Schistosoma mansoni. Anti-egg monoclonal antibodies protect against cercarial challenge in vivo.

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.

Influence of Olfactory Function on Appetite and Nutritional Status in the Elderly Requiring Nursing Care.

OBJECTIVE: To investigate olfactory function in elderly subjects requiring nursing care to clarify its association with appetite and nutritional status. SETTING: 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. MEASUREMENTS: 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. RESULTS: 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<0.01). The mean score was 8 or lower in both groups, demonstrating lower olfactory function in both groups. Regarding factors that may be associated with olfactory function, a significant difference was noted in the HDS-R (p<0.01), confirming significantly lower cognitive function in the elderly subjects requiring nursing care. No significant difference was noted in the CNAQ or BMI. Experiment II: On a univariate linear regression analysis, an association with the OSIT-J was noted for age and HDS-R. Age was inversely correlated and the HDS-R was positively correlated. Factors associated with lower olfactory function in the elderly subjects requiring nursing were age and cognitive function, whereas appetite, nutritional status, and food intake were not associated. CONCLUSION: Olfactory function in elderly subjects requiring nursing care was poorer than that in those not requiring nursing care, suggesting that aging and cognitive decline are associated with lower olfactory function. In addition, no association of lower olfactory function with appetite, nutritional status, or food intake was noted in the elderly subjects requiring nursing care.

Decreased capacity of aged mice to produce interferon-gamma in Legionella pneumophila infection.

We investigated the difference in natural resistance to Legionella pneumophila infection between aged (18-20-month-old) and young (3-month-old) mice of ddY strain. Aged mice were more susceptible to the bacterial infection than young mice; 50% lethal doses of L. pneumophila for aged and young mice were 2.2 x 10(7) and 8.5 x 10(7) colony forming units (CFU), respectively, after intraperitoneal injection of the bacteria. The bacterial burden in the livers was larger in aged than young mice after a challenge with a sublethal dose of L. pneumophila. However, peritoneal macrophages of aged mice paradoxically had a greater capacity to kill intracellular L. pneumophila than those of young mice. Interferon-gamma (IFN-gamma) production from naive spleen cells was compared after an in vitro stimulation with formalin-killed L. pneumophila. Spleen cells of aged mice produced significantly less IFN-gamma than those of young mice. When anti-murine IFN-gamma monoclonal antibody was administered before the bacterial infection, the subsequent bacterial burden in the livers significantly increased in young but not in aged mice. These data suggest that, in aged mice, IFN-gamma production is depressed at an early phase of L. pneumophila infection and it renders aged mice more susceptible to the infection.

Identification by monoclonal antibody of a major (28 kDa) surface membrane antigen of Schistosoma mansoni.

Monoclonal antibody M.1 was generated from mice immunized with membrane enriched extracts of mechanically transformed schistosomula. M.1 bound to the surface membranes of cercariae and young (0-24 h post-transformation) schistosomula but did not bind to older schistosomula or cultured worms. M.1 immunoprecipitated an antigen of approximate molecular weight 27-28 kDa from schistosomula. Experiments using metabolic labeling showed that the antigen was actively synthesized by developing schistosomula. Further M.1 immunoprecipitated a similar 27-28 kDa antigen from membrane-enriched extracts of miracidia, lung and adult worms as well as from schistosomula.

A low degree of requirement for Ia-positive macrophages and IL 2 in the induction phase of Listeria monocytogenes-specific T cells in vitro.

