine production in Meth A fibrosarcoma (Meth A)-bearing BALB/c mice were examined. Intrapleural (i.pl.) administration of LC 9018 was effective in prolonging the survival of Meth A-bearing mice, and frequently cured mice of the tumor. However, the results also indicated that the effect of LC 9018 was in part inhibited in mice treated with anti-CD3 or anti-CD8 antibody, but not affected in anti-CD4 antibody-treated mice. In contrast, LC 9018 had little effect on Meth A-bearing SCID or nude mice. These results demonstrated that CD8⁺ T cells participated in prolonging the survival of Meth A-bearing mice. Moreover, the examination of the production of several cytokines revealed that the production of interferon-γ and interleukin-6 was, in particular, augmented in the exudated fluid of the thoracic cavity in BALB/c mice injected with LC 9018 i.pl. These results suggested that i.pl. administration of LC 9018 induced those cytokines which had the potential to activate the thoracic macrophages or proliferate the thoracic lymphocytes to the cytotoxic T cells. Taken together, these findings demonstrated that the prolonging effects on survival by i.pl. administration of LC 9018 depended on CD8⁺ T cells, and the i.pl. administration of LC 9018 into i.pl. Meth A-bearing mice induced several cytokines which participated in the subsequent immunoresponses. Received: 24 June 1996     

Effects of oral administration of Lactobacillus casei on antitumor responses induced by tumor resection in mice

The effects of an oral administration of BLP, a preparation of viable Lactobacillus casei YIT 9018, upon tumor growth and the mitogenic responses of splenocytes from tumor-bearing mice were studied. BALB/c mice were insufficiently pre-immunized by resecting a Colon 26 tumor mass (primary tumor) grown for 5 days intradermally. Thereafter, mice were rechallenged by injecting Colon 26 tumor (secondary tumor) into the hind footpad. The secondary tumor grew progressively in control mice, but was markedly suppressed by oral administration with BLP at a dose of 100 or 200 mg/kg/day for 7 consecutive days. The suppression was a primary tumor-specific response. Viable L. casei, not heat-killed L. casei, could suppress the secondary tumor.Although the lymphoproliferative responses of splenocytes from the secondary tumor-bearing mice with T-cell mitogens (concanavalin A and phytohaemagglutinin) and cytokines (interleukin-1 and interleukin-2) were lower than those of normal mice, this suppression of mitogenic responses under tumor-bearing conditions was abolished by oral administration with BLP.Thus we concluded that oral BLP potentiated systemic immune responses that modified T-cell functions in tumor-bearing mice.     

Increased production of cytotoxic macrophage progenitors by Lactobacillus casei in mice

Heat-killed Lactobacillus casei YIT9018 (LC9018), when administered intravenously to normal mice, induced increase in Mac-1+ cells and Mac-2+ cells but not in Mac-3+ cells in spleen. The number of both populations changed in the same time course and was maximal 14 d after the administration. To know the effect of LC9018 on hematopoietic progenitor level, we examined the number of macrophage colony-forming cells (M-CFC), granulocyte-macrophage CFC (GM-CFC), and colony-forming units in spleen (CFU-S) in bone marrow 3 d after the administration. LC9018 stimulated the proliferation of M-CFC but not that of GM-CFC and CFU-S. LC9018-induced M-CFC were similar to normal M-CFC in dependence on macrophage colony-stimulating factor (M-CSF) and buoyant density. M-CFC-derived macrophages cultured in the presence of M-CSF expressed Mac-1 and Mac-2 but not Mac-3. They showed cytotoxic activity against syngenic tumor cells, Meth A, via direct contact, when assayed by using an in vitro colony inhibition assay or an in vivo Winn test. These results indicate that LC9018 stimulates the proliferation of cytotoxic macrophage progenitors in bone marrow and induces their differentiation in spleen. These effects may be one of the ways in which LC9018 suppresses tumor growth.     

