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.

Modification of allergic inflammation in murine model of rhinitis by different bacterial ligands: involvement of mast cells and dendritic cells.

BACKGROUND: It has been suggested that airway bacterial infections exacerbate allergic disorders, and bacterial components in the air affect allergic inflammation via Toll-like receptors expressed on mast cells and dendritic cells in the airway mucosa. OBJECTIVE: Peptidoglycan (PGN) is a major component of the bacterial cell wall. We investigated the effect of PGN on the effector phase of allergic inflammation, in comparison with the effect of CpG-oligodeoxynucleotides (CpG), which is known to be a Th1 adjuvant. METHODS: Ovalbumin (OVA)-sensitized mice were challenged intranasally with OVA alone or OVA together with PGN or CpG. Nasal allergic symptoms and eosinophilia were scored, and the OVA-specific cytokine response was examined in the cells of cervical lymph nodes and nasal mucosa. Bone marrow-derived mast cells (BMMCs) and dendritic cells (BMDCs) were stimulated with PGN or CpG in vitro, and the expression level of cytokines and chemokines was examined by RT-PCR. In addition, the expression level of chemokines was examined by RT-PCR in mast cells of OVA-sensitized mice challenged with OVA alone or OVA together with PGN or CpG. RESULTS: PGN exposure exacerbated the nasal allergic symptoms and eosinophilia, whereas CpG exposure suppressed them. In addition, PGN exposure increased the OVA-specific IL-4 response in the cells, whereas CpG exposure decreased it. On the other hand, there were no significant differences in the OVA-specific IFN-gamma response. PGN but not CpG induced the expression of thymus and activation-regulated chemokine (TARC) and macrophage/monocyte-derived chemokine (MDC) in both BMMCs and mast cells of mice sensitized and challenged with OVA. CpG but not PGN induced the expression of IFN-beta and interferon-inducible protein-10 (IP-10) in BMDCs, and histamine did not influence this effect. CONCLUSION: These results demonstrate that PGN exposure exacerbates allergic inflammation mainly via mast cells, whereas CpG exposure suppresses allergic inflammation mainly via dendritic cells.

Listeriolysin O derived from Listeria monocytogenes inhibits the effector phase of an experimental allergic rhinitis induced by ovalbumin in mice.

Listeriolysin O (LLO) derived from Listeria monocytogenes is highly capable of inducing interleukin (IL)-12, IL-18 and interferon (IFN)-gamma, and facilitates the generation of Th1 cells. We have recently shown that recombinant LLO (rLLO) inhibits generation of ovalbumin (OVA)-specific Th2 immune response by skewing maturation of antigen-specific T cells into Th1 cells. In the present study, we investigated the effect of rLLO on the effector phase of Th2-dependent allergic rhinitis in BALB/c mice sensitized with OVA. In mice sensitized intraperitoneally and challenged intranasally with OVA, nasal allergic symptoms such as sneezing and nose-scratching were observed at a high frequency. A high titre of anti-OVA IgE antibody was detected in sera and a large number of eosinophils migrated into the nasal tissue. However, rLLO treatment during the intranasal challenge inhibited the allergic symptoms, production of anti-OVA IgE antibody and eosinophil infiltration. Though rLLO did not affect antigen-specific cytokine production from splenic CD4(+) T cells, rLLO significantly suppressed OVA-specific IL-4 and IL-5 production from nasal mononuclear cells. We further found that rLLO inhibited the recruitment of CD4(+) T cells in nasal mucosa, and diminished the transcription and cell surface expression of CCR4 on splenic CD4(+) T cells. Moreover, rLLO was able to inhibit the passive cutaneous anaphylaxis reaction mediated by anaphylactic antibodies (IgE and IgG(1)) and mast cells. Taken together, these data showed that rLLO suppresses the effector phase of allergic rhinitis by inhibition of Th2 cell recruitment to nasal mucosa and type I allergic reaction.

New computer-based cognitive function test for the elderly.

We developed a modified trail-making test using a PC and touch panel and compared it with the mini mental state examination (MMSE). 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 +/- 7 years) were diagnosed as having Alzheimer's disease and the others had cerebrovascular dementia. Sixty-two of 134 patients (23 male, 39 female; average age, 77.6 +/- 8 years; MMSE score, 21.5 +/- 5.6 points) completed the test. The correlation coefficient between test performance time and MMSE score was -0.534. 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.

Listeriolysin O, a cytolysin derived from Listeria monocytogenes, inhibits generation of ovalbumin-specific Th2 immune response by skewing maturation of antigen-specific T cells into Th1 cells.

