Effect of a high-fat diet on resistance to Listeria monocytogenes.

Effects of a high-fat diet on macrophage (M phi) functions were investigated. Eight-week-old ddN mice were fed a high-fat diet and carbon clearance was tested. Remarkable suppression of phagocytic activity (K16) was observed in mice fed such a diet for 1 or 2 weeks. Resistance against Listeria monocytogenes inoculated intravenously (iv) with a lethal, a sublethal, or a non-pathogenic dose was observed in the liver of mice fed a high-fat diet. When mice were infected with a lethal dose of bacteria, the number of listeria increased progressively in the liver to kill both control and a high-fat diet fed mice by day 4. The number of listeria revealed no significant difference between the group in the case of low dose inoculation. High-fat diet fed mice given a sublethal dose of bacteria showed a rise in the number of viable bacteria during the first 3 days after infection while a decline in the number of bacteria was observed in control mice during such a period. Suppression of M phi activity induced by a high-fat diet may account for the reduced resistance.

Contradictory responses in induction of delayed type hypersensitivity in orally immunized mice.

Induction of delayed type hypersensitivity (DTH) in mice fed with sheep red blood cells (SRBC) and live Vibrio cholerae (V. cholerae) both forcedly and ad lib was comparatively investigated. Although suppression of DTH to SRBC or V. cholerae was induced in mice fed forcedly for 1 or 2 weeks, mice fed ad lib could produce the positive footpad reactions to antigens. Furthermore, the suppression of DTH in forcedly fed mice showed an antigenic specificity. These observations indicated that the induction of unresponsiveness to DTH in orally immunized mice was markedly influenced by the oral administration.

Cholera toxin-induced tolerance to allografts in mice.

When C3H/HeN (C3H) mice were primed with viable C57BL/6 (B6) spleen cells and treated with cholera toxin (CT) on the same day, a profound tolerance to tumour allografts of B6 origin was induced. The tolerant state was sustained for as long as 6 weeks or more. Skin allografts of B6 were rejected by such tolerant C3H mice, although the survival times were prolonged very slightly. Generation of cytotoxic T lymphocytes was reduced markedly in the tolerant mice, whereas delayed footpad reaction to B6 cells was maintained at the normal immune level or higher. There is a possibility that a T-cell subset responsible for delayed footpad reaction is resistant to CT-induced tolerance and participates in the rejection of skin allografts in tolerant mice.

Differing resistance to polymorphonuclear cells of halophilic- and hypotonic-types of Vibrio vulnificus.

Bacterial growth and lethality of 4 strains of Vibrio vulnificus infection of mice were enhanced by gamma-irradiation but not by treatment with carrageenan. Therefore, protection against V. vulnificus, at least in the early phases, probably depends mainly on polymorphonuclear cells (PMN), since carrageenan depletes macrophages but not PMN. PMN dependent protection against infection differs for 2 types of V. vulnificus strains. Halophilic- and hypotonic-type were distinguished from the corresponding parent strain. The hypotonic-type of the strains had capsular materials, as clarified by electron microscopic observation of the organisms stained with ruthenium red. On the other hand, halophilic-types either had no observable capsular materials, or incomplete materials, in contrast to the corresponding hypotonic-type. The corresponding halophilic- and hypotonic-types of the strains were compared for virulence in mice. The strains with capsular materials acquired resistance to phagocytic activity and were highly lethal. Capsular materials of V. vulnificus are no doubt important for the expression of virulence.

Mechanism of protection during the early phase of a generalized viral infection. II. Contribution of polymorphonuclear leukocytes to protection against intravenous infection with influenza virus.

The contribution of phagocytes to the early protection of mice inoculated intravenously with influenza virus was investigated in phagocyte-depleted mice. Following the inoculation of a sublethal dose of influenza virus, virus titres in the liver and lung of both untreated and carrageenan-treated mice were reduced rapidly by day 1 and decreased slowly to reach an undetectable level by day 7. The titres in gamma-irradiated mice decreased transiently by day 1 and increased progressively thereafter to kill all of the hosts by day 8. The clearance of virus from blood at the early stage of infection was retarded by gamma-irradiation but not by carrageenan treatment. In addition, increase in virus titres in the liver and lung in the early stage of the infection was prevented by adoptive transfer with syngeneic polymorphonuclear leukocytes into gamma-irradiated mice. No significant rise of neutralizing antibody was detectable by day 3 after the inoculation, in any of the three groups of mice. These observations imply that gamma-sensitive and carrageenan-resistant polymorphonuclear leukocytes play a protective role at the early stage in the infection, whereas fixed macrophages or natural killer cells, both of which are carrageenan-sensitive and gamma-resistant, scarcely participate in the early phase.