Comparison of antitumor activity ofLactobacillus casei with other bacterial immunopotentiators

Antitumor activity ofLactobacillus casei YIT 9018 (LC9018) was demonstrated by intralesional (i. 1.) or intravenous (i. v.) administration into tumor-bearing mice which were inoculated with methylcholanthrene-induced fibrosarcoma (Meth A) or Kirsten murine sarcoma virus-transformed tumor (K234) cells. Its activity was significantly superior to the activity of two other species of lactobacilli but was nearly the same as that ofCorynebacterium parvum orMycobacterium bovis Bacille Calmette-Guérin (BCG). I. l. or i. v. administration of LC9018 into the tumor bearers caused local transient swelling or hepatosplenomegaly but did not cause other pronounced lesions. There was no significant difference in the degree of hepatosplenomegaly in LC9018 and that in other immunopotentiators. In mice whose tumors had regressed as a result of administration of LC9018 or the other immunopotentiators, the phytohemagglutinin P (PHA-P) response of the spleen cells was less than that of mice whose tumors progressed, and approached the normal level. The PHA-P response of popliteal lymph node cells proximal to the tumor lesion was fairly low compared with the splenic PHA-P response and there was no difference between the lymphocytes from mice whose tumors had regressed or progressed. Adjuvant activity of LC9018 in inducing tumor immunity was demonstrated by administering a mixture of LC9018 and Meth A cells to mice. This adjuvant activity was of the same efficiency as that ofC. parvum and BCG. The presence of the antitumor activity of LC9018 in cell wall components was deduced from fact that removal of its cell wall by endo-N-acetylmuramidase (M-1 enzyme) abolished the activity. The possible availability of LC9018 for immunotherapy of tumors is discussed.     

Lactobacillus casei subspecies casei endocarditis--a case report

Lactobacillus sp., generally considered to be a harmless indigenous bacteria of the mucous membrane, occasionally causes serious infections. Lactobacillus endocarditis is a very rare disease, and no case has been reported in Korea. Gram-positive bacilli were isolated from blood cultures of a 41-year-old man with clinically suspected subacute bacterial endocarditis. The patient had a dental procedure 3 months prior to the infection. The isolate was identified as L. casei subsp. casei based on the cultural characteristics and gas liquid chromatography of metabolic products. The patient was treated with ampicillin and improved. When Lactobacillus is isolated from the blood of an endocarditis patient, the significance should be seriously considered.     

Protective effect of Lactobacillus casei on Pseudomonas aeruginosa infection in mice.

The protective effect of heat-killed Lactobacillus casei YIT9018 (LC 9018) against Pseudomonas aeruginosa infection in mice was compared with that of Corynebacterium parvum. Survival of mice after intraperitoneal (i.p.) infection with P. aeruginosa was augmented in mice that had been pretreated i.p. with LC 9018 5 days previously. Similar treatment of mice with C. parvum, however, was not effective at all. Moreover, mice became more susceptible to infection with P. aeruginosa after such treatment. Growth of P. aeruginosa in the peritoneal cavity and spleen was markedly inhibited in LC 9018-pretreated mice, whereas such inhibition of bacterial growth was not observed in C. parvum-treated mice. The protective effect of LC 9018 was observed in mice subjected to 800 rads of whole body irradiation but was abrogated when mice were treated with carrageenan. These results suggest that augmentation of the resistance of mice to P. aeruginosa was caused by the induction of activated macrophages. The number of macrophages detectable in the peritoneal cavity was almost the same in LC 9018- and C. parvum-treated mice. Growth of Listeria monocytogenes was inhibited by pretreatment with LC 9018. Inhibition of L. monocytogenes was also observed after the same pretreatment with C. parvum. It was suggested that macrophages activated with LC 9018 were involved in the protective immunity to P. aeruginosa.     

Lactobacillus casei Zhang 对扑热息痛诱导的小鼠急性肝损伤的保护作用

目的:研究益生菌Lactobacillus casei Zhang(Lcz)对扑热息痛(APAP)所致小鼠急性肝损伤的保护作用及其机制。方法:C57BL/6 小鼠被随机分为空白组(Ctrl)、APAP 诱导急性肝损伤模型组(Acetaminophen ,APAP)、阳性药物组(N-Acetylcysteine,NAC)、Lcz 预防组(Lcz/ APAP)和Lcz 对照组(Lcz)。Lcz(1伊109 CFU/ ml)连续灌胃30 d 后,NAC 组在APAP 处理前1 h 腹腔注NAC(150 mg/ kg)。APAP、NAC 以及Lcz/ APAP 组均腹腔注射APAP(300 mg/ kg)。APAP 作用18 h 后,采集血液和收集肝脏组织,检测血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)的水平。通过Western blot 检测肝脏组织中血红素氧化酶(HO-1)、超氧化物歧化酶2(SOD2)、Bcl-2 以及TLR4 的表达水平。结果:Lcz 能抑制APAP 诱导的急性肝损伤血清中ALT 和AST 水平。Lcz 提高了HO-1、SOD2 和Bcl-2 的蛋白表达水平,而降低了APAP 诱导的TLR4 的表达。结论:益生菌Lcz对APAP 诱导的小鼠急性肝损伤有保护作用,其保肝作用机制可能与其抗氧化和抗炎活性有关。     