Listeriolysin O (LLO), a cholesterol-dependent cytolysin derived from Listeria monocytogenes, is a potent inducer of interleukin (IL)-12, IL-18 and interferon (IFN)-gamma. We have shown that LLO facilitates development of T cells mediating protective immunity against L. monocytogenes through the induction of IFN-gamma production at an early stage. Based on this finding, it is postulated that LLO inhibits differentiation of Th2 cells and the Th2 immune response. By using a murine model of ovalbumin (OVA)-induced allergic rhinitis, we investigated whether LLO has an ability to modulate the Th2-type immune disorder. In mice sensitized intraperitoneally with ovalbumin (OVA)/alum and challenged intranasally with OVA, a large number of eosinophils migrated into the nasal tissue, and high titres of anti-OVA IgE and IgG(1) antibodies were detected in sera. However, LLO treatment during sensitization markedly inhibited the eosinophil infiltration and production of these anti-OVA antibodies. A large number of T cells from mice sensitized and challenged with OVA produced high level of IL-4 and IL-5 but not IFN-gamma after stimulation with OVA. In contrast, OVA-specific IFN-gamma-producing T cells were preferentially induced in mice treated with LLO at the time of sensitization. In the absence of LLO administration, the expression level of GATA-3 and SOCS-3 in CD4(+) T cells was enhanced after sensitization with OVA. LLO treatment resulted in a reduction of GATA-3 and SOCS-3 expressions but induced the transcription of T-bet instead. Taken together, these data show clearly that LLO is capable of inhibiting Th2 immune response by skewing differentiation of antigen-specific T cells into Th1 cells.

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.

Sporothrix schenckii thermo-intolerant mutants losing fatal visceral infectivity but retaining high cutaneous infectivity.

Four mutants of the Sporothrix schenckii lung isolate IFM 41598 were isolated by their inability to form colonies on a Sabouraud glucose agar plate incubated at 37 degrees C for 4 days. In contrast to the parent IFM 41598, these thermo-intolerant mutants were all defective in producing fatal visceral infections in mice, even though they retained infectivity in footpad tissues with a small fungal inoculum (approximately equals 10 cfu).

[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.

Essential role of domain 4 of pneumolysin from Streptococcus pneumoniae in cytolytic activity as determined by truncated proteins.

Pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae, is one of the members of thiol-activated cytolysins (TACYs) consisting of four domains. TACYs commonly bind to membrane cholesterol and oligomerize to form transmembrane pore. We have constructed full-length and various truncated PLYs to study the role of domains of PLY in the cytolytic activity. Full-length PLY had binding ability to both cell membrane and immobilized cholesterol. A truncated PLY which comprised only domain 4 molecule, the C-terminal domain of PLY, sustained the binding ability to cell membrane and cholesterol, whereas domain 1-3 molecule had no binding ability to them. Furthermore, the domain 4 molecule inhibited both the membrane binding and the hemolytic activity of full-length PLY. Accordingly, the present results provided the direct evidence that domain 4 was essential for the initial binding to membrane cholesterol and the interaction led to the subsequent membrane damage process.

Role of macrophage scavenger receptors in response to Listeria monocytogenes infection in mice.

Type I and type II macrophage scavenger receptors (SR-A I/II) recognize a variety of polyanions including bacterial cell-wall products such as lipopolysaccharide, suggesting a role for SR-A I/II in immunity against bacterial infection. SR-A I/II-deficient (MSR-A-/-) mice were more susceptible to infection with listeriolysin-O (LLO)-producing Listeria monocytogenes. After infection, Kupffer cells in wild-type (MSR-A+/+) mice phagocytized larger numbers of Listeria than those in MSR-A-/- mice. The number and the diameter of hepatic granulomas were larger in MSR-A-/- mice than MSR-A+/+ mice. L. monocytogenes replicated at higher levels in the liver of MSR-A-/- mice compared with MSR-A+/+ mice, and macrophages from MSR-A-/- mice showed impaired ability to kill Listeria in vitro. However, macrophages from MSR-A+/+ and MSR-A-/- mice showed similar levels of listericidal activity against isogenic mutant L. monocytogenes with an inactivated LLO gene. The listerial phagocytic activities of MSR-A+/+ macrophages treated with an anti-SR-A I/II antibody (2F8) and MSR-A-/- macrophages were significantly impaired compared with untreated MSR-A+/+ macrophages, indicating that SR-A I/II function as a receptor for L. monocytogenes. Electron microscopy revealed that most L. monocytogenes had been eliminated from the lysosomes of MSR-A+/+ macrophages in vivo and in vitro. In contrast, L. monocytogenes rapidly lysed the phagosomal membrane and escaped to the cytosol in MSR-A-/- macrophages and in MSR-A+/+ macrophages treated with 2F8 before phagosome-lysosome fusion. These findings imply that SR-A I/II plays a crucial role in host defense against listerial infection not only by functioning as a receptor but also by mediating listericidal mechanisms through the regulation of LLO-dependent listerial escape from the macrophages.

[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.

[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.

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.

The role of Kupffer cells and regulation of neutrophil migration into the liver by macrophage inflammatory protein-2 in primary listeriosis in mice.

Depletion of mouse Kupffer cells and splenic macrophages following intravenous administration of liposome-entrapped clodronate severely reduced host resistance to primary infection with Listeria monocytogenes. Infection of clodronate-treated mice with a sublethal dose of L. monocytogenes resulted in death of the mice within 3 days. The macrophage depletion resulted in marked increases in bacterial growth in the liver and spleen, but not in other tissues. The proliferation of L. monocytogenes was observed in a large number of hepatocytes that underwent apoptosis. Infiltration of neutrophils in the liver and rapid formation of microabscesses were observed in the control mice after L. monocytogenes infection. However, there was less accumulation of neutrophils in the liver of Kupffer cell-depleted mice than in the control mice. Expression of macrophage inflammatory protein-2 (MIP-2) was enhanced in the livers of both the control and Kupffer cell-depleted mice after L. monocytogenes infection. MIP-2 was also induced in a murine hepatocyte cell line following L. monocytogenes infection. The administration of neutralizing anti-interleukin-8 receptor homolog antibody severely abrogated neutrophil infiltration into the Listeria-infected mouse liver. Anti-MIP-2 antibody moderately reduced neutrophil infiltration and microabscess formation in the liver. These findings indicate that Kupffer cells protect hepatocytes from L. monocytogenes infection and the resultant apoptosis. Moreover, MIP-2 and its related molecules produced by the infected hepatocytes regulate neutrophil infiltration and microabscess formation in primary listeriosis.

Induction of protective T cells against Listeria monocytogenes in mice by immunization with a listeriolysin O-negative avirulent strain of bacteria and liposome-encapsulated listeriolysin O.

Only listeriolysin O (LLO)-producing strains of Listeria monocytogenes generate protective immunity in mice. Based on the findings that endogenous gamma interferon (IFN-gamma) production was induced only by such strains and that purified LLO could induce IFN-gamma from NK cells, we have postulated that LLO may play a pivotal role in the induction of Th1-type protective T cells, which are highly dependent on IFN-gamma. In this study, mice were immunized with L. monocytogenes ATCC 15313, an LLO-nonproducing avirulent strain, along with LLO encapsulated in liposome (LLO-liposome). LLO-liposome was highly potent in the induction of various cytokines, including IFN-gamma. Immunization of mice with either LLO-liposome or the viable strain ATCC 15313 alone did not induce protection against challenge infection. In contrast, the combination of LLO-nonproducing bacteria plus LLO-liposome induced a significant level of protective immunity mediated mainly by Th1-type cells capable of producing a large amount of IFN-gamma in an antigen-specific manner. The protection afforded by the combination was not dependent on LLO-specific cytotoxic T cells. These results support the idea that the inability of an LLO-nonproducing avirulent strain or killed bacteria to induce the generation of protective T cells is due not to the lack of a central T-cell epitope(s) but to the lack of ability to induce the production of endogenous cytokine during the early stage of immunization; the results also suggest that an appropriate use of LLO at least in an animal model may be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria.

Involvement of CD4+ T cells and macrophages in acquired protection against infection with Sporothrix schenckii in mice.

The cell-mediated immune response against infection with Sporothrix schenckii was investigated in vivo and in vitro. Following primary infection, mice were protected against a secondary subcutaneous inoculation with S. schenckii as determined by the local growth of fungi, and subcutaneously immunized mice were able to survive a lethal intravenous infection. Protection could be transferred to naive congenitally athymic nude mice using lymph node cells taken from immune mice. When immune lymph node cells had CD4+ cells depleted by antibody plus complement, the ability to transfer protection was significantly reduced. Treatment of mice with carrageenan, a macrophage blocker, before and after secondary inoculation abolished protection. An in vitro system, using either immune lymph node cells alone or macrophages alone, failed to kill the organism. However, inhibition of fungal growth was observed when both immune lymph node cells and macrophages were combined. Growth inhibition in vitro was reduced after depletion of CD4+ cells but not CD8+ cells. Addition of anti-interferon (IFN)-gamma monoclonal antibodies (MAb) also reduced growth inhibition. By using reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was shown that immune lymph node cells expressed message for IFN-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 after stimulation with heat-killed S. schenckii. These results suggest that acquired immunity against S. schenckii is expressed mainly by macrophages activated by CD4+ T cells.

[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.

Characteristic infectivity of Sporothrix schenckii to mice depending on routes of infection and inherent fungal pathogenicity.

Isolates of Sporothrix schenckii were examined for their infectivity in BALB/c mice. The mice were injected with yeast forms of S. schenckii isolates differing in clinical source (human cutaneous lesions and pulmonary lesions), and fungal growth was determined at intervals in the footpad and visceral organs. After subcutaneous injection of approximately 10 colony forming units (cfu) of S. schenckii into the footpad, locally restricted fungal infection developed gradually. At the peak of the infection (3-4 weeks post-inoculation), viable fungal counts reached 102-106 cfu/footpad. Dissemination to other tissues and visceral organs was not observed. After intravenous or intraperitoneal injection of 106 cfu of yeast forms, three of four isolates from cutaneous sporotrichosis were unable to establish infection and were eliminated from blood and visceral organs. The development of systemic infection was observed only with S. schenckii isolates obtained from the human lung lesion. Thus, inherent properties of each clinical isolate and routes of infection were shown to be critical for the establishment of systemic infection in spite of the remarkably strong infectivity of S. schenckii to the cutaneous tissue.