Signature-tagged mutagenesis of Vibrio vulnificus.

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.

Pantoea agglomerans lipopolysaccharide maintains bone density in premenopausal women: a randomized, double-blind, placebo-controlled trial.

Lipopolysaccharide fromPantoea agglomerans (LPSp) facilitates Ca and P turnover in chicken calvaria and femurs. This study investigated osteoporosis prevention by the oral administration of LPSp in mice and in double-blind clinical tests. Using ovariectomized (OVX) osteoporosis mice model, we investigated the effects of LPSp on the bone density and Ca concentration after ingesting LPSp-containing water for 4 weeks. Oral administration of LPSp tended to suppress the decline in the bone density and the cortical bone thickness in the OVX mice. Moreover, the Ca concentrations were maintained in the OVX-LPSp mice. The effects of LPSp on bone turnover were tested in randomized and double-blind clinical test subjects, who were healthy women aged 40-79 years. The subjects ingested either soy milk without LPSp (control group) or with LPSp (LPSp group) for 3 months. The results showed that the LPSp group on premenopause maintained their bone density compared with the control group pre- and postmenopause. Moreover, these effects were maintained for 2 months postobservation. LPSp maintains bone volume and density in vivo. Thus, a combination of soy milk and LPSp may be useful for osteoporosis prevention.

Effects of interactions between intestinal microbiota and intestinal macrophages on health.

Macrophages reside in every tissue of the body and play an important role in maintaining homeostasis. The intestinal mucosa is the largest immune organ and harbors macrophages in abundance. Dysfunction of intestinal macrophages is characteristic of patients with certain inflammatory bowel diseases. Although intestinal macrophages exhibit hyporesponsiveness to foreign substances, including various bacterial products, their physiological functions are unknown, but may be related to the contribution of intestinal bacteria to the maintenance of various physiological functions of the host. Moreover, recent reports suggest that there are associations between intestinal microbiota and the onset of pathologies, such as diverse metabolic syndromes, depression, and cancer. Evidence indicates that the host's immune response to intestinal microbiota may be etiologically-linked to these diseases; however, the mechanisms are poorly understood. In the present review, we discuss the possibility that intestinal microbiota influence health through the function of intestinal macrophages.

A mixture of Salacia oblonga extract and IP-PA1 reduces fasting plasma glucose (FPG) and low-density lipoprotein (LDL) cholesterol levels.

At present, lifestyle-related diseases are one of the most critical health issues worldwide. It has been reported that lipopolysaccharide derived from a Gram-negative bacteria (IP-PA1) symbiotic with wheat exhibited several advantageous biological effects, such as the reduction of plasma glucose levels in NOD mice and low-density lipoprotein (LDL) levels in WHHL rabbits. In this study, the beneficial effects on plasma glucose and lipids of a tea (SI tea) consisting of IP-PA1 and Salacia (which contains an inhibitor of α-glucosidase) were investigated in the KK-Ay/TaJcl type 2 diabetic model mice and in human subjects with premetabolic syndrome in a double-blind, randomized study. SI tea significantly decreased plasma glucose levels in KK-Ay/TaJcl mice. A clinical trial of SI tea was performed with 41 subjects between the ages of 40 and 69, who belonged either to a high plasma glucose group (HG: FPG 100-125 mg/dl) or to a hyperlipidemia group (HL: TG ≥ 150 mg/dl, or LDL ≥ 120 mg/dl, or HDL < 40 mg/dl). These subjects ingested either Salacia without IP-PA1 (the control) or SI tea. Blood samples were collected at 0, 30, and 60 days after initiating SI tea treatment, and were measured for FPG, HbA1c, TG, LDL, and HDL. These results showed that SI tea reduced FPG and HbA1c more rapidly than the control in the HL group, and also significantly improved LDL and HDL levels in the HG group. Thus, SI tea may be helpful in preventing lifestyle-related diseases.

Lipopolysaccharide IP-PA1 from Pantoea agglomerans prevents suppression of macrophage function in stress-induced diseases.