Aseptic addition method for Lactobacillus casei assay of folate activity in human serum

An ;aseptic addition' method is described for microbiological assay with Lactobacillis casei of folate activity in human serum. It has the following advantages over the previously reported ;standard' method. 1 The manipulations involved in the assay are halved, by deleting autoclaving of serum in buffers. 2 The use of 1 g.% ascorbate better preserves serum folates than the lower amounts of ascorbate which are the maximum quantities usable in the standard methods. 3 Only 0.3 ml. of serum is required (0.1 ml. for one sample; 0.2 ml. for its duplicate).     

Inhibitory effect of oral administration of Lactobacillus casei on 3-methylcholanthrene-induced carcinogenesis in mice

The present study was designed to determine whether tumor induction by 3-methylcholanthrene (MC), a carcinogenic hydrocarbon, can be inhibited by oral administration of Lactobacillus casei strain Shirota (LC). C3H/HeN mice were divided into four groups and assigned to the following treatments: treated with MC and given control or LC-containing diet; treated with vehicle only and given control or LC-containing diet. MC (1 mg) was injected intradermally at 7 weeks of age and the tumor incidence was monitored; LC was mixed into a diet at a concentration of 0.05% (w/w) and the diet was fed from the day of MC injection throughout the study. Spleen cells were analyzed for the immune parameters at 12 and 16 weeks after the MC injection. Oral feeding of mice with LC reduced tumor incidence (P < 0.05). MC treatment lowered the in vitro response to concanavalin A (Con A) of spleen cells, the secretion of interleukin-2 in spleen cell culture after stimulation of the cells with Con A and the proportions of CD3⁺, CD4⁺ and CD8⁺ splenic cells. However, the analysis of the spleen cells obtained from the mice treated with MC and given the LC-containing diet revealed that these disrupted host immune parameters were maintained at the level of normal controls. These results suggest that oral feeding of mice with LC inhibits MC-induced tumorigenesis by modulating the disrupted host immune responses during MC carcinogenesis. Received: 14 April 1999     

Enhancement of hematopoietic response of mice by subcutaneous administration of Lactobacillus casei.

Mice that had received heat-killed Lactobacillus casei (LC 9018) subcutaneously (s.c.) showed enhanced resistance to systemic (i.e., intravenous) infection with Listeria monocytogenes, but the antilisterial resistance of mice was less augmented by s.c. administration of Propionibacterium acnes ("Corynebacterium parvum"). Though there was little change in the total number of splenic leukocytes after s.c. administration of LC 9018, the monocyte-macrophage ratio increased after treatment, reaching its peak on day 5 to 7 after injection. The number of progenitor cells that form macrophage colonies under the stimulus of L-cell-conditioned medium in a semisolid agar culture system increased in the spleens of mice pretreated s.c. with LC 9018, showing a peak response on day 5 after injection. The increase corresponded to the increase in the dose administered, and increased numbers were detected even 10 days after treatment. The number of macrophage colonies in the femurs of mice pretreated s.c. with LC 9018 showed a temporary increase on day 3 after injection but then a decrease until day 10. Colony-stimulating activity was detected in the sera of mice administered LC 9018 s.c. 18 h previously, and the colonies produced were of three types: granulocyte (8%), macrophage (56%), and granulocyte-macrophage (36%). Administration of C. parvum s.c. had little effect on these hematopoietic responses of mice.     

Modulatory activity of a Lactobacillus casei strain on 1,2-dimethylhydrazine-induced genotoxicity in rats

The present study was designed to investigate the putative antigenotoxic effects of supplementing the diet of rats treated with the colon carcinogen 1,2-dimethylhydrazine hydrochloride (DMH) with a Lactobacillus casei strain using an in vivo approach. The antigenotoxic response was evaluated in colon and liver cells using the alkaline comet assay. Since the balance between the bioactivation and detoxification metabolic pathways is crucial for the formation of toxic and genotoxic metabolites, alterations in the level of some xenobiotic metabolizing enzymes (XME) were studied in liver preparations. In the challenge group (L. casei + DMH), lactobacilli-supplemented diet, there was a decrease in the extent of DMH-induced DNA damage, especially in colon cells. Compared with control rats, there was less basal DNA damage in colon cells of rats fed on a lactobacilli-supplemented diet. These findings are the first to give clear evidence of DNA-protective effects of lactobacilli against basal DNA damage. Moreover, the chemopreventive effects were accompanied by changes in the activities of several XME. The observed decrease in the concentration of nonenzymatic antioxidants (i.e. GSH) and the reduced activity of enzymatic antioxidants (i.e., GST, GPx, and SOD) in liver could reflect an overall reduction in the level of oxidative stress in rats on a diet supplemented with the L. casei suspension compared with control rats (basal state). Thus, the concentrations of GSH and the activities of GST, GPx, and SOD could be downregulated by supplementing the diet with L. casei as a response to an improved antioxidant status.     

Systemic augmentation of the immune response in mice by feeding fermented milks with Lactobacillus casei and Lactobacillus acidophilus.

This study investigates the effect of feeding fermented milks with Lactobacillus casei, Lactobacillus acidophilus and a mixture of both micro-organisms on the specific and non-specific host defence mechanisms in Swiss mice. Animals fed with fermented milk for 8 days (100 micrograms/day) showed an increase in both phagocytic and lymphocytic activity. This activation of the immune system began on the 3rd day, reached a maximum on the 5th, and decreased slightly on the 8th day of feeding. In the 8-day treated mice, boosted with a single dose (100 micrograms) on the 11th day, the immune response increased further. The feeding with fermented milk produced neither hepatomegaly nor splenomegaly. These results suggest that L. casei and L. acidophilus, associated with intestinal mucosae, can influence the level of activation of the immune system. The possible clinical application of fermented milks as immunopotentiators is also discussed.     

The treatment of mice with Lactobacillus casei induces protection against Babesia microti infection

In this study, we report that administration of Lactobacillus casei confers protection to mice against the intracellular protozoan Babesia microti. Mice treated with L. casei orally or intraperitoneally were inoculated 7 days later with an infectious dose of B. microti. Mice treated with lactobacilli showed significant reduction in the percentage of parasitized erythrocytes (PPE) compared to untreated mice. When mice were inoculated intraperitoneally with L. casei 3 or 0 days before challenge with B. microti, the PPE was significantly lower compared to untreated mice and there were no differences between treated mice and mice immune to B. microti infection. When mice treated with live or dead L. casei were compared to mice inoculated with Freund Complete Adjuvant before a B. microti infection, a significant reduction of PPE was observed. These results show the protective effect of L. casei administered to mice against a B. microti infection and suggest that it might act by stimulating the innate immune system.     

Influence of feeding fermented milk and non-fermented milk containing Lactobacillus casei on immune response in mice

The effect of feeding Lactobacillus casei in the form of fermented and non-fermented milk on non-specific as well as humoral immune response in mice was investigated. Feeding of L. casei fermented milk (LcF), its cell free supernatant (LcS), and L. casei non-fermented milk (LcNF) to mice for period of two, five and eight days resulted in increased activity of β-galactosidase and β-glucuronidase in peritoneal macrophages in comparison to skim milk (control). The phagocytic activity of peritoneal macrophages also increased significantly (p<0.01) in mice fed on LcF, LcS and LcNF compared to skim milk-fed mice. The maximum rise in β-galactosidase, β-glucuronidase and phagocytic activity was on day 5 post-feeding. In challenge studies with Shigella dysenteriae, it was observed that colonization of S. dysenteriae in the intestine, liver and spleen was significantly less in mice fed on LcNF and LcF in comparison to mice fed on skim milk for a period of seven days before challenge. Levels of secretory antibodies were higher in groups fed LcNF and LcF. The results suggest the immunomodulatory potential of L. casei.     

Lactobacillus casei prevents the development of dextran sulphate sodium-induced colitis in Toll-like receptor 4 mutant mice

Probiotics, defined as live or attenuated bacteria or bacterial products, confer a significant health benefit to the host. Recently, they have been shown to be useful in the treatment of chronic inflammatory bowel disease and infectious colitis. In this study, we investigated the effect of probiotics on the development of experimental colitis using Toll-like receptor 4 (TLR-4) mutant (lps-/lps-) mice. TLR-4(lps-/lps-) and wild-type (WT) mice were given 2.5% dextran sulphate sodium (DSS) in drinking water to induce colitis with or without Lactobacillus casei pretreatment. Clinical and histological activity of DSS-colitis was attenuated markedly both in TLR-4(lps-/lps-) and WT mice pretreated with L. casei. Interestingly, histological activity was less severe in TLR-4(lps-/lps-) mice than in WT mice. The levels of myeloperoxidase activity and interleukin (IL)-12p40 were attenuated in pretreated TLR-4(lps-/lps-) mice after DSS administration. By contrast, transforming growth factor (TGF)-beta and IL-10 mRNA and protein expressions were increased markedly in pretreated TLR-4(lps-/lps-) mice. The current results suggest that L. casei has a preventive effect in the development of acute DSS-induced colitis and its action depends largely upon TLR-4 status. L. casei modulates the expression of inflammatory cytokines and down-regulates neutrophilic infiltration in the case of incomplete TLR-4 complex signalling.     

Pleiotropic effects of lactate dehydrogenase inactivation in Lactobacillus casei

In lactic acid bacteria, conversion of pyruvic to lactic acid through the activity of lactate dehydrogenase (Ldh) constitutes the final step of the homofermentative pathway. Lactobacillus casei has two characterized genes encoding Ldh activities. The ldhL gene codes for an L-Ldh, which specifically catalyzes the formation of L-lactate, whereas the hicD gene codes for a D-hydroxyisocaproate dehydrogenase (HicDH), which catalyzes the conversion of pyruvate into D-lactate. In L. casei cells fermenting glucose, a mixture of L-/D-lactate with a 97:3% ratio was formed. Inactivation of hicD led to undetectable D-lactate levels after glucose fermentation, while L-lactate levels remained constant. Inactivation of ldhL did not abolish the production of L-lactate, but the lactate final concentration decreased about 25% compared to the wild type, suggesting the presence of at least a second L-Ldh. Moreover, part of the pyruvate flux was rerouted and half of the lactate produced was in the D-isomer form. ldhL inactivation in L. casei showed additional interesting effects. First, the glycolytic flux from pyruvate to lactate was redirected and other fermentation products, including acetate, acetoin, pyruvate, ethanol, diacetyl, mannitol and CO(2), were produced. Second, a lack of carbon catabolite repression of lactose metabolism and N-acetyl-glucosaminidase activity was observed. This second effect could be partly avoided by growing the cells under aeration, since NADH oxidases could account for NAD+ regeneration.     

Natural killer cell activities of synbiotic Lactobacillus casei ssp. casei in conjunction with dextran

We have reported previously that Lactobacillus casei ssp. casei, together with specific substrate dextran, exhibited an adjuvant effect of stimulating humoral immune responses against bovine serum albumin (BSA) as a model antigen in BALB/c mice. In the present study, among the Lactobacillus species tested, L. casei ssp. casei with dextran significantly elevated the natural killer (NK) cell activities in spleen mononuclear cells from BALB/c mice in comparison to L. casei ssp. casei alone or other Lactobacillus species with or without dextran. Oral administration of L. casei ssp. casei together with dextran also resulted in a significant increase of NK cell activities in healthy human volunteers. Further, L. casei ssp. casei induced significant production of interleukin (IL)-12 in human peripheral blood mononuclear cells and IL-15 mRNA expression in the human intestinal epithelial cell line Caco-2. L. casei ssp. casei with dextran in food also significantly elevated the survival rate of BALB/c mice bearing Meth-A cells. Taken together, these results demonstrate that dietary synbiotic supplementation which is a combination of the L. casei ssp. casei used as a probiotic together with the dextran, a specific substrate as a prebiotic, efficiently elicits murine and human NK cell activities.