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To investigate olfactory function in elderly subjects requiring nursing care to clarify its association with appetite and nutritional status. Facility for the elderly requiring nursing care. Participants: The subjects were 158 elderly people requiring nursing care and 37 elderly people not requiring nursing care. Experiment I: Olfactory function and factors (cognitive function, appetite, and nutritional status) that may be associated with it were compared between the elderly subjects requiring nursing care and those not requiring nursing care using covariance analysis in consideration of age. For evaluation, the OSIT-J was used for olfactory function, the HDS-R for cognitive function, the CNAQ for appetite, and BMI for nutritional status. Experiment II: The subjects were the same elderly subjects requiring nursing care in Experiment I, and food intake was surveyed in addition to the OSIT-J, HDS-R, CNAQ, and BMI. A univariate linear regression analysis was performed with OSIT-J as the response variable, and age, HDS-R, CNAQ, BMI, and food intake as the explanatory variables. Experiment I: On covariance analysis, the OSIT-J score was significantly lower for the elderly subjects requiring nursing care than for those not requiring nursing care (p

Viable and killed BCG were compared for the ability to induce nitric oxide (NO) production in normal spleen cells and peritoneal exudate macrophages. Stimulation of spleen cells with viable BCG resulted in a strong expression of inducible NO synthase followed by an enhanced production of nitrite. However, those responses were not induced after stimulation with killed BCG or when macrophages were stimulated with killed and viable BCG. The same level of TNF-alpha mRNA expression was observed in reverse transcription-PCR after stimulation of spleen cells with viable and killed BCG. However, IFN-gamma production was induced only when spleen cells were stimulated with viable BCG. Concurrent stimulation of rIFN-gamma with either viable or killed BCG resulted in a strong nitrite production by macrophages. Neutralization of IFN-gamma and TNF-alpha caused a complete inhibition of nitrite production. Furthermore, anti-asialo GM1 Ab plus complement treatment abolished IFN-gamma production after stimulation with viable BCG, indicating that the NK cell was the major source of IFN-gamma, and its production was triggered only by stimulation with viable BCG. The present study showed that the ability of viable BCG to induce NO production is superior to that of killed BCG, and both IFN-gamma and TNF-alpha are essential for BCG-induced NO production by spleen cells. NK cells appeared to be important as a source of IFN-gamma, and the insufficient NO induction by killed BCG was due to the inability to induce IFN-gamma from NK cells.

We searched for a functional marker with which T cells mediating acquired cellular resistance (ACR) can be discriminated from those mediating delayed-type hypersensitivity (DTH) in mice immunized with Mycobacterium bovis BCG. Four wk after injection of the mice with 10(5) viable BCG (vBCG) cells emulsified in IFA, a passive transfer experiment revealed that T cells mediating DTH as well as ACR, which we designated TACR, were generated in the spleen. In contrast, T cells mediating only DTH (TDTH) were generated by immunization with 10(7) killed BCG cells along with IFA. The transferring ability of both TACR and TDTH was substantially reduced by treatment with anti-Thy-1.2 or anti-CD4 mAb plus complement, whereas anti-CD8 treatment had no effect. To determine the functional differences between TACR and TDTH, we assessed IL-2 and IFN-gamma production from TACR and TDTH after stimulation with PPD in vitro. Similar levels of enhanced IL-2 activity were detected in the culture supernatants of both groups of T cells. However, augmented production of IFN-gamma was observed only in TACR. This finding was confirmed by Northern blot analysis for detection of IFN-gamma-specific mRNA. In addition, a significant increase in the number of IFN-gamma-producing cells was observed only in T cells from mice immunized with vBCG. The production of IFN-gamma was also totally abolished by treatment with anti-Thy-1.2 and anti-CD4 mAb plus complement in vitro, whereas anti-CD8 mAb treatment had no effect. These results suggest that CD4+ protective T cells generated by immunization with vBCG are characterized by the ability to produce IFN-gamma after stimulation with specific Ag.

Listeriolysin O produced by a hemolytic strain of Listeria monocytogenes was purified from the ammonium sulfate precipitate of a culture supernatant through the steps of ion-exchange chromatography and gel filtration. The purified hemolysin finally gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 58,000. When peritoneal exudate macrophages were stimulated with purified hemolysin, we found a high level of IL-1 activity as determined by thymocyte costimulator assay in the culture supernatant. Cell-associated and intracellular IL-1 activity was also detected. The activity in the supernatant or membrane was blocked by polyclonal antibody to murine IL-1α. Moreover, IL-1-specific mRNA expression could be detected in the macrophages stimulated with listeriolysin O by Northern blot analysis. Possible contamination by LPS of the listeriolysin O preparation did not seem to contribute to the induction of macrophage IL-1 production.

