Intestinal microflora are necessary for development of spontaneous adenocarcinoma of the large intestine in T-cell receptor beta chain and p53 double-knockout mice.

This study was conducted to confirm the hypothesis that intestinal microflora are required for the development of adenocarcinoma in the colon of the TCRbeta and p53 double-knockout (TCRbeta-/- p53-/-) mouse. Germ-free TCRbeta-/- p53-/- mice were produced. At 7 weeks of age, the animals were divided into two groups (n = 10/group), and one of these groups was conventionalized. Animals of both groups were subjected to histopathological examination for adenocarcinoma of the colon at 4 months of age. There was no development of adenocarcinoma of the colon among the germ-free mice, whereas in the conventionalized group, adenocarcinomas of the ileocecum and cecum were detected in 70% of animals. These results indicate the usefulness of the TCRbeta-/- p53-/- mouse as a colon cancer animal model that develops spontaneous adenocarcinoma of the colon early in life, and suggest that intestinal microflora play a major role in the development of adenocarcinoma of the colon in this animal model.

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.

Studies on lectins of amago (Oncorhyncus rhodurus). I. Amago ova lectin and its receptor on homologous macrophages.

A lectin was found in the ova of amago, a Japanese trout (Oncorhyncus rhodurus), which agglutinates rabbit, rat and human B-type erythrocytes. The hemagglutination was specifically inhibited by monosaccharides, L-rhamnose, D-galactose, and their C2 and C4 analogs, and p-nitrophenyl-alpha-D-galactoside and melibiose, indicating a binding specificity for alpha-L-rhamnosyl or alpha-D-galactosyl type sugar moiety. To study its interaction with homologous cells, amago peritoneal macrophages were isolated from corn starch-stimulated peritoneal exudates. The lectin-rabbit erythrocyte complexes were found to adhere onto the macrophages harvested on the 4th day or later after the stimulation, but not to those obtained within 3 days; the latter macrophages acquired the complex-binding capacity when cultured for 3 to 4 days in vitro. These findings indicated that a lectin receptor is expressed on peritoneal macrophages after inflammatory stimulation. Similar lectin receptor-bearing macrophage-like-cells were also detected during in vitro amago head kidney culture. This suggested that the inflammatory induced peritoneal macrophages may be differentiated from the head kidney macrophage precursor cells and during this process the ova lectin receptors also become expressed.

Survival of a probiotic, Lactobacillus casei strain Shirota, in the gastrointestinal tract: selective isolation from faeces and identification using monoclonal antibodies.

Lactobacillus casei strain Shirota (LCS) is a probiotic bacterium used in the production of fermented milk products and lactic acid bacteria preparations. To investigate the survival of LCS in the gastrointestinal tract, we have developed a selective medium and specific monoclonal antibodies to isolate and identify this strain. Selective LLV agar medium was prepared by modifying LBS medium, a selective medium for lactobacilli, through the replacement of glucose with lactitol as a carbon source and vancomycin as a selective antibiotic. Culture in LLV agar medium followed by ELISA using monoclonal antibodies specific for LCS was able to detect the organism in faeces. Using this method, we studied the faecal recovery of LCS in individuals who drank 125 ml of fermented milk which contained 10(10) live LCS for 3 days. The mean recovery was about 10(7) live bacteria per gram of faeces, indicating that LCS survived transit through the gastrointestinal tract after ingestion of the fermented milk.

Establishment of a Tcrb and Trp53 genes deficient mouse strain as an animal model for spontaneous colorectal cancer.

A congenic C57BL/6JJcl-Tcrbtm1MomTrp53tm1 (Tcrb-/-:Trp53-/-) mouse lacking T-cell receptor beta chain (TCR beta) and transformation related protein 53 (p53) has been established at the N8th generation of backcrossing male Tcrb-/-:Trp53-/- mice, which had been obtained by mating a Tcrb-/- mouse with a Trp53-/- mouse, with female C57BL/6JJcl mice. In the mice deficient for the both genes, occurrence of tumor masses was observed mostly in the cecum with high frequency as examined at 3 months of age. The majority of the masses had histologic features of hyperplasia or dysplasia while occasional lesions were noted to be adenocarcinomas invading the submucosa (invasive adenocarcinoma). As examined at 4 months of age and thereafter, all mice had 4-5 colorectal tumors per animal, the lesions being located mainly in the cecum and, histopathologically, all the obvious neoplastic growths in the regions examined were invasive adenocarcinomas. The Tcrb and Trp53 genes deficient mouse strain which develops spontaneous colorectal carcinoma with fairly high frequency at early age would be useful as an animal model for colorectal cancer.