Chronic psychological stress impairs health and induces various diseases by causing an imbalance in the immune, neuropsychiatric and endocrine systems. The primary reason for the development of stress-induced disease is suppression of macrophage function, which plays a pivotal role in innate immunity. In fact, surgical stress has been shown to exacerbate opportunistic infections by significantly suppressing macrophage function. Conversely, administration of macrophage activating substances before surgery, such as tumor necrosis factor (TNF)-α or Picibanil (OK-432), has been shown to protect against macrophage suppression and the resulting exacerbation of infectious diseases, and against tumor metastasis in the lungs. Thus, if suppression of macrophage function by stress could be safely prevented by use of a macrophage activating substance, the detrimental side effects of stress could be reduced. Recently, we identified a lipopolysaccharide, IP-PA1, derived from Pantoea agglomerans, a symbiotic Gram-negative bacteria found in wheat and other food plants. Oral administration of IP-PA1 demonstrated macrophage activation (priming) and protective effects against infection, allergy and cancer, without any side-effects. In this review, the possibility of using IP-PA1 as a safe, macrophage activating substance for prevention of stress-induced impairments is discussed.

Mechanism for maintaining homeostasis in the immune system of the intestine.

Every organism possesses a mechanism for maintaining homeostasis. We have focused on the immune system as a system that helps maintain homeostasis of the body, and particularly on the intestine as the largest organ of immunity in the body. We have also focused our research on the mechanism that responds to foreign substances in the intestine, especially the toll-like receptors (TLR). The activation of myeloid differentiation primary response gene 88 (MyD88) signal transduction as a response to TLR in the intestine is believed to contribute to the maintenance of homeostasis of the body through the homeostasis of the intestine. Furthermore, significant findings were reported in which signal transduction from TLR4 was essential for the maintenance and regulation of the intestine. These results strongly suggest the possibility that homeostasis in the intestine is maintained by TLR4, and signaling by TLR4 after exposure to lipopolysaccharide (LPS) probably has a role in regulating homeostasis. It is expected that the prevention and treatment of various diseases using TLR4 will continue to develop. As LPS is a substance that enhances the activity of TLR4, it will also attract attention as a valuable substance in its own right.

Intestinal macrophages involved in the homeostasis of the intestine have the potential for responding to LPS.

Recently, there has been interest in the tertiary functions of food, those that maintain human health. Moreover, lipopolysaccharides (LPS), which are components of Gram-negative bacteria, have been found to be highly effective in activating innate immunity and have been rediscovered as new functional food materials. In this review, we discuss the significance of LPS as a food component with reference to these tertiary functions based on recent findings. There is special emphasis on the plasticity of responses to LPS by intestinal macrophages. According to the macrophage-network theory, local macrophages cooperate with other tissue macrophages. For this reason, this review also discusses the possibility that information is transferred throughout the body from intestinal macrophages.

Intracellular localization of CD14 protein in intestinal macrophages.

BACKGROUND: Our research is focused on intestinal macrophages, which are believed to contribute to the maintenance of intestinal homeostasis. In addition, intestinal macrophages are unique in that there is an impairment of expression of tumor necrosis factor (TNF) from lipopolysaccharide (LPS). This characteristic can be attributed to the lack or poor level of expression of toll-like receptor 4 (TLR4) or CD14 on the membrane of intestinal macrophages. We therefore decided to identify where CD14 was localized in intestinal macrophages. MATERIALS AND METHODS: The endoplasmic reticulum and Golgi apparatus were double stained and the intracellular localization in the intestinal macrophages was observed using a confocal laser microscope. RESULTS: CD14 of peritoneal macrophages was expressed both in the endoplasmic reticulum and Golgi apparatus. By contrast, intestinal macrophages expressed very little CD14 on the cellular membrane. CD14 was present in the endoplasmic reticulum of intestinal macrophages, but was rare in the Golgi apparatus. CONCLUSION: The lack of expression of CD14 on the cell membrane of intestinal macrophages may be caused by transport interference from the endoplasmic reticulum to the Golgi apparatus.