We have previously reported that the lethal toxicity of 5-fluorouracil (5-FU) in specific-pathogen-free mice is due to an intestinal infection with indigenous Escherichia coli induced by the drug (K. Nomoto, T. Yokokura, Y. Yoshikai, M. Mitsuyama, and K. Nomoto, Can J. Microbiol. 37:244-247, 1991). In the present study we demonstrate that nonspecific immunostimulation is effective in the protection of mice from the lethal indigenous infection induced by 5-FU. Intravenous or subcutaneous injection of a preparation of heat-killed Lactobacillus casei YIT 9018, a potent nonspecific immunostimulant, into BALB/c mice reduced the lethal toxicity of 5-FU at doses ranging from 338 to 800 mg/kg of body weight if YIT 9018 was injected 7 to 40 days before administration of 5-FU. Systemic infection with E. coli developed in all of the 5-FU-treated control mice 7 days or more after administration of 5-FU in large doses and was accompanied by overgrowth of the bacteria in the intestinal tract. Pretreatment of mice with YIT 9018 resulted in a decreased occurrence of systemic infection with E. coli to levels of 0 to 20% and no significant changes in the population levels of E. coli in the intestinal tract during the 14 days after administration of 5-FU. The levels of leukopenia in the spleen and peripheral blood were lower, and recovery of granulocyte-macrophage precursor cells in the spleen and femur began earlier in the treated animals than in the 5-FU-treated controls. Intravenous transfusion of syngeneic normal bone marrow cells or spleen cells into the mice at an early period after administration of 5-FU diminished markedly the occurrence of the lethal indigenous infection, suggestion that an earlier recovery from chemotherapy-induced myelosuppression is important in the mechanisms of protection of the host from the infection.

mAb M.1 was previously shown to recognize a 28-kDa Ag in all stages of the human helminth parasite, Schistosoma mansoni, and to bind to the surface membranes of newly transformed schistosomula in a transient manner. Here we demonstrate that M.1 passively transfers partial resistance (41-49%) to cercarial challenge in naive mice. Thus, the 28-kDa Ag recognized by M.1 is a putative vaccine candidate. After immunoaffinity purification, tryptic digests of the 28-kDa Ag were prepared and individual peptides were sequenced. Amino terminus sequences of tryptic peptides of the 28-kDa Ag had high (79-87%) sequence homology with the mammalian glycolytic/gluconeogenic enzyme triosephosphate isomerase (TPI). Purified, native 28-kDa Ag from adult parasites was shown to function enzymatically in an analogous manner to yeast and mammalian TPI in the reverse reaction. Addition of M.1 antibody to the enzyme reaction altered the catalytic activity of schistosome TPI. To determine the immunologic cross-reactivity of this vaccine candidate with mammalian TPI, Western blot analysis was performed and demonstrated that M.1 was immunologically specific for the schistosome enzyme.

CD4+ T cells mediating both delayed-type hypersensitivity (DTH) and acquired cellular resistance (ACR) were generated in mice after immunization with viable Listeria monocytogenes. In contrast, CD4+ T cells from mice immunized with killed L. monocytogenes in complete Freund's adjuvant were capable of mediating only DTH but not ACR. To determine the functional difference between T cells mediating DTH and T cells mediating ACR, we examined two different populations of T cells for profiles of lymphokine production after stimulation with a specific antigen in vitro. The production of interleukin-2 (IL-2) and IL-3 but not IL-4 was observed in both T cells mediating only DTH and those mediating DTH and ACR. In this respect, both types of T cells could be categorized into the TH1 population, and they produced macrophage chemotactic factor equally well. However, the production of gamma interferon (IFN-gamma) was observed only in T cells capable of mediating both DTH and ACR. This result was confirmed not only by an enzyme immunoassay specific for murine IFN-gamma but also by Northern (RNA) analysis for the detection of IFN-gamma mRNA. These results suggested that the TH1 population may be subdivided further into two distinct subsets and that the ineffectiveness of the killed bacterial vaccine may be partly explained by the dissociated development of T cell function.

We developed a modified trail-making test using a PC and touch panel and compared it with the Mini Mental State Examination (MMSE). Methods and Patients: The test consisted of a series of numbers from 1 to 36, randomly arranged across the display. The object of the test was for the subject to touch the numbers in order, beginning with 1 and ending with 36, in as little time as possible. The system consisted of a PC and a liquid crystal display (LCD) touch-panel screen. One hundred and thirty-four patients with dementia performed the test. Sixty of the 134 patients (15 male, 45 female; average age, 81.1 r 7 years) were diagnosed as having Alzheimer's disease and the others had cerebrovascular dementia. Results: Sixty-two of 134 patients (23 male, 39 female; average age, 77.6 r 8 years; MMSE score, 21.5 r 5.6 points) completed the test. The correlation coefficient between test performance time and MMSE score was -0.534. Discussion: This test may also be a useful indicator of focal frontal lesions and can be used as an early screening test for Alzheimer's disease. I. INTRODUCTION n an aging society, the number of dementia patients is increasing. A major problem of dementia, especially of the Alzheimer's type, is that it is fatal, and early diagnosis is necessary for both patient and caregiver. There are several treatments available, either with or without medication. Early diagnosis is essential to initiate early treatment. Computed tomography, magnetic resonance imaging, single photon emission computed tomography, and positron emission tomography are all used as diagnostic tools. Qualitative tests, such as the Mini Mental State Examination (MMSE), are commonly used to screen for the disease. A computer-based MMSE has been developed in which questionnaire results are converted to a digital format. MMSE is a widely used method for assessing cognitive mental status. The evaluation of cognitive functioning is important in a clinical setting because of the recognized high prevalence of cognitive impairment. As a clinical instrument, the MMSE has been used to detect impairment, follow the course of an illness, and monitor response to treatment. The MMSE has also been used as a research tool to screen for cognitive disorders in epidemiological studies and follow cognitive changes in

Intracellular parasitic bacteria are capable of escaping from the intracellular killing inside macrophages by virtue of highly sophisticated molecular mechanism. Because of the escape from phagocytic defense, infection caused by these bacteria is difficult to be controlled even in the presence of normal host defense system. In order to understand the mechanism of intracellular parasitism of particular types of bacteria, the basic mechanism of intracellular killing inside phagocyte and the strategy of escape by some representative intracellular bacteria are reviewed. Based on the mechanism of T-cell dependent protective immunity, some possible approaches to the infection by intracellular bacteria, especially to tuberculosis, have been discussed.

Mycobacterium tuberculosis is one of the intracellular parasitic bacteria escaping the intracellular killing inside macrophages. The aim of this symposium was to get some insight into the mechanism of pathogenicity and host defense in M. tuberculosis infection, which has not yet been elucidated well, by the presentation of up-to-date knowledge on these aspect in infection with different intracellular parasitic microbes. Dr. Yoshikai (Nagoya Univ.) indicated that TLR is involved in the initial response of host against S. choleraesuis. Among the cytokines contributing to the induction of specific immunity, the importance of IL-15 was emphasized, based on their own experimental data using IL-15 transgenic mice and the application of anti-IL-15 antibody in vivo. Dr. Yoshida (Kyushu Univ.) reviewed the mechanisms of intracellular growth of Legionellae. Several genes so far identified as essential genes in intra-macrophage growth appeared to be similar to those encoding type 3 secretion system observed in Shigellae. There is a significant strain difference in the growth of L. pneumophila inside macrophages and such difference seemed to be under the control of a gene at chromosome 13, Lgn 1. The investigation of difference in the mode of escape among various Legionella. spp. may provide a novel mechansim in bacterial invasion and escape. Dr. Kawamura (Kyoto Univ.) summarized some new reports on the molecular mechanism of the inhibition of P-L fusion by M. tuberculosis. He emphasized the importance of the alteration in phagosomal maturation as indicated by the accumulation of TACO protein. The possible involvement of TLR in the recognition of Mycobacterial cells and its LAM was discussed. Dr. Kawakami (Ryukyu Univ.) first discussed the possibility that Cryptococcus neoformans, a fungal pathogen, could be regarded as one of the intracellular parasitic microbes. His presentation mainly focused on the TH1-Th2 balance in the expression of host defense against C. neoformans in mice. From their experimental infection using attenuated strain TC-13 in various cytokine-knock out mice, the pivotal role of both IL-12 and IL-18 was clearly indicated.

The induction of anti-tuberculous immunity highly depends on the cytokines produced endogenously at the initial stage of immunization. Among several cytokines, IFN-gamma appears to be the most important to generate antigen-specific Th1 type of protective T cells in mice. IL-12 and IL-18, which are produced by macrophages in response to virulent mycobacteria, are responsible for stimulating NK cells to produce IFN-gamma. Once antigen-specific Th1 cells are generated, Th1-dependent macrophage activation was effective in the elimination of infected bacteria through enhanced production of reactive oxygen intermediates and reactive nitrogen intermediates. In Listeria monocytogenes, one of the intracellular bacteria, listeriolysin O (LLO) appeared to be responsible for the induction of endogenous IFN-gamma from NK cells. The possible mechanisms operating in the induction and expression of anti-tuberculous immunity are discussed with special reference to cytokine responses. An application of LLO to the induction of protective immunity is also discussed.

It is known that only listeriolysin O (LLO)-producing Listeria monocytogenes strains are able to induce protective immunity, but the underlining relationship between LLO produced by virulent strains and generation of protective immunity in the infected host remains poorly understood. In the present study, it was found that LLO gene expression was only detected in the mice infected with virulent strain which was able to induce protective immunity, while non-virulent strains or killed bacteria were not able to generate protective immunity. When mice were immunized with LLO plus killed bacteria in the presence of incomplete Freund's adjuvant, the protective immunity was partially generated, and adoptive transfer experiment confirmed that this protection was antigen specific. Reverse-transcription polymerase chain reaction revealed that LLO plus killed bacteria induced the expression of interferon-gamma (IFN-gamma) and interleukin-12 (IL-12). Our results also showed CD4+ T cells were the principal cells constituting protective immunity. Taken together, it may be concluded that LLO produced from virulent strains of L. monocytogenes was essential for the generation of protective immunity, and that LLO plus killed bacteria induced IFN-gamma and IL-12 expression which resulted in the generation of protective immunity.

Skin lesions of atopic dermatitis were examined for cytokine expression by reverse transcription-polymerase chain reaction. The profile of mRNA for various cytokines revealed that both T(H1) and T(H2) types of CD4+ T cells, probably including T(H0) type, infiltrate into the skin lesion. We observed that atopic skin lesions improved after varicella infection. In such lesions, expression of T(H1) type cytokines predominated. The peripheral blood T cells from atopic patients exhibited a differentiation into T(H2) type cells upon in vitro stimulation with mite antigen. In contrast they differentiated into T(H1) type cells upon stimulation by varicella antigen. Since IL-12 has been reported to switch the in vitro recall response of allergen-specific T cells of atopic donors from a T(H2)- to a T(H1)-like phenotype, we examined its local production in varicella lesions. IL-12 p35 and p40 mRNA were expressed in fresh lesions. Peripheral blood mononuclear cells from atopic patients expressed p40 mRNA upon in vitro stimulation with live varicella zoster virus, but they did not show p40 mRNA without stimulation. This finding suggested that in atopic skin lesions containing the virus, IL-12 was produced and the cell type was changed to T(H1) type-predominance. These results suggested that patients with atopic dermatitis always have highly reactive CD4+ T cells infiltrating into their skin, and that the switch to T(H1) or T(H2) dominance is related to whether the lesion is improved or exacerbated.

Listeria monocytogenes-specific, IFN-gamma-producing CD4+ T cells are induced in 5-day in vitro cultures of naive spleen cells with viable L. monocytogenes (VLM) but are not induced in cultures with heat killed L. monocytogenes (HKLM). Anti-IL-12 Abs abrogated the induction of the IFN-gamma-producing CD4+ T cells by VLM, suggesting an importance of IL-12 in the induction of IFN-gamma-producing CD4+ T cells. Accordingly, IL-12p40 was expressed by macrophages (M phi) from the cultures of whole spleen cells with VLM but not with HKLM, although HKLM induced IL-12p40 expression on M phi when nonadherent cells were removed before culture. In vivo analysis also showed that VLM induced IL-12p40 expression by M phi, whereas HKLM failed to induce it. On the other hand, the addition of anti-IL-10 mAb into cultures of whole spleen cells and HKLM resulted in an increase of IL-12p40 expression by M phi. Furthermore, we detected an expression of IFN-gamma by both adherent and nonadherent spleen cells at the early stage of stimulation with VLM before the appearance of IFN-gamma-producing CD4+ T cells. These data suggest that HKLM induce nonadherent spleen cells to produce IL-10 which may down-regulate IFN-gamma-producing CD4+ T cells by blocking IL-12 production by M phi. In contrast, VLM support IFN-gamma production by CD4+ T cells by stimulating M phi to produce IL-12 and IFN-gamma.

The mechanism of induction of protective T cells mediating the anti-tuberculous immunity is not well elucidated. Using viable and killed cells of Mycobacterium bovis BCG and Listeria monocytogenes, which differ in the ability to induce specific protection in mice, we have found that the difference is mainly due to the different ability to induce several cytokines especially IFN-rho not due to the difference in antigenic property. It was shown that the induction of protective T cells is highly dependent upon various cytokines produced by the host at the early stage of immunization. Based on our own experimental result in mice, the relative contribution of several cytokines to the induction and expression of cell-mediated immune protection was discussed.

Various types of defects in the host defense system are involved in bacterial infection especially in so-called compromised host. Among the humoral factors of defense system, complement system and immunoglobulins are indispensable. Phagocytes are the most important cellular factor in the effective phagocytosis and intracellular killing of ingested bacteria through the formation of ROI, RNI and by the release of antibacterial peptides including defensin or serprocidin family. T lymphocytes are specially required when the infection is caused by intracellular parasitic bacteria for activation of macrophages through the production of several cytokines or expression of cytotoxic effect. gamma delta T cell may be involved in the first line defense. The functional significance of each factor of host defense was discussed with special reference to the escape mechanisms of bacteria.

To obtain some insight into the interaction between listeriolysin O (LLO) and the macrophage membrane, we examined the effect of purified Listeria monocytogenes hemolysin on the viability and functions of mouse peritoneal exudate macrophages. The study showed that purified LLO impaired a variety of functions of the macrophages. First, it suppressed the luminol-dependent chemiluminescence response of macrophages. Second, it suppressed the phagocytic ingestion of opsonized sheep erythrocytes and latex beads. Third, exposure of macrophages to LLO resulted in an increase in dead cells, as determined by the trypan blue dye exclusion method. An interesting observation of this study is that the LLO-induced production of interleukin-1 from macrophages could not be blocked by preincubation with cholesterol, while the membrane-damaging ability could be blocked by cholesterol. The dissociation of the blocking effects of cholesterol suggests that the interleukin-1-inducing ability of LLO may be distinct from its membrane-damaging ability.

T-cell-mediated immunity to Listeria monocytogenes in mice, as determined by delayed-type hypersensitivity and acquired resistance, was induced by immunization with viable bacteria but not with killed bacteria, even when killed cells were injected in a high dose or repeatedly. T cells obtained from mice immunized with viable L. monocytogenes were readily stimulated with killed-bacterial antigens, resulting in T-cell proliferation in vitro and expression of a delayed footpad reaction in vivo. After immunization with killed-bacterial vaccine, T-cell responsiveness to interleukin 2 (IL-2) never developed but a lower level of responsiveness to IL-1 appeared later than with T cells from mice immunized with viable bacteria. When IL-1 production by macrophages was examined in vitro, viable L. monocytogenes stimulated a high level of IL-1 release while killed bacteria did not. Avirulent strains which were ineffective in the induction of T-cell mediated immunity were incapable of inducing IL-1 production as well. The impaired ability of killed bacteria to stimulate IL-1 production was confirmed by the level of IL-1 mRNA expression. These results suggested that the ineffectiveness of killed L. monocytogenes vaccine is not due to loss or lack of antigenic epitopes but may be ascribed to insufficient induction of IL-1 production in the initial stage of the immune response in vivo.

Monoclonal antibody M.2 binds to the surface membranes of cercariae and developing schistosomula. This antibody was generated from mice immunized with membrane-enriched extracts of mechanically transformed schistosomula. The antigen detected by M.2 was shown to persist on developing schistosomula for at least 96 hr post-transformation. M.2 also bound to the surface of living, cultured lung worms but not to freshly harvested lung worms. The ability of M.2 to bind to cultured lung worms coincided with the loss of host H-2 from the parasite surface. The apparent m.w. of the antigen was 22,000; an antigen with the same apparent m.w. was immunoprecipitated from cercariae, schistosomula, lung worms, and adult worms.

We investigated the pregnancy-induced changes in macrophage activity which are important in the expression of host defense against infections. Several macrophage functions were examined by using Listeria monocytogenes. In pregnant mice, prolonged survival and enhanced in vivo elimination of bacteria were observed in the early phase of primary infection. Functions of peritoneal macrophages, including in vitro phagocytosis intracellular killing, glucose consumption, generation of superoxide anion, and intracellular beta-glucuronidase activity were shown to be enhanced in pregnant mice. These findings indicate that pregnancy enhances macrophage functions qualitatively. Possible mechanisms for this enhancement and the significance of macrophage activation for pregnant hosts are discussed.

By the use of a culture supernatant containing a lymphokine capable of inducing Ia-rich peritoneal macrophages in vivo, we investigated the effect of increased macrophage Ia expression on the induction of antibody response to sheep erythrocytes (SRBC) in mice. Peritoneal macrophages from mice injected intraperitoneally (i.p.) with lymphokine 3 and 4 days earlier contained a high proportion of Ia-bearing macrophages without any significant increase in their phagocytic activity for SRBC. In lymphokine-treated mice, slight enhancement was observed in the primary IgG plaque-forming cell (PFC) response only at an early stage after i.p. immunization with SRBC. When primed mice were injected with lymphokine before secondary i.p. immunization with SRBC, the secondary IgG PFC response was greatly enhanced. On the other hand, lymphokine treatment did not affect either the primary or the secondary IgM PFC response. This effect was dependent on both timing of lymphokine injection and route of secondary immunization with SRBC. Secondary immunization via several routes other than the i.p. route never resulted in such an enhanced secondary IgG PFC response. These results suggest that lymphokine-induced Ia-rich peritoneal macrophages, which first encounter the antigen in the peritoneal cavity, play an important role in the modification of the subsequent antibody response.

Release of O2- and H2O2 from isolated rat liver Kupffer cells was studied by making use of the methods of SOD sensitive ferricytochrome c reduction and horseradish peroxidase catalysed scopoletin oxidation, respectively. Kupffer cells from BCG treated rats showed a 1.8 times significantly higher O2- release and a 2.4 times higher H2O2 release as compared to the controls. Moreover the yield of Kupffer cells was also increased with administration of BCG. These results suggest that Kupffer cells can be immunologically activated to secrete larger amounts of O2- and H2O2.

The protective mechanisms against Listeria monocytogenes were analysed in mice. Listeria immune mice were rechallenged with viable listeria, and the degree of activation of macrophages, the degree of accumulation of macrophages to the infected sites, and these mechanisms were studied. Enhanced acquired resistance to the reinfection became detectable from the early stage of immunization. It was observed that activation of macrophages was mediated by pretreatment with antigen from the early stage of immunization. Enhanced accumulation of macrophages at the infected sites was observed only in the early stage. Enhanced accumulation and activation of macrophages in the early stage were generated by mediators from listeria antigen sensitized T lymphocytes which were different from classical tuberculin type. Enhanced activation of macrophages in the later stage was generated by MAF and/or MIF from sensitized T lymphocytes of classical tuberculin type. It was suggested that acquired cellular resistance to listeria in the early stage depends on both activation and accumulation of macrophages, and that in the later stage depends on activation of macrophages.

Legionella pneumophila is known to grow intracellularly in resident peritoneal macrophages of guinea pigs. The present study was done to determine what kinds of macrophage stimulants are able to activate guinea pig macrophages to inhibit intracellular growth of the organism. Peritoneal macrophages were harvested from healthy guinea pigs, from guinea pigs injected intraperitoneally with proteose peptone (PP) or thioglycolate medium, from guinea pigs injected intraperitoneally with live Mycobacterium bovis BCG or killed Propionibacterium acnes (Corynebacterium parvum), and from guinea pigs surviving infection with live L. pneumophila. After in vitro phagocytosis, the L. pneumophila CFU in each well were counted on charcoal-yeast extract agar plates. In the macrophages elicited by PP or thioglycolate medium, the organism grew as well as it did in resident macrophages. In BCG-activated and immune macrophages, growth was inhibited almost completely. In P. acnes-activated macrophages, the initial growth of L. pneumophila was inhibited to some extent, but its growth reached the same level as in the resident and PP-induced macrophages after 3 or 4 days of culture. In the lethal challenge experiments in vivo, the superior protection provided by BCG over P. acnes was ascertained and the importance of macrophages in resistance to L. pneumophila was confirmed. Difference of activation by BCG and P. acnes in relation to the inhibition of intracellular growth of L. pneumophila in guinea pig macrophages is discussed.

Monoclonal antibodies that bind to surface membranes of developing schistosomula and/or cercarial tails were generated from mice immunized with living schistosome eggs or soluble egg antigen. These monoclonal antibodies detected at least three different surface epitopes. One surface antigen detected by anti-egg monoclonal antibody EG1C4B1 (E.1) persisted on the surface of developing schistosomula for 96 h posttransformation . The same or a cross-reactive antigen was also detected on the surfaces of S. japonicum and S. haematobium schistosomula and cercarial tails. Monoclonal antibody E.1 killed schistosomula in vitro as well or better than infected mouse sera and transferred immunity to naive mice when administered in vivo. The monoclonal antibody reduced the number of lung worms recoverable on day 4 postchallenge by up to 85% and reduced the adult worm burden up to 41% as compared with controls. The data also show that the molecular weights of the egg antigens detected by monoclonal antibody E.1 were different from those detected on schistosomula.

Protective mechanisms against Listeria monocytogenes were studied in young (3-month-old) and old (15-month-old) mice of C3H/He strain. Cumulative mortality rates of old mice were lower than those of young mice after intravenous inoculation of the same doses of bacteria. The numbers of bacteria in the liver and spleen on days 1 and 3 were larger in young than old mice. Bacterial growth at this stage of infection is suppressed by accumulation of non-immune macrophages. On day 7, however, the numbers of bacteria in the liver were smaller in young than old mice. Bacterial elimination at this stage depends upon immune macrophages. These results suggest that the enhanced resistance to lethal effects of bacteria in old mice may be ascribed to activation of non-immune macrophages in the presence of depressed capacities to raise cell-mediated immunity.

Using a local passive transfer system, we found that effector T cells mediating delayed-type hypersensitivity (DTH) but not acquired cellular resistance (ACR) to Listeria monocytogenes (strain EGD) were generated in mice immunized with killed Listeria, although immunized mice did not express DTH or ACR. When non-adherent cells of peritoneal, lymph node, or spleen cells from mice immunized with killed Listeria were transferred into the footpad of naive recipient mice along with eliciting antigen, positive delayed footpad reaction (DFR) was elicited. However, there was no evident protection against challenge at the site of the local transfer. Cells from mice immunized with viable Listeria conferred significant degrees of DFR and ACR on the recipients. DFR transferred by cells immunized with killed Listeria was mediated by L3T4+ T cells in an antigen-specific manner. The antigen-specific proliferative response of T cells from mice immunized with killed Listeria was much lower than that of T cells from mice immunized with viable Listeria. The production of macrophage chemotactic factor (MCF) by cells from killed Listeria-immune mice was much the same as that by cells from viable Listeria-immune mice. In contrast, the production of interleukin-2 (IL-2) and macrophage activating factor (MAF) was much lower in cells from killed Listeria-immune mice. The elimination of L. monocytogenes (strain L461), a strain of low virulence, was enhanced at the site of DFR transferred with cells from killed Listeria-immune mice. These results suggest that stimulation with killed bacteria is effective for the generation of DTH-mediating effector T cells, and that different effector T cells mediating DTH or ACR are involved in cell-mediated immunity to L. monocytogenes.

To determine whether the presence of bacterial flora contributes to the ontogenic development of macrophage function, the ability of macrophages to release superoxide anion (O2-) in response to stimulation with phorbol myristate acetate was compared in conventional and germfree mice of various ages after birth. One-week-old conventional mice showed a very low level of O2- release by their macrophages, and gradual increases were observed in 2-, 3-, and 4-week-old mice in an age-dependent manner. Macrophages from germfree mice always showed a significantly lower level of O2- release compared with conventional mice of the same age; however, age-dependent functional development was seen also in the germfree group. The poor level of O2- release by macrophages from adult germfree mice could be restored to more than the level by conventional mice when the mice were conventionalized for 3 weeks. These results suggested that the ontogenic development of macrophage function is not controlled by the presence of bacterial flora but that the full-scale expression of function at each age is under the influence of microflora.

The protective roles of alveolar macrophages and polymorphonuclear cells were analyzed against intratracheal challenge with Candida albicans in mice. When mice were treated with carrageenan, a known cytotoxic agent for macrophages, there was no change in susceptibilities to the challenge in terms of the survival and the progressive elimination of fungi from the lung and kidney, in spite of a decreased in vitro phagocytosis of Candida albicans by their alveolar macrophages. On the other hand, irradiated mice (whole body irradiation with 800 rads) showed an enhanced mortality and a progressive growth of Candida albicans in their lungs and kidneys, although no change was observed in the in vitro phagocytic activity of alveolar macrophages until day 6 after irradiation. In normal and carrageenan treated mice, there was a progressive increase in the recruitment of polymorphonuclear cells into the lung after the challenge as shown by bronchoalveolar lavage and histological examination. In irradiated mice, on the other hand, there was a decreased recruitment of polymorphonuclear cells at 24 hr after the challenge, and a complete impairment at a late stage. When phagocytes were obtained from normal mice and examined for in vitro phagocytic activity to Candida albicans, polymorphonuclear cells showed higher activity than that of alveolar macrophages. These results suggest that polymorphonuclear cells play a very important role in the protection against intratracheal infection with Candida albicans.

The comparative effect of immunization by intratracheal and intraperitoneal routes was assessed against intratracheal challenge with Candida albicans. When mice were immunized intratracheally with 5 X 10(6) cfu of viable or heat-killed C. albicans 2 weeks before challenge, they showed an enhanced resistance against intratracheal challenge with C. albicans, in spite of a decrease in phagocytosis of C. albicans in vitro by alveolar macrophages from intratracheally immunized mice. In these mice, 100% survival and an accelerated elimination of C. albicans from their lungs were observed after intratracheal challenge with C. albicans. In contrast, intratracheal immunization with same high dose of viable or heat-killed C. albicans 1 week before challenge, and intraperitoneal immunization with same high or low doses of viable or heat-killed C. albicans 1 or 2 weeks before intratracheal challenge with C. albicans, could not induce such high resistance against intratracheal challenge with same high dose of viable C. albicans. Comparing to normal and intraperitoneally immunized mice, number of C. albicans recovered from the lung 48 hr after challenge was approximately 100 fold smaller in mice immunized by intratracheal route 2 weeks before challenge. When cellular response was determined by bronchoalveolar lavage after intratracheal inoculation of C. albicans, a prominent recruitment of polymorphonuclear cells was observed 12 and 36 hr after challenge in control mice, intraperitoneally and intratracheally immunized mice. Among these groups, sustained polymorphonuclear cell recruitment by 72 hr after challenge was seen only in the group immunized by intratracheal route with high dose of heat-killed C. albicans 2 weeks before challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatally thymectomized (NTx) mice, whose ability to mount antigen-specific cell-mediated immunity is reported to be generally defective, were found to be capable of mounting a normal level of acquired cellular resistance (ACR) and delayed footpad reaction (DFR) to Listeria monocytogenes. The present study was done in order to determine the functional differences of T cells contributing to the protection against L. monocytogenes between NTx and sham-operated mice. In mice immunized with viable L. monocytogenes, the absolute number of splenic T cells was significantly lower in NTx mice compared with sham-operated mice. When the ability of immune T cells to transfer ACR and DFR was examined by passive transfer, lymphocytes from immune NTx mice conferred a higher level of ACR and DFR on naive recipient mice, despite the marked difference in total number of T cells compared with immune Sham mice. Antigen-specific proliferation and interleukin-2 (IL-2) production by splenic T cells from immune NTx mice were significantly lower than in those from immune Sham mice. The proliferative response of T cells to exogenous IL-2 was also lower in NTx group. These results suggest that the requirement for the IL-2-driven T-cell proliferation system is basically low in the generation of effector T cells specific for L. monocytogenes.

Meth A tumor-bearing mice were examined for changes in the host defense mechanism against infection with Listeria monocytogenes. The resistance of tumor-bearing mice to systemic, i.e., intravenous, infection in an early phase of the infection was suppressed for 1-3 days after tumor implantation (5 x 10(5) cells/mouse, subcutaneously), but was augmented thereafter even on the 35th day as compared with normal mice. Suppression and enhancement of the resistance of tumor-bearing mice to primary infection with L. monocytogenes was also observed in tumor-bearing athymic nude mice. Splenic macrophages from tumor-bearing mice on the 14th day after tumor implantation exerted potent bactericidal activity against L. monocytogenes in vitro as compared with those from normal mice. The function of macrophages as nonimmune scavenger cells seemed to be activated in mice bearing a progressing tumor. However, some of the tumor-bearing mice challenged with a sublethal dose of L. monocytogenes showed diminished resistance in the late phase of the infection; moreover listeria-immunized tumor-bearing mice showed less resistance to a secondary challenge with the bacteria than did normal immunized mice. This suppression of the specific immune response of tumor-bearing mice to L. monocytogenes was shown also in the assay of the delayed-type footpad reaction. The bacterial growth-inhibiting function of listeria-immune T lymphocytes, determined by the effect of adoptive transfer of the cells on the growth of Listeria, was also reduced in tumor-bearing mice as compared with normal mice.