Enhancement of immune response in Peyer's patch cells cultured with Bifidobacterium breve.

Bifidobacterium breve, included in fermented milk, was tested for adjuvanticity and mitogenicity using cells of mouse Peyer's patch, one of the gut-associated lymphoid tissues. Addition of B. breve enhanced antilipopolysaccharide antibody production in Peyer's patch cells and also anti-sheep red blood cell plaque-forming cells in Peyer's patch cells cultured with sheep red blood cells. Furthermore, addition of B. breve accelerated proliferation of Peyer's patch cells, particularly B cells. In BALB/c mice, enhancement of proliferation by B. breve was also found in Peyer's patch cells from nude mice and a B cell-enriched fraction, including both the B cell fraction and plastic-adherent cells. Enhancement was not found in the fraction in which Sephadex G10-adherent and carbonyl-iron phagocytic cells were excluded from Peyer's patch cells or in a pure B cell fraction in which plastic-adherent cells were excluded from the B cell-enriched fraction of Peyer's patch cells. The proliferation of B cells was enhanced when the supernatant of plastic-adherent cells cultured with B. breve was added. It is concluded that B. breve activated plastic-adherent cells and that these cells secreted a soluble factor that enhanced proliferation of B cells.

Dose-dependent induction of immunologic enhancement and suppression after oral administration of antigen.

The effects of feeding various quantities of a particulate antigen, sheep red blood cells (SRBC), on plaque-forming cells (PFC) in the spleen were determined. Mice were given various numbers of SRBC orally daily for 14 days, then injected with SRBC intravenously. Splenic IgA PFC responses to SRBC were enhanced in the mice fed 5 X 10(8) SRBC and splenic IgG PFC responses to SRBC were depressed in the mice fed 5 X 10(9) SRBC. Adoptive transfer experiments showed that enhancement of splenic IgA PFC responses and suppression of splenic IgG PFC responses were induced by the T-cell rich fraction from Peyer's patches (PP) and the spleen in 5 X 10(8) SRBC- and 5 X 10(9) SRBC-fed mice, respectively. Kinetic studies revealed that IgA helper cells or IgG suppressor cells appeared in PP 2 days after oral administration and 4 days after it in the spleen.

Induction by Lactobacillus casei of increase in macrophage colony-forming cells and serum colony-stimulating activity in mice.

The content of macrophage colony-forming cells (M-CFC) and the serum colony-stimulating activity (CSA) were investigated in mice after intravenous administration of Lactobacillus casei YIT9018 (LC9018). In normal BALB/c mice, 500 micrograms of LC9018 increased both femoral and splenic M-CFC; the highest levels were found a few days and a week, respectively, after the administration. LC9018 also induced an increase in splenic M-CFC in C3H/HeJ mice as well as in C3H/HeN mice, unlike lipopolysaccharide (LPS), which was ineffective in C3H/HeJ mice. In Meth A-bearing BALB/c mice, LC9018 (250 micrograms X 5) suppressed the growth of tumor cells and increased femoral and splenic M-CFC to much greater extents than Lactobacillus plantarum YIT0102 (250 micrograms X 5) did. LC9018 induced a rise of serum granulocyte-macrophage CSA in the same way as LPS. Sera taken 6 hr after LPS administration, when transferred to normal mice, induced increases in femoral and splenic M-CFC. However, sera taken 6 hr after LC9018 administration increased neither femoral nor splenic M-CFC. These results indicate that LC9018 modulates myelopoiesis at least at the stage of the proliferation of M-CFC in a different way from LPS, and this ability may be related to its antitumor activity.

Immunogenicity of Bacteroides isolated from mice: relationship between immunogenicity and cell wall antigens.

Three different strains of Bacteroides were isolated from feces and cecal contents of mice. The immunogenicity of the strains was determined by measuring the serum agglutinin titers after intraperitoneal antigen injection. There were marked differences in quantity and quality of produced antibodies among the three strains. One strain (2-2) induced low antibody titers in both the primary and secondary responses, and a significant 2-mercaptoethanol (2-ME)-resistant antibody production occurred. Another strain (Y) induced low antibody titers in the primary response and high titers in the secondary response, but 2-ME-resistant antibody production did not occur. The third strain (2-4) induced very high antibody titers in both the responses, and a large amount of 2-ME-resistant antibody production occurred. Further, heat-ethanol-treated strain Y induced only immunoglobulin M antibody, but periodate-treated strain Y induced no antibody. Heat-ethanol- or periodate-treated strain 2-4 induced immunoglobulin M or G antibody, respectively. These observations suggest that the surface antigens of the two strains are distinctly different: the antigen of strain Y would be mostly O-antigen, whereas those of strain 2-4 would be O-antigen and protein moieties.