We have analyzed the priming process of Listeria-specific T cells using an in vitro primary culture system. Listeria-specific T cells mediating delayed footpad reaction (DFR) and acquired cellular resistance (ACR) upon passive local transfer into naive recipients were generated from non-immune mouse spleen cells when stimulated with viable Listeria monocytogenes primarily in vitro. The effectors were detected on the third day of culture, and culturing for 5 days was sufficient for the generation of effectors mediating the peak level of DFR and ACR. The requirement of T cell subsets, Ia antigen and interleukin 2 (IL 2) for inducing effectors was studied. Presence of macrophages (M phi) and their contact to T cells were required for priming of Listeria-specific T cells in vitro. The presence of Ia antigens on macrophages was absolutely required for priming, but this requirement was lower than that in secondary immune response of Listeria-specific T cells. Effectors could not be generated when L3T4+ cells were depleted, but effectors capable of conferring a full level of DFR and ACR were induced even after the depletion of Lyt2+ cells. Contribution of IL 2 to the generation of effectors during early phase of priming was not observed. IL 2 was not produced in the supernatant of the in vitro primary culture. Precursor cells of the effectors did not respond to exogenously added recombinant IL 2 (rIL 2). Some mechanisms operating in the induction phase of Listeria-specific T cells were clarified in this study.

Macrophage Ia expression in athymic nude versus neonatally thymectomized mice.

The requirement of the thymus for the production of Ia-inducing lymphokine was studied in athymic nude, neonatally thymectomized (NTx), and sham-operated (Sham) mice. The peritoneal macrophages from NTx mice immunized with viable Listeria monocytogenes 14 days previously contained as high a proportion of Ia-bearing macrophages as those from Sham mice, while those from athymic nude mice contained only a small proportion. Intraperitoneal injection of a culture supernatant derived from immune spleen cells of NTx mice induced Ia-rich exudates in recipient normal mice just as well as did a corresponding supernatant from cells of Sham mice, but that from cells of athymic nude did not. The production of Ia-inducing lymphokine in culture supernatants of immune spleen cells from both NTx and Sham mice was abolished by pretreatment of cells with anti-Thy-1.2 antibody plus complement. These results suggest that a T cell subset responsible for the production of Ia-inducing lymphokine requires the presence of the thymus for just a short period in the ontogenic development.

In vitro primary induction of T cells mediating delayed footpad reaction and acquired cellular resistance to Listeria monocytogenes.

We established an in vitro system generating L. monocytogenes-specific T cells primarily from unprimed spleen cells of mice. Normal spleen cells were cultured for 5 days in the presence of L. monocytogenes in vitro. Viable cells were harvested and assessed for their capacity to confer acquired cellular resistance (ACR) and delayed footpad reaction (DFR) upon local passive transfer to naive syngeneic recipient mice. When normal spleen cells were stimulated with viable L. monocytogenes, the viable cells that were recovered after 5 days of culture conferred a high level of ACR and DFR. Negative selection revealed that the effector cells obtained in primary in vitro culture were Thy 1+, L3T4+, Lyt2- cells. T cells mediating ACR could not be generated in the culture of normal spleen cells with heat-killed bacteria; however, cells mediating only DFR were generated in the presence of a large number of killed L. monocytogenes. The expression of DFR and ACR by T cells generated in this primary culture system was Listeria-specific; reactions were not observed against unrelated bacterial antigens including S. typhimurium, S. aureus, E. coli and PPD. FACS analysis of the cells in culture showed that L3T4+ and Lyt2- T cells were being enriched during culture. The primary generation of antigen-specific T cells in vitro was also possible with spleen cells from NTx mice but not with cells from nude mice, suggesting the presence of Listeria-specific precursors in NTx mice.

Ultraviolet-B irradiation alters cytokine production by immune lymphocytes in herpes simplex virus-infected mice.

Previous studies from our laboratories have shown that UV-B irradiation at the site of an intradermal infection of herpes simplex virus (HSV) resulted in a higher incidence of zosteriform lesions and suppressed cellular immune responses to HSV in mice. In order to determine whether the production of T-cell-derived cytokine (IFN-gamma, IL-2 and IL-4) by immune cells from irradiated mice is also suppressed, we examined the production of cytokines by lymph node cells and spleen cells taken from UV-B irradiated, HSV type 1 (HSV-1)-infected mice. UV-B irradiation (120 mJ/cm2) prior to HSV-1 infection was found to markedly suppress IFN-gamma production compared to that of the non-irradiated control. IL-4 production was enhanced compared to IL-2 in the UV-B irradiated mice. These results suggest that alteration(s) in the cytokine production profile may therefore be involved in the development of severe skin lesions caused by HSV infection in UV-B irradiated mice.