Protective mechanisms against pulmonary infection with influenza virus. I. Relative contribution of polymorphonuclear leukocytes and of alveolar macrophages to protection during the early phase of intranasal infection.

The relative contribution of polymorphonuclear leukocytes and macrophages in the early protection against intranasal infection of mice with influenza virus was investigated. Virus multiplication in the lung in the early phase of infection with less than 1.5 X 10(3) plaque-forming units was enhanced by X-ray irradiation. The intranasal administration of carrageenan did not influence the titre of virus. However, when mice were infected with 1.5 X 10(4) plaque-forming units, the virus titre was elevated by intranasal administration of carrageenan as well as by X-ray irradiation, but not by intraperitoneal administration of carrageenan. The intranasal administration of carrageenan not only inhibited the phagocytic activity of alveolar macrophages but also enhanced susceptibility to the virus. On the other hand, polymorphonuclear leukocytes were capable of phagocytosing the virus in vitro and were non-permissive for virus infection. Neutralizing antibody and interferon were not detectable in the early stage of the infection. These results suggested that polymorphonuclear leukocytes (X-ray-sensitive, carrageenan-resistant) were the cells primarily responsible for early protection in influenza virus infection and that after infection with a high dose of the virus alveolar macrophages (X-ray-resistant, carrageenan-sensitive) also played a protective role in the early phase.

Oral immunization in adult mice to live and heat-killed Vibrio cholerae.

Effects of systemic and intestinal local immune responses in mice fed ad libitum and forcedly with live or heat-killed Vibrio cholerae on the elimination of vibrios from the intestine were investigated. In mice fed with live vibrios, ad libitum feeding could induce potential delayed-type hypersensitivity and rapid production of vibriocidal antibody in the serum whereas forcedly feeding suppressed the delayed-type hypersensitivity and retarded the antibody production. In contrast, when killed microorganisms were used as antigens, significant delayed-type hypersensitivity and rapid response of vibriocidal antibody were induced in forcedly fed mice although ad libitum feeding suppressed the induction of the delayed-type hypersensitivity and retarded the production of vibriocidal antibody. The elimination of vibrios from the intestine of mice was promoted in both mice groups fed ad libitum and forcedly with live vibrios but not with killed microorganisms. Total IgA in the intestinal contents of mice fed with live vibrios both ad libitum and forcedly were higher than those of mice fed with killed antigens. In addition, when the extracts of intestinal contents were absorbed by live antigens, IgA contents in mice fed with live vibrios were reduced more markedly than those in mice immunized orally by the feeding with killed antigens. These findings suggested that the elimination of vibrios from the organ was closely related to local IgA antibody response to heat-labile substance of live Vibrio cholerae.

Immune protective mechanisms during pregnancy. I. Cell-mediated immunity against Listeria monocytogenes in pregnant mice.

Characteristics of protective mechanisms during pregnancy were investigated using neonatally thymectomized (NTx) and/or pregnant mice infected with sublethal doses of Listeria monocytogenes, of which the explosive growth at an early phase of 2 or 3 days after infection is prevented by non-immune macrophages, and complete elimination at a late phase from 4 to 10 days after infection is attributed to the augmented functions of macrophages in co-operation with lymphokine-producing sensitized T lymphocytes. Although in virgin control mice there was a gradual decline of bacteria from the day after infection, viable bacteria in pregnant mice showed an increase in number until Day 3. In such pregnant mice, carbon clearance was suppressed. Thus, the enhanced bacterial growth in pregnant mice within 3 days may be attributable to the suppressed functions of non-immune macrophages. Complete elimination of Listeria from Day 4 was observed in pregnant sham-operated mice as well as in non-pregnant and pregnant NTx mice. Twenty-four hour reaction of delayed-type in normal mice induced by sheep red blood cells (SRBC) in incomplete Freund's adjuvant (IFA) was not affected by pregnancy, while 48 hr reaction in mice immunized with SRBC in complete Freund's adjuvant (CFA) was suppressed by pregnancy. We have reported previously that macrophage migration inhibitory factor (MIF) was produced in the latter but not in the former, and that the tuberculin type of delayed hypersensitivity accompanied by MIF production scarcely participated in acquired resistance to Listeria. Effective elimination of Listeria in pregnant and/or NTx mice at a late phase may be attributable to the activity of cellular immunity comparable to 24 hr reaction. These results suggest that T cells showing a low degree of thymus dependency in the ontogenic development may be the major component required for acquired protective immunity against Listeria and may account for the protection in pregnant mice.

Response of bacterial antigen in palmoplantar pustulosis.

Motilities of leukocytes in response to bacterial antigens or sera were examined in tissues from patients with palmoplantar pustulosis (PPP). Accelerated migration towards bacterial antigens was detected in the case of Staphylococcus epidermidis in 9 of 19 patients, to Propionibacterium acnes in 5 of 19, to Proteus mirabilis and Staphylococcus aureus in 3 of 19, while no acceleration was found in neutrophils from the controls. A significantly accelerated migration of normal lymphocytes in response to both patients' and control sera was nil. Inhibition of migration of guinea pig peritoneal exudate cells mixed with lymphocytes from PPP patients was detected in 12 out of 14 patients with the addition of S. epidermidis antigen and 8 of 14 with the addition of P. acnes antigen, while no such inhibition was detected in all 7 controls. The accelerated migration of neutrophils and inhibition of macrophages may participate in the development of PPP.

Depression of protective mechanism during the early phase of a viral infection in tumor-bearing mice and prevention by PSK.

Effector mechanisms responsible for resistance against ectromelia virus including antiviral activity of non-immune macrophages, antiviral antibody, delayed footpad reaction to viral antigen, and interferon induction after viral infection were depressed in BALB/c mice bearing syngeneic Meth A tumor. The degree of viral growth correlated well with the depression of delayed footpad reaction, antibody production, and interferon induction. Therefore, modification of macrophage functions by a tumor-bearing state and treatment with PSK may contribute to this modification of antiviral resistance, at an early phase of infection. Cytotoxic activity may not be the principal effector, since the cytotoxicity was induced in normal and tumor-bearing mice to almost the same extent yet an extensive viral growth occurred only in the latter.

Demonstration and characterization of immunosuppressive factors in sera from patients with Crohn's disease.

The effect of sera from 17 patients with Crohn's disease, 8 with ulcerative colitis or 5 with intestinal tuberculosis on the proliferative response of mouse spleen cells induced by phytohemagglutinin (PHA) was studied. Sera from patients with Crohn's disease markedly suppressed the blastogenesis of mouse spleen cells (S.I. = 6.8 +/- 2.0, % suppression = 83%), as compared with normal sera (S.I. = 41.0 +/- 5.2, p less than 0.001, % suppression = 0). Conversely, ulcerative colitis sera did not suppress the blastogenesis of mouse spleen cells (S.I. = 43.5 +/- 8.7, % suppression = -6%), nor the sera of intestinal tuberculosis (S.I. = 38.9 +/- 4.0, % suppression = 6%). Thus, we confirmed the possible existence of immunosuppressive factors in Crohn's disease. Moreover, immunosuppressive factors in Crohn's disease were characterized for biochemical properties. The approximate molecular weight is 45,000 estimated by diafiltration and gel filtration on a Sephadex G-75 column. Analytical isoelectric focusing showed an increased amount of acidic protein in fractionated sera (m.w. ranging 30,000-50,000) from patients with Crohn's disease and ulcerative colitis, in comparison with that in normal sera. Furthermore, the main peak of this acidic protein in Crohn's disease was an isoelectric point (pI) of 2.8, while the pI of that from ulcerative colitis was 3.0. These results suggest that qualitative differences of such acidic protein may serve to discriminate between the sera of Crohn's disease and ulcerative colitis.

Mechanisms of in vivo generation of cytotoxic activity against syngeneic tumours. I. Local differentiation of mature cytotoxic T lymphocytes in the rejection of tumours.

Mature cytotoxic T lymphocytes (CTL) were detected in the peritoneal cavity of syngeneic mice immunized intraperitoneally (i.p.) with mitomycin C (MC)-treated EL-4 or X5563 cells, but were not found in their spleens or lymph nodes. Mature CTL appeared among PE cells after transfer of spleen cells from those immune mice, along with MC-treated tumour cells, to the peritoneal cavity of syngeneic mice. These results lead us to the hypothesis that immature CTL primed in the spleen and lymph nodes may migrate to the site of tumour inoculation and differentiate into mature CTL after antigenic or non-specific stimulation at that site. Inability of primed CTL to differentiate to mature CTL in the spleen might be explained by the effect of splenic suppressor cells, since mature CTL became detectable in the spleen of immune mice by treatment with cyclophosphamide.

[Augmentation of resistance against metastatic tumor cells after local administration of PSK].

Local administration of PSK augmented the generation of cytotoxic lymphocytes and the induction of resistance against metastatic tumor. Augmented generation of cytotoxic lymphocytes may be ascribed to local effects of PSK in the lymph nodes, since this is mediated by Lyt-1+2+ cells. Local administration of PSK increased the threshold number of metastatic tumors eliminated by hosts. This finding seems to be important in relation to augmentation of resistance against metastasis or local implantation with a limited number of tumor cells.

Effect of PSK on interferon production in tumor-bearing mice.

The effect of PSK on the depressed interferon (IF) production in tumor-bearing mice was studied. In tumor-bearing mice, in vitro IF production by spleen cells treated with polyinosinic-polycytidylic acid (poly I:C) was remarkably inhibited. However, these inhibitions were prevented by the intraperitoneal (ip) administration of PSK. Mice were inoculated intravenously (iv) with poly I:C-treated spleen cells, administered with PSK ip at 3 days after the tumor inoculation. When PSK was not given, poly I:C-treated spleen cells did not show an inhibitory effect on tumor growth. In mice given PSK ip, poly I:C-treated spleen cells exerted slightly inhibiting effects on tumor growth. These results suggest that PSK prevented such a modulation in tumor-bearing mice.

[Depression of protective mechanisms against ectromelia virus infection in tumor-bearing mice and its prevention by PSK].

Effector mechanisms responsible for protection against ectromelia virus (EMV) including antiviral activity of non-immune macrophages, cytotoxic T cells, antiviral antibody, delayed footpad reaction to viral antigen and interferon induction after viral infection were depressed in BALB/c mice bearing syngeneic Meth A tumors. The degree of viral growth correlated well with the depression of delayed footpad reaction, antibody production and interferon induction. But a control level of these elements could be obtained by pretreatment of tumor-bearing mice, with PSK Cytotoxic activity may not be the principal effector, since cytotoxicity was induced in both normal and tumor-bearing mice to almost the same extent but an explosive viral growth was observed only in the latter. These results suggest that PSK was responsible for restoring the depressed antiviral protective immunity to normal levels in tumor-bearing animals.

Entrance of cholera enterotoxin subunits into thymus cells.

Analysis of the staining of cholera enterotoxin on the surface of cells with specific antibodies against each subunit of cholera enterotoxin, using a fluorescence-activated cell sorter and electron microscopy, showed that not only subunit A but also subunit B penetrates the cell membrane. The detection of subunits inside the cell was facilitated by the use of saponin, an agent that increases membrane permeability.

[Effect of PSK on the recovery of macrophage function and T cell-mediated immunity in tumor-bearing mice].

The effect of PSK on the depressed bactericidal activity of macrophages and delayed-type hypersensitivity (DTH) to Listeria monocytogenes in BALB/c mice bearing transplantable Meth A fibrosarcoma was studied. In tumor-bearing mice pretreated with PSK, L. monocytogenes was cleared rapidly from the circulating blood and bacterial growth in the liver was inhibited effectively in the early phase of infection. This resistance to the infection could be transferred with peritoneal exudate cells (PEC) but not with non-adherent PE cells of PSK-treated mice. In the early phase of infection, tumor-bearing mice developed a lower level of DTH to L. monocytogenes than did nongrafted control mice. However, the control levels of DTH could be obtained by pretreatment of tumor-bearing mice with PSK. These results suggest that the restoration of resistance to L. monocytogenes in tumor-bearing mice by PSK may be ascribed to both prevention of depression or activation of macrophage function and prevention of depression of T cell-mediated immunity.

Induction of impaired activation of lymphocytes by suppressive factor in Crohn's disease patients.

Lymphocyte proliferative response to phytohemagglutinin (PHA) and relevance of serum factors to the response were studied in 12 patients with Crohn's disease (CD). The lymphocyte proliferative response was markedly reduced in patients with Crohn's disease (S.I. = 38.8 +/- 36.8) (mean +/- SD), as compared with normal controls (S.I. = 100.6 +/- 28.6) (p less than 0.01). In addition, the effect of sera from patients with CD or 10 patients with ulcerative colitis (UC) who had extremely impaired lymphocyte responsiveness to PHA on the proliferative response of normal lymphocytes to PHA was also measured. Sera from CD patients had a marked suppressive effect on the blastogenesis of normal lymphocytes (S.I. = 46.4 +/- 28.5), as compared with normal sera (S.I. = 126.2 +/- 14.7) (p less than 0.001). On the other hand, UC sera did not suppress the blastogenesis of normal lymphocytes (S.I. = 114.9 +/- 27.7). Moreover, serum immunosuppressive acidic protein (IAP) levels in patients' sera were measured by single radial immunodiffusion assay. A marked increase in serum IAP levels was revealed both in CD patients (780 +/- 470 micrograms/ml) and in UC patients (601 +/- 278 micrograms/ml), as compared with normal controls (376 +/- 92 micrograms/ml) (p less than 0.001). But there was no precise correlation between the suppressive effect of sera and serum IAP levels in patients with CD. Thus, we demonstrated an impairment of the lymphocyte responsiveness to PHA in CD patients and the possible existence of immunosuppressive factors which is not identical with IAP in the sera from patients with CD.