Suppression of response to foreign substances by intestinal macrophages.

Macrophages play an important role in the maintenance of homeostasis by changing their function according to the tissue and environment everywhere in the body. We have proposed that intestinal macrophages, which exist in the front line receiving environmental information, have an important function in forming a macrophage network for biophylaxis. In this review, we introduce intestinal macrophages as an example of the highly plastic and flexible cells adaptable to environmental information. Intestinal macrophages are hyporesponsiveness to foreign substances, especially lipopolysaccharide (LPS), and less expression of CD14 and TLR4/MD-2, receptors for LPS. However, those proteins expression was observed in the cytoplasm of intestinal macrophage. We also found that intestinal macrophages treated with IgA could restore in response to LPS. In conclusion, intestinal macrophages possess the plasticity to respond sensitively to change in their environment and are considered to be involved inflammatory bowel disease development.

Treatments for the activating macrophages that reduces surgical stress and postoperative mortalities from bacterial infections and tumor metastases.

BACKGROUND: Some of the mortalities caused by infectious diseases and/or distant metastases following surgery are thought to be due to immunological suppression. For this reason, techniques that reduce immunological suppression following surgery may reduce mortalities and/or incidences of micrometastases in distant organs. MATERIALS AND METHODS: Mice were anesthetized and their peritoneal cavities were opened for 30 min. Immunological suppression was estimated by the presence of tumor necrosis factor-a (TNF) after injection with OK-432 (dead bacterial bodies). The mice were administered with either Staphylococcus aureus or cancer cells of Meth A fibrosarcoma. Survival times and lung metastastic foci were then observed at 3 weeks. Results were compared for mice with or without treatment by OK432 or TNF prior to surgery. RESULTS: While significant suppression of TNF production was observed after laparotomy, administration of a macrophage-activating agent (TNF or OK-432) 3 h prior to laparotomy prevented immune suppression after the laparotomy. Laparotomy increased mortalities from bacterial infections and promoted the number of lung metastases. By contrast, administration of TNF or OK-432 3 h prior to the laparotomy decreased mortalities and metastases after the laparotomy. CONCLUSION: These results suggest that appropriate activation of macrophages prior to surgery is a method to reduce some of the detrimental effects caused by surgical operations.

Inherent potential for production of tumor necrosis factor-alpha by human intestinal macrophages.

BACKGROUND AND AIMS: Tumor necrosis factor (TNF) production by the macrophages in intestines appears to play a critical role in the pathogenesis of Crohn's disease (CD). However, it is reported that resident intestinal macrophages (both colonic and small-bowel) do not produce TNF after lipopolysaccharide (LPS) stimulation. It has not yet been proven whether or not intestinal macrophages have an inherent potential to produce TNF. The purpose of this study is to answer this question. MATERIALS AND METHODS: Colonic macrophages were isolated from lamina propria of human large intestine and stimulated with a variety of substances: LPS, a lipid A derivative (ONO-4007), killed Streptococcus bacterial body (OK-432), phorbol 12-myristate 13-acetate, and lectins (pokeweed mitogen and Sarcophaga lectin). RESULTS: Colonic macrophages were phenotypically negative for CD14 and positive for CD68 and produced very little TNF in response to LPS, as reported previously. Of the substances tested, only Sarcophaga lectin, which is a defense protein of fleshflies (Sarcophaga peregrina), induced TNF production by the intestinal macrophages. In addition, when the colonic macrophages were cultured on immunoglobulin-A-coated dishes, their characteristic response to LPS was altered, and they produced TNF at a level 6.6 times higher than when on collagen-coated dishes. CONCLUSION: Colonic macrophages have an inherent ability to produce TNF. Activation of colonic macrophages by unknown substances may contribute to the induction of TNF production, which causes the intestinal inflammation of CD.

Unique molecular characteristics of the environmental responses of mucosal macrophages.

Macrophages are thought to be the cells that initially respond to environmental information and transmit this information to other immune cells. We hypothesize that there is a "network system" consisting of various tissue macrophages; the macrophages respond to stimulation and transmit secondary information to neighboring cells, which is important for the maintenance of homeostasis. Macrophages exist in all animal organs as tissue macrophages, and their cellular characteristics may change as an adaption to tissue-specific environments. It is believed that mucosal macrophages are particularly important in the macrophage network system because mucosa exist where there is regular exposure to foreign substances. However, the molecular mechanism by which intestinal mucosal macrophages respond to the external environment is not yet clear. In this review the biological characteristics of mucosal macrophages are introduced and how they recognize and eradicate various foreign substances is discussed.

Innate-immune therapy for lung carcinoma based on tissue-macrophage activation with lipopolysaccharide.

BACKGROUND: Over the last decade, tumor-specific antigens have been discovered, but so far it has not been possible to use them as part of an effective acquired immunotherapy. This failure may be due to the fact that the expression of the MHC class 1 is low and in lung cancer cells is heterogeneous. Therefore, it may be advantageous to develop techniques that activate the antitumor mechanism of the innate immune system. An experimental model was developed for testing lung cancer therapies that are based on the stimulation of macrophages, which then activate innate immunity. MATERIALS AND METHODS: A549, a human lung adenocarcinoma cell line, was co-cultured with a rat macrophage cell line (NR8383), or a human macrophage cell line (THP 1) at the ratios of 1:1 or 1:5. The experiments were performed with lipopolysaccharide (LPS) or in its absence. The cytotoxicity rate to A549 cells was estimated over time using a dye-uptake method and the amount of lactate dehydrogenase released was measured. The amount of nitric oxide (NO) induced in the medium was assayed, because it may be a candidate as a useful cytotoxic factor. RESULTS: High cytotoxicity was observed to A549 cells when co-cultured with NR8383 cells in the presence of LPS. This effect was not observed in the absence of LPS. Similar results, although to a lesser extent, were observed when A549 cells were co-cultured with THP-1 cells. A high concentration of NO was measured in the co-culture medium of A549 cells and NR8383 cells when LPS was present. CONCLUSION: The induction of cell death in lung cancer cells occurred after contact with macrophages that had been activated by LPS. The NO that was produced by macrophages in response to LPS was responsible for some of this effect.

Establishment of an in vitro model using NR8383 cells and Mycobacterium bovis Calmette-Guerin that mimics a chronic infection of Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis infection affects one-third of the world's population and causes the death of three million people each year. To clarify details of M. tuberculosis survival strategies, it is important to establish a suitable in vitro model that mimics a chronic infection in alveolar macrophages by M. tuberculosis. For this reason, we established a new in vitro model using a rat alveolar macrophage cell line, NR8383. MATERIALS AND METHODS: Basic characteristics, including phagocytotic ability and production of nitrogen oxide and tumor necrosis factor in response to several stimuli, of NR8383 cells were compared with those of primary alveolar macrophages. The course after phagocytosis of live or killed M. bovis bacilli Calmette-Guerin (BCG) was examined over 21 days using NR8383 cells as the host. RESULTS: The characteristics that have been examined to date were nearly the same for both primary alveolar macrophage and NR8383 cells. Live BCG phagocytosed by NR8383 cells had successfully begun to grow in the cells within 7 days, while killed BCG were almost completely destroyed by 21 days. CONCLUSION: BCG-infected NR8383 cells are potentially a suitable in vitro model that mimics a chronic infection with M tuberculosis.

Utilization of macrophages in anticancer therapy: the macrophage network theory.

Appropriate and rational modulation of innate immunity may enhance the therapeutic efficacy of emerging immune therapies for treating cancer. One of the crucial cells of innate immunity is the macrophage. The purpose of this article was to review those issues that suggest ways of exploiting macrophage local functions in immune therapy, and to discuss the suitability of low molecular-weight lipopolysaccharides as potent modulators of macrophage functions for immune therapy of cancer.