Immunogenicity of Bifidobacterium breve and change in antibody production in Peyer's patches after oral administration.

Immunogenicity in the intestine of Bifidobacterium breve, included in fermented milk, was compared with that of Bacteroides thetaiotaomicron, also predominant in human intestine. In vivo, serum antibody to B. breve was detected first in mice fed the organism for 33 d; antibody decreased in mice fed these for more than 33 d. Serum antibody to Bact. thetaiotaomicron was detected in mice fed the organism for 7 d and was maintained at the same level in mice fed these for more than 7 d. From in vitro tests, the optimal doses of B. breve and Bact. thetaiotaomicron to induce antibody production by Peyer's patch cells, intestinal lymphoid tissue cells, were 5 x 10(8) and 5 x 10(7) bacteria/ml, respectively. Therefore, it was suggested that immunogenicity of B. breve is weaker than that of Bact. thetaiotaomicron. Furthermore, the change of antibody production to the organism by Peyer's patch cells in the mice administered B. breve orally was tested by the Peyer's patch cell culture method. Antibody production against B. breve by Peyer's patch cells in mice given B. breve for 25 and for 33 d increased and decreased, respectively, in comparison with the control. These results suggest that when serum antibody to B. breve increases significantly, anti-B. breve antibody production by Peyer's patch cells is suppressed, and thereafter, serum antibody to B. breve decrease and is not detected. These findings favor the view that serum antibody production to B. breve is regulated in Peyer's patches.

A comparative study on the humoral immune responses in germ-free and conventional mice.

The plaque-forming cell (PFC) responses to sheep red blood cell (SRBC) dinitrophenyl-lysine-Ficoll (DNP-lys-Ficoll), and dinitrophenylated bovine serum albumin (DNP-BSA) have been studied in both germ-free and conventionally reared ICR mice. In germ-free mice, the IgG response to SRBC and the IgM and IgG responses to DNP-BSA were lower than in conventional mice, but no difference was observed in the IgM response to SRBC or the IgM and IgG responses to DNP-lys-Ficoll. Further, the number of 0-bearing cells in the spleen was smaller, and the mitogenic response of spleen cells to PHA was lower in germ-free mice than in conventional mice. These observations suggest that T cells of germ-free mice remain functionally immature.

Comparison of antitumor activity of Lactobacillus casei with other bacterial immunopotentiators.

Antitumor activity of Lactobacillus casei YIT 9018 (LC9018) was demonstrated by intralesional (i.l.) 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 of Corynebacterium parvum or Mycobacterium 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 of C. 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.

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

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

Measurement of staphylokinase by enzyme-linked immunosorbent assay using monoclonal antibodies.

Hybridoma clones producing monoclonal antibodies specific for staphylokinase were isolated. A competitive assay revealed that the monoclonal antibodies studied could be divided into at least two groups. Representatives of these groups, AS22 and B3E6, recognized quite different epitopes on staphylokinase. This finding led us to develop an assay system for the quantitative analysis of staphylokinase by enzyme-linked immunosorbent assay using AS22 as the capturing antibody and biotinylated B3E6 as the "detector". The lower limit of sensitivity of the assay was 20 pg of staphylokinase per ml. The assay exhibited good reproducibility, with values of 5.8 and 3.8% for the intra- and inter-assay coefficients of variation, respectively. Staphylokinase could be assayed in the presence of human plasma when the plasma was diluted more than 320-fold, and the measurement was unaffected by the presence of physiological concentrations of human plasminogen. Hence, this assay was considered useful for the detection and quantification of staphylokinase in clinical samples.

Antibacterial effect of bovine milk antibody against Escherichia coli in a mouse indigenous infection model.

A skim-milk fraction and a whey-protein concentrate (WPC) fraction were prepared from the cows that had been immunized with E. coli isolated from the mouse intestine. The antibacterial effect of these fractions against E. coli was examined. They contained antibody with a high affinity for E. coli strain 48, a representative strain in the mouse intestine, which is composed of a large amount of IgG and smaller amounts of IgA and IgM. Although these fractions showed no bactericidal or bacteriostatic activity against E. coli 48 directly in vitro, they exhibited strong agglutination and opsonization activities against the bacteria in vitro. The bacteria opsonized with the WPC fraction were taken up more effectively by liver macrophages in vivo, compared with unopsonized E. coli, after an intravenous injection into mice. Oral administration of the skim-milk fraction to mice significantly reduced the susceptibility to the lethal toxicity of 5-fluorouracil (5 FU). The increase in the population levels of E. coli in the intestinal tract after administration of 5 FU was inhibited by oral administration of the skim-milk fraction. These results strongly suggest that specific antibody may be effective in the prophylaxis against the indigenous infection with gram-negative bacteria such as E. coli after a period of chemotherapy in cancer patients.

Activation of T-cell receptor-gammadelta+ cells in the intestinal epithelia of KN6 transgenic mice.

We analysed the properties of intraepithelial lymphocytes of small intestine (SI-IEL) in KN6-transgenic (Tg) mice expressing cDNA of T-cell receptor (TCR)-gammadelta specific for the T22b molecule. While most splenic Tg TCR-gammadelta+ cells from KN6-Tg mice with H-2d/d background (Tgd/d mice) were Thy-1+ CD8alpha- CD44dull+ CD45RB+ CD69-, Tg TCR-gammadelta+ cells in SI-IEL (Tg gammadelta-IEL) were heterogeneous in the expression of Thy-1, CD8alpha and CD44 molecules and predominantly CD45RB+ CD69+. Tg gammadelta-IEL exhibited a much reduced proliferative response to the antigen (irradiated H-2b splenocytes) than splenic Tg TCR-gammadelta+ cells; the CD44+ subset, but not the CD44- subset, in Tg gammadelta-IEL responded to the antigen. Furthermore, Tg gammadelta-IEL, but not splenic Tg TCR-gammadelta+ cells, displayed cytolytic activity whether they were prepared from conventional or germ-free KN6-Tg mice. Comparative analysis of young and aged KN6-Tg mice revealed that the proportion of CD44+ cells in Tg gammadelta-IEL increased but the proliferative response of Tg gammadelta-IEL to the antigen attenuated in association with ageing. Moreover, although Tg gammadelta-IEL from Tgb/d mice contained a higher proportion of CD44+ cells than Tgd/d mice, they did not respond to the antigen. These results demonstrate that Tg TCR-gammadelta+ cells lose the ability to recognize the antigen following activation in the intestinal epithelia.

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.

Host-mediated antitumor activity of Lactobacillus casei in mice.

Antitumor activity of Lactobacillus casei YIT 9018 ( LC9018 ) was studied in BALB/c mice by using two syngeneic tumors; methylcholanthrene-induced tumor (Meth A fibrosarcoma) and Kirstein murine sarcoma virus-transformed BALB/3T3 ( K234 tumor). Administration of an LC9018 -Meth A cell mixture induced complete suppression of the tumor growth, while simultaneous injections of LC9018 and Meth A cells into different sites had no suppressive effect on the tumor growth. Administration of the mixture subsequently induced specific transplantation immunity to the challenge tumor, which started to be generated on about the 5th day after the administration and continued to at least the 30th day. Administration of an LC9018 - K234 cell mixture also induced suppression of the tumor growth and generated specific antitumor immunity. Neutralization (Winn type) tests showed that T lymphocytes possessed tumor cytotoxicity but humoral immune serum did not, suggesting that the T cells with LC9018 -potentiated antitumor immunity functioned in the suppression of the tumor growth.

Source of prolactin in human follicular fluid.

To analyze whether prolactin (PRL) in human follicular fluid (FF) is synthesized locally or derived from the circulation, PRL concentrations of plasma and FF were determined in the patients after ovarian stimulations. The amounts of PRL messenger ribonucleic acid (mRNA) in the follicular tissues during different menstrual phases were also determined. The FF PRL concentration was correlated positively with plasma PRL and highest estradiol levels during the stimulatory cycle. No PRL mRNA sequence was detected in the RNAs extracted from follicles at any stage in the menstrual cycle, although beta-actin mRNA was detected in all samples. In a comparison with pituitary RNA, the PRL mRNA concentration in ovarian follicular tissues seemed to be 10,000 times less than that in the pituitary. These results suggest that FF PRL may not be synthesized locally, but derived from the pituitary via the circulation through passive diffusion, and thus regulated by estrogen.