Difference in cholesterol-binding and cytolytic activities between listeriolysin O and seeligeriolysin O: a possible role of alanine residue in tryptophan-rich undecapeptide.

We have constructed recombinant listeriolysin O (rLLO) and seeligeriolysin O (rLSO) from Listeria monocytogenes and Listeria seeligeri, respectively. In hemolysis and cholesterol-binding assays, the specific activity of recombinant toxin was lower for LSO as compared to LLO. To understand the molecular basis of this difference, in particular with respect to the conserved Trp-rich undecapeptide, a naturally occurring Ala to Phe substitution in LSO was introduced into rLLO. The rLLO:A488F hemolysin exhibited a reduced activity in both hemolysis and cholesterol-binding. The reverse mutation, inserted into rLSO, also increased the hemolytic activity of this mutant LSO. These results suggested that the natural replacement of Ala to Phe is involved in the weak cytolytic activity of LSO.

Killing mechanism of Listeria monocytogenes in activated macrophages as determined by an improved assay system.

Exposure of Listeria monocytogenes to gentamicin 5 mg/L for 4 h resulted in the killing of most extracellular bacteria, but had no effect on the survival of bacteria inside macrophages. Higher concentrations of gentamicin caused a reduction in the number of intracellular bacteria. This effect was associated with cellular uptake of gentamicin, but was unaffected by activation of macrophages by interferon-gamma and lipopolysaccharide. In experiments in which exposure to gentamicin 5 mg/L for 4 h was used to kill extracellular bacteria, killing by activated macrophages was impaired when O2- production was inhibited by superoxide dismutase, but not when nitric oxide production was blocked by NG-monomethyl-L-arginine. These data suggest that the reactive oxygen intermediates are more important than nitric oxide in the killing of L. monocytogenes, at least in macrophages activated in vitro.

Antibody-independent protection against Pseudomonas aeruginosa infection in mice after treatment with a homologous strain vaccine.

Formalin-killed cells of Pseudomonas aeruginosa strain M-24 elicited an antibody-independent protective effect against P. aeruginosa infection in mice. The effect was observed as early as 6 h after administration and 100% protection was obtained by 48 h. The protective effect could not be attributed to the production of specific antibody. In M-24-treated mice, the bacteria in the peritoneal cavity, blood and liver were eliminated 12 h after P. aeruginosa infection. This suggested that the protective effect was due to enhanced bacterial elimination. The percentage of macrophages in the peritoneal cavity was increased after M-24 administration. Furthermore, the enhanced bacterial elimination was abrogated by treatment of mice with 60Co-irradiation or carrageenan. These findings suggest the involvement of macrophages in the enhanced bacterial elimination observed. The chemiluminescence of peritoneal exudate cells from M-24-treated mice was markedly increased when compared with that of cells from untreated mice. The ability to kill P. aeruginosa in vitro was also greater in macrophages from mice treated with killed M-24 than in cells from proteose-peptone-treated mice. The M-24-treated mice showed enhanced nonspecific protection against infection with lethal doses of P. aeruginosa, Escherichia coli or Listeria monocytogenes. However, susceptibility to LPS in mice was not increased by M-24 treatment. These results suggest that macrophage activation without increasing LPS susceptibility was responsible for the antibody-independent protection induced by killed M-24.

Involvement of various combinations of endogenous inflammatory cytokines in Listeria monocytogenes-induced expression of inducible nitric oxide synthase in mice.

Using a in vitro infection of spleen cells with Listeria monocytogenes, the relationship between endogenous cytokines and the expression of inducible nitric oxide synthase (iNOS) was examined. When all interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha, or the combination of IFN-gamma with either TNF-alpha or IL-1 alpha were neutralized by antibodies, there was a significant reduction of iNOS expression and nitrite production in culture. However, there was no reduction of iNOS expression and nitrite production when these cytokines were individually neutralized. After the depletion of natural killer cells, there was no change in the expression of Listeria-induced iNOS and nitrite production although the IFN-gamma production was abrogated. Neutralization of TNF-alpha and IL-1 alpha in natural killer cell-depleted culture resulted in the reduction of iNOS expression. Thus, various combinations of cytokines to play an important role in iNOS induction by L. monocytogenes.

Overgrowth and translocation of Escherichia coli from intestine during prolonged enteral feeding in rats.

Our objective was to examine changes in bacterial flora in the gastrointestinal tract that might lead to their translocation during the prolonged enteral administration of an elemental liquid diet. Eleven rats (experimental group) received a feeding gastrostomy and were administered an intragastric infusion of a liquid elemental diet for 17 days, while 9 rats (control group) received chow and water ad libitum for 17 days. The animals were then killed and the mesenteric lymph nodes (MLNs), liver, jejunum, ileum, and cecum were aseptically removed, homogenized, and cultured for isolation of bacteria. A statistically significant increase in the population of a single species of Enterobacteriaceae, Escherichia coli, was observed in the ileum and cecum of the experimental group fed the liquid diet as compared with the control group. Five of the MLNs from the 11 experimental rats (45%) were positive for E. coli, as compared with none from the 9 control rats, a statistically significant difference (P = 0.038). The E. coli isolated from the MLNs and the ileum of the experimental group exhibited the O168:H serotype. Our results suggest that E. coli isolated from the MLNs of the experimental group had translocated from the intestine. The animal model described here may be useful in screening for drugs to reduce the problem of translocation of intestinal bacteria.

Specific immune response to the 40-kDa outer-membrane protein of Porphyromonas gingivalis in mice.

Whether B-cell activation to Porphyromonas gingivalis in periodontal diseases is the consequence of a specific immune response or is due to non-specific polyclonal activation is still not clear. Here the immune response of mice to a purified 40-kDa recombinant protein antigen, a major outer-membrane protein specific to P. gingivalis, was investigated. Patients' sera strongly reacted to this recombinant antigen. Purified splenic T and B cells from mice immunized with 40-kDa antigen or from normal mice were reconstituted in vitro and cultured in the presence or absence of the P. gingivalis 40-kDa protein antigen and antigen-presenting cells (APCs). In vitro antibody production to this particular antigen was analysed with respect to the requirement of helper T cells and APCs by enzyme-linked immunosorbent assay. The results clearly showed that an effective secondary response required the presence of B cells, T cells and APCs. In the absence of CD4+ helper T cells, an antibody response to the 40-kDa protein was not observed. In addition, the requirement of H-2 restricted, Ia-positive APCs was evident for an adequate response to the 40-kDa protein of P. gingivalis. Thus the antibody response to the 40-kDa protein of P. gingivalis was generated in an immunologically antigen-specific manner and was not simply the result of polyclonal B-cell activation. This in vitro system could be used in the detection of antigen-specific memory B or T cells in patients with periodontal diseases.

[The mechanism of induction and expression of protective immunity with special reference to the role of cytokines].

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.

[Mechanisms of induction and expression of anti-tuberculous immunity].

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.

[Mechanisms of pathogenicity and host defense in infections by intracellular parasitic microbes].

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.

[Problems in the host defense system against bacterial infection].

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.

[Escape mechanism of intracellular parasitic bacteria and prospect of new approach to infection control].

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.

[Protective immunity against intracellular parasitic bacteria].

Facultative intracellular bacteria are resistant to the killing mechanism inside macrophages by virtue of various escape mechanisms. Activation of macrophages by cytokines is the key event to overcome of bacterial escape in macrophages of the infected host. Generation of TH1 type of antigen-specific T cell is the essentially required for the macrophage activation. This short review summarizes the escape mechanism of activated macrophages and the mechanisms involved in the generation of TH1 cells.