Utility of In Vitro Cellular Models of Low-Dose Lipopolysaccharide in Elucidating the Mechanisms of Anti-Inflammatory and Wound-Healing-Promoting Effects of Lipopolysaccharide Administration In Vivo.

Lipopolysaccharide (LPS) is a bacterial component that activates intracellular signaling pathways upon binding to the Toll-like receptor (TLR)-4/MD-2 complex. It is well known that LPS injected into animals and high-dose (100 ng/mL to 1 µg/mL) LPS treatment to innate immune cells induce an inflammatory response. In contrast, LPS is naturally present in the gastrointestinal tract, respiratory tract, and skin of humans and animals, and it has been shown that TLR-4-deficient animals cannot maintain their immune balance and gut homeostasis. LPS from commensal bacteria can help maintain homeostasis against mucosal stimulation in healthy individuals. Oral LPS administration has been shown to be effective in preventing allergic and lifestyle-related diseases. However, this effect was not observed after treatment with LPS at high doses. In mice, oral LPS administration resulted in the detection of LPS at a low concentration in the peritoneal fluid. Therefore, LPS administered at low and high doses have different effects. Moreover, the results of in vitro experiments using low-dose LPS may reflect the effects of oral LPS administration. This review summarizes the utility of in vitro models using cells stimulated with LPS at low concentrations (50 pg/mL to 50 ng/mL) in elucidating the mechanisms of oral LPS administration. Low-dose LPS administration has been demonstrated to suppress the upregulation of proinflammatory cytokines and promote wound healing, suggesting that LPS is a potential agent that can be used for the treatment and prevention of lifestyle-related diseases.

Suppression of Inflammatory Cytokine Genes Expression in Vascular Endothelial Cells by Super-low Dose Lipopolysaccharide-activated Macrophages.

BACKGROUND/AIM: Vascular endothelial cells play an important role in regulating immune responses and in keeping the balance between blood coagulation and fibrinolysis. Inflammatory cytokines produced by activated macrophages and vascular endothelial cells excessively activate vascular endothelial cells, leading to an imbalance in the expression of blood coagulation- and fibrinolysis-related factors. The dysfunction of vascular endothelial cells can lead to development of various diseases. In a previous study the increased expression of inflammatory cytokines in adipocytes was shown to be suppressed by the culture medium of macrophages activated by low-dose lipopolysaccharide (LPS). Suppressing inflammatory cytokine gene expression of low-dose LPS-activated macrophages may allow for the regulation of the dysfunction in vascular endothelial cells. MATERIALS AND METHODS: Human monocytes THP-1 cells were differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and were activated with LPS. The culture medium of the LPS-activated THP-1 was added to human aortic endothelial cells (HAoEC). After five days, the expression of inflammatory cytokine genes interleukin (IL)1B, IL6, IL8, and tumor necrosis factor (TNF)A, blood coagulation-related genes SERPINE1, tissue factor (TF), and thrombopoietin (TM), and fibrinolysis-related gene tissue-type plasminogen activator (t-PA) was analyzed using quantitative real-time PCR. RESULTS: IL1B, IL8, SERPINE1, TF, and TM expression in HAoEC was significantly reduced in the culture medium of super-low dose (0.1 ng/ml) LPS-activated macrophages. CONCLUSION: Super-low dose LPS-activated macrophages can suppress vascular endothelial cell inflammation and may be useful in preventing various diseases caused by the dysfunction of activated vascular endothelial cells.

Regulation of Plasminogen Activator Inhibitor-1 in Adipocytes by Macrophages Activated by Low-dose Lipopolysaccharide.

BACKGROUND/AIM: Increased expression of inflammatory cytokine genes through cell interactions in tissues may cause chronic inflammation, leading to the development of lifestyle-related diseases. Since the activation of inflammatory cytokine genes in monocytes/macrophages by co-culturing with cancer cells or adipocytes was suppressed by pre-treatment with low-dose lipopolysaccharide (LPS), we hypothesized that low-dose LPS-activated macrophages may regulate the expression of immune response-related genes in other cells. MATERIALS AND METHODS: Phorbol myristate acetate-treated human monocytes (THP-1) were activated by LPS. The conditioned medium of LPS-activated THP-1 cells was added to human adipocytes. After 5 days, the expression of genes encoding interleukin (IL)-6 (IL6), IL-8 (IL8), monocyte chemotactic protein (MCP)-1 (CCL2), adiponectin (ADIPOQ), and plasminogen activator inhibitor (PAI)-1 (SERPINE1) was analyzed using quantitative real-time PCR. RESULTS: The increased expression of inflammation-related genes and SERPINE1 in adipocytes was suppressed by the conditioned medium of THP-1 cells activated by low-dose LPS, whereas the expression of ADIPOQ was significantly increased. CONCLUSION: Low-dose LPS-activated macrophages convert adipocytes to anti-inflammatory phenotypes.

Enhanced Effect of Hyaluronan and Elastin Synthesis in Fibroblasts Through Lipopolysaccharide-activated Macrophages.

BACKGROUND/AIM: The functions of macrophages change in response to environmental factors such as lipopolysaccharide (LPS). LPS derived from Pantoea agglomerans (LPSp) is involved in macrophage activation and tissue repair when administered dermally. LPSp-activated macrophages may be useful for restoring and maintaining homeostasis of the skin. MATERIALS AND METHODS: Phorbol myristate acetate-treated human monocytes (THP-1 cells) were activated with LPSp. The medium of LPSp-activated THP-1 cells was added to normal human dermal fibroblasts (NHDF cells). After 24 h, the expression of hyaluronan (HA) synthase (HAS)2, hyaluronidase (HYAL)1, and tropoelastin in NHDF cells was analyzed using quantitative real-time PCR. RESULTS: The expression of HAS2 and tropoelastin was significantly increased, but that of HYAL1 was significantly decreased. It was demonstrated that the abilities of HA and elastin synthesis in NHDF cells increased through LPSp-activated THP-1 cells. CONCLUSION: LPSp-activated macrophages may be useful for enhancing the abilities of HA and elastin synthesis in fibroblasts, subsequently improving dysfunction and reducing various age-related disorders.

Usefulness of Monocytes/macrophages Activated With Low-dose Lipopolysaccharide in Tumor Tissue and Adipose Tissue of Obesity.

Chronic inflammation is involved in the development of cancer, lifestyle-related diseases, and autoimmune diseases. It also influences the severity of these diseases. Macrophages that accumulate in tumor tissues and adipose tissues of obesity have been shown to increase expression of inflammatory cytokines, thereby inducing inflammatory changes in these tissues. The macrophage phenotype is believed to be important in mediating inflammatory changes in tissues. Recently, monocytes/macrophages activated with low-dose lipopolysaccharide (LPS) were demonstrated to suppress increased expression of monocyte chemotactic protein (MCP)-1 and inflammatory cytokines (interleukin (IL)-1 ß, IL-8, and tumor necrosis factor (TNF)-α). By suppressing the increased expression of chemotaxis-related and inflammation-related factors, monocytes/macrophages activated with low-dose LPS are considered to suppress the migration of macrophages into tissues and to regulate inflammatory changes in these tissues, respectively. The effects of macrophages activated with low-dose LPS were different from those of macrophages activated with high-dose LPS. In this review, we discuss the usefulness of monocytes/macrophages activation by low-dose LPS.

Gene Expression in Lipopolysaccharide-treated Human Monocytes Following Interaction with Hepatic Cancer Cells.

BACKGROUND/AIM: Monocytes migrate into the tissue where they differentiate into various types of macrophages with tissue-specific characteristics. When human monocytes are co-cultured with colon cancer cells they exhibit increased mRNA expression of angiogenesis- and signaling pathway-related genes; however, this increase is suppressed by pretreatment with low-dose lipopolysaccharide (LPS). Thus, LPS-treated human monocytes may be useful in suppressing tumor invasion and proliferation in colon cancer. However, it is suggested that the characteristics of tumor-associated macrophages may differ depending on the type of cancer. The function of human tumor-associated macrophages in hepatic cancer remains unclear. In this study, we investigated mRNA expression of various genes in LPS-treated human monocytes following interaction with hepatic cancer cells. MATERIALS AND METHODS: The human monocyte cell line THP-1 was treated with LPS and subsequently co-cultured with the human hepatic cancer cell line HepG2. mRNA expression of various factors were then analyzed using quantitative real-time polymerase chain reaction (PCR) and DNA microarray. RESULTS: The mRNA expressions of monocyte chemotactic protein-1, vascular endothelial growth factor-A, tumor necrosis factor-α, interleukin (IL)-1ß, IL-8, nuclear factor-κB, RelB, signal transducer and activator of transcription 3, IL-10 and transforming growth factor-ß in THP-1 cells following interaction with HepG2 cells, were suppressed by pretreatment with LPS. CONCLUSION: LPS-treated human monocytes may be useful in suppressing tumor invasion and proliferation of hepatic cancer, as well as colon cancer. The co-culture system of monocytes and cancer cells may be beneficial for evaluating antitumor effects in LPS-treated monocytes.

Molecular Response of Human Monocytes Following Interaction with Colon Cancer Cells by Pre-treatment with Low-dose Lipopolysaccharide.

BACKGROUND/AIM: The increased mRNA expression of chemotaxis- and angiogenesis-related factors in human monocytes following interaction with colon cancer cells has been shown to be suppressed by pre-treatment with low-dose lipopolysaccharide (LPS) (100 pg/ml). It has been demonstrated that low-dose LPS reduced the expression of RelB, a member of the nuclear factor (NF)-κB transcription factor family, in mouse macrophages and the NF-κB signaling pathway was important for tumor initiation and growth in tumor-associated macrophages. In addition, the signal transducer and activator of transcription 3 (STAT3) regulated innate immunity via Toll-like receptor (TLR)4 signaling. In the present study, the mRNA expression of signaling pathway- and suppression-related genes in human monocytes following a low-dose LPS treatment and subsequent interaction with colon cancer cells was investigated, in order to assess the molecular response. MATERIALS AND METHODS: The human monocyte cell line THP-1 was treated with LPS and, subsequently, co-cultured with the human colon cancer cell line DLD-1. The mRNA expression of various genes was then analyzed using quantitative real-time polymerase chain reaction (PCR). RESULTS: The mRNA expression of RelB, STAT3, interleukin (IL)-10 and transforming growth factor (TGF)-ß in THP-1 cells following interaction with DLD-1 cells was suppressed by pre-treatment with low-dose LPS (100 pg/ml). CONCLUSION: Treating human monocytes with low-dose LPS may be useful for suppressing tumor progression and may be valuable for maintaining homeostasis.

Expression of chemotaxis- and angiogenesis-related factors in human monocytes following interaction with colon cancer cells is suppressed by low-dose lipopolysaccharide.

BACKGROUND: We have previously reported that mRNA expression of chemotaxis- and angiogenesis-related factors in human monocytes increased following interaction with colon cancer cells. Recently, it was also reported that mRNA expression of the chemotaxis-related factor, monocyte chemotactic protein (MCP)-1, in mouse macrophages following treatment with low-dose lipopolysaccharide (LPS) was significantly lower compared to that following treatment with high-dose LPS, and that low-dose LPS failed to activate the classical nuclear factor (NF)-κB pathway. In the present study, we examined changes in mRNA expression of chemotaxis- and angiogenesis-related factors in human monocytes following low-dose LPS treatment and subsequent interaction with colon cancer cells. MATERIALS AND METHODS: The human monocyte cell line THP-1 was treated with LPS and subsequently co-cultured with the human colon cancer cell line DLD-1. mRNA expression was analyzed by quantitative real-time PCR. RESULTS: mRNA expression of MCP-1, vascular endothelial growth factor (VEGF)-A, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-8 in THP-1 cells treated with low-dose LPS (100 pg/ml) decreased compared to untreated THP-1 cells after five days of co-culture with DLD-1 cells. CONCLUSION: mRNA expression of chemotaxis- and angiogenesis-related factors in human monocytes following interaction with colon cancer cells is suppressed by prior treatment with low-dose LPS. Thus, low-dose LPS treatment of human monocytes may be useful for prevention and therapy of colon cancer.

Angiogenesis-, metastasis- and signaling pathway-related factor dynamics in human colon cancer cells following interaction with monocytes.

BACKGROUND: In tumors, monocytes differentiate into tumor-associated macrophages following interaction with cancer cells. We have previously reported that angiogenesis- and chemotaxis-related factors are associated with human monocyte differentiation following interaction with colon cancer cells. However, the exact nature of factors remains unknown. We investigated factors associated with differentiation of human colon cancer cells following interaction with monocytes. MATERIALS AND METHODS: The human colon cancer cell line DLD-1 was co-cultured with the human monocyte cell line THP-1. mRNA expression was analyzed by quantitative real-time PCR. RESULTS: Expression of interleukin-1ß, matrix metalloproteinase (MMP)-1, MMP-2, and MMP-3 increased in human colon cancer cells after co-culture with monocytes. Conversely, the expression of monocyte chemotactic protein-1, tumor necrosis factor-α, and signal transducer and activator of transcription-3 did not increase. CONCLUSION: Differentiation of human colon cancer cells following interaction with monocytes may be associated with angiogenesis and metastasis but not chemotaxis and signaling pathways. Thus, angiogenesis- and metastasis-related factors associated with differentiation of human colon cancer cells may constitute important targets for colon cancer therapy.

Differential expression of mRNA in human monocytes following interaction with human colon cancer cells.

UNLABELLED: Monocytes are known to differentiate into tissue-specific macrophages in response to the tissue environment, and it has been suggested that tumor-associated macrophages might promote angiogenesis. Therefore, the factors associated with monocyte differentiation into tumor-associated macrophages may become new targets for cancer therapy. However, these factors remain unclear in human colon cancer. The aim of this study was to identify the factors associated with human monocyte differentiation into tumor-associated macrophages at human colon cancer sites. MATERIALS AND METHODS: A human monocyte cell line (THP-1) was co-cultured with a human colon cancer cell line (DLD-1) and mRNA expression was analyzed by quantitative real-time PCR. RESULTS: In THP-1 cells, monocyte chemotactic protein (MCP)-1 mRNA expression increased in a time-dependent manner from day 3 after co-culture with DLD-1 cells; furthermore, expression of vascular endothelial growth factor (VEGF)-A, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-8 mRNA was increased from day 5. This increase in mRNA expression in the THP-1 cells was attributable to the presence of the DLD-1 cells. Therefore, MCP-1, VEGF-A, TNF-α, IL-1ß, and IL-8 are suggested to be associated with differentiation of human monocytes into tumor-associated macrophages at human colon cancer sites.

The relationship between clinicopathological factors and the reduction of pyrimidine nucleoside phosphorylase activity after preoperative administration of 5'-deoxy-5-fluorouridine.

AIM: The response to fluoropyrimidine chemotherapeutic drugs is different in individual tumors. Predictive biomarkers of antitumor effects by these drugs are unknown. 5'-Deoxy-5-fluorouridine (5'-DFUR), a fluoro-pyrimidine chemotherapeutic drug, is converted to 5-fluorouracil (5-FU) by pyrimidine nucleoside phosphorylase (PyNPase). It is suggested that 5'-DFUR will efficiently exert antitumor effects via PyNPase in tumor tissues. The change of PyNPase activity in tumor tissues following 5'-DFUR administration may reflect antitumor effects, and may be useful for detecting predictive factors of antitumor effects. The aim of this study was to search for predictive factors of antitumor effects by analyzing the relationship between clinicopathological factors and the change of PyNPase activity in colorectal tumor tissues after preoperative 5'-DFUR administration. PATIENTS AND METHODS: PyNPase activity in colorectal tissues from 45 patients with colorectal tumors was measured using an ELISA method. RESULTS: The reduction rate of PyNPase activity in colorectal tumor tissues after preoperative 5'-DFUR administration was correlated with significant differences in lymphatic invasion, stage, and histologic classification. It is suggested that lymphatic invasion, stage (distant metastasis), and histologic classification may be predictive factors for evaluating antitumor effects and selecting 5-FU-based chemotherapeutic drugs for patients with colorectal tumors.

Development and potential use of a monoclonal antibody to the lipopolysaccharide of Pantoea agglomerans (IP-PA1).

BACKGROUND: The lipopolysaccharide of Pantoea agglomerans (IP-PA ) has been shown to be effective and safe in the prevention of various diseases, such as bacterial or viral infection, lifestyle-related diseases, when administered transdermally or orally. To clarify the mechanisms of the preventive or therapeutic effect induced by IP-PA1, we tried to establish a monoclonal antibody to detect IP-PA1. The enzyme-linked immunosorbent assay (ELISA) was used to measure the amount of IP-PA1. MATERIALS AND METHODS: Antibodies were raised by immunization using heat-killed Pantoea agglomerans and screening was conducted to isolate monoclonal antibodies specific to IP-PA1. RESULTS: Six kinds of IP-PA1 specific monoclonal antibodies with different epitopes were established. An ELISA using the monoclonal antibodies was successfully established which could specifically detect IP-PA1. CONCLUSION: By use of this ELISA, the staple food content and pharmacodynamic analysis of IP-PA1 could be conveniently estimated.

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.

Cloning and characterization of a LPS-regulatory gene having an LPS binding domain in kuruma prawn Marsupenaeus japonicus.

LPS is known as an effective stimulator of the immune system in various animals, including mammals and horseshoe crabs (HSC). Both of these animal groups have suppressive regulatory proteins for the LPS response, e.g. the bactericidal/permeability increasing protein in mammals and anti-LPS factor (ALF) in HSC. Prawns are a valuable aquaculture species, but the regulatory molecules and/or mechanisms that respond to LPS are largely unknown. To investigate the molecular mechanism of the LPS response in kuruma prawns, we cloned a cDNA having a LPS binding domain. A full-length cDNA gene, denoted as M-ALF (Marsupenaeus japonicus ALF-like peptide) was cloned that consisted of 746bp and encoded 123 amino-acid residues. The 3' non-translated region of this gene had the pentamer of ATTTA repeated four times; this is known as sequences for messenger RNA stabilization. Deduced amino-acid sequences showed a 42% homology with Japanese HSC-ALF. In particular, both have clusters of basic and hydrophobic amino acids, indicating that the region is probably binding to lipid A. The mRNA expression was determined for hemocytes, lymphoid organs, hearts, intestines and gills by RT-PCR. The mRNA expression was augmented 1.5-3h after LPS administration in lymphoid organs, but then decreased to normal level at 6h. Synthetic peptides containing Cys30 to Cys51 had LPS neutralizing activity to the Limulus reaction and NO production in RAW264.7 cells. These data suggest that in kuruma prawns, M-ALF acts as a LPS regulator during the acute phase response after invasion of pathogens.

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.

Molecular cloning and expression analysis of a macrophage-colony stimulating factor receptor-like gene from rainbow trout, Oncorhynchus mykiss.

The M-CSF and its receptor (M-CSFR, CSF-1R or c-fms proto-oncogene) system were initially implicated as essential in mammals for normal monocyte development as well as for pregnancy. To allow a comparison with the M-CSF and M-CSFR system of an oviparous animal, we cloned a M-CSFR-like gene from rainbow trout (Oncorhynchus mykiss). The gene was cloned from a cDNA library of head kidney. It contained an open reading frame encoding 967 amino acids with a predicted size of 109 kDa. The putative amino acid sequence of rainbow trout M-CSFR showed 54% amino acid identity to fugu (Takifugu rubripes) M-CSFR, 52% to zebrafish (Danio rerio) M-CSFR and 40% to mouse (Mus musculus) and human (Homo sapiens) M-CSFR. The M-CSFR-like gene was constitutively expressed in head kidney, kidney, intestine, spleen and blood. The gene was detected especially in the ovary of immature female rainbow trout. These results suggest that a M-CSFR-like receptor may be involved in female reproductive tracts even in an oviparous animal like fish.

Antitumor effects of isolated hypoxic hepatic perfusion (IHHP) with high-dose TNF against colonic liver metastases in a rat model.

Although various treatment methods have been applied to unresectable metastatic liver tumors, an effective way to treat this disease has yet to be established. Recently, the combined treatment with tumor necrosis factor-alpha (TNF-alpha) and melphalan has been reported to be effective in 75% of cases in Western countries. In the present study, the antitumor effect of isolated hypoxic hepatic perfusion (IHHP) with TNF-SAM2 and an anticancer agent (5-FU), which is widely used for gastrointestinal cancers, was investigated using the F344 rat model with colonic liver metastases. The inhibitory effect on tumor growth in rats administered with either 40 microg/rat of TNF-SAM2 (89%) or 5 mg/kg or 10 mg/kg of 5-FU (56% or 19%) was significantly greater than that in non-treated rats (176%). On the other hand, the inhibitory effect on tumor growth in rats administered with both TNF-SAM2 and 5-FU (10 mg/kg) was 65%, which was equal to, or less than, that in rats administered with only TNF-SAM2 or 5-FU. There was no additive/synergistic effect of the combined treatment with these drugs. These results indicate the kinds and dose of anticancer agents which, used in combination with TNF, need to be evaluated for the most appropriate antitumor effect in the IHHP method.

Development and characterization of a monoclonal antibody with cross-reactivity towards uracil and thymine, and its potential use in screening patients treated with 5-fluorouracil for possible risks.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in catabolism of pyrimidines including 5-fluorouracil. There have been efforts to isolate a monoclonal antibody that will bind selectively to pyrimidine and can be used to measure the concentration of pyrimidine in blood and/or in urine that may reflect the activity of dihydropyrimidine dehydrogenase. However, the monoclonal antibodies selective to pyrimidine have not been available. METHODS: Using 1-carboxymethyl-uracil as a hapten, in which steric conformation of uracil is thought to be well maintained, extensive screening was done to isolate a monoclonal antibody specific to uracil. RESULTS: We established the first monoclonal antibody that reacted with uracil and thymine but not with pseudouridine, dihydrouracil, dihydrothymine, cytosine, uridine, or N-carbamyl-beta-alanine at the concentration of 100 microg/ml. CONCLUSIONS: The monoclonal antibody can be used to develop a simple screening assay for patients with dihydropyrimidine dehydrogenase deficiency. This may increase the safety of 5-fluorouracil treatment.

Cloning and characterization of the homolog of mammalian lipopolysaccharide-binding protein and bactericidal permeability-increasing protein in rainbow trout Oncorhynchus mykiss.

We cloned two cDNAs denoted as RT-LBP/BPI-1 and RT-LBP/BPI-2, respectively, which were derived from the mRNA of head kidney from rainbow trout. They showed structural homology with LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) in mammals. The full-length cDNA of RT-LBP/BPI-1 and RT-LBP/BPI-2 is 1666 and 1741 bp, respectively. Both cDNAs encoded 473 aa residues, including the amino acids conserved in mammalian LBP and BPI proteins that were assumed to be involved in LPS binding. The overall coding sequence of RT-LBP/BPI-1 has 33% amino acid homology to human LBP and 34% to human BPI, and RT-LBP/BPI-2 has 32% amino acid homology to human LBP and 33% to human BPI. Three-dimensional structure analysis by three-dimensional/one-dimensional (3D-1D) methods also demonstrated that RT-LBP/BPI-1 and RT-LBP/BPI-2 proteins showed significant similarity to human BPI, having a boomerang shape with N-terminal and C-terminal barrels. Phylogenetic analysis showed that the LBP and BPI genes seemed to be established after the divergence of mammals from teleosts. These results suggested that RT-LBP/BPI-1 and RT-LBP/BPI-2 may be a putative ortholog for mammalian LBP and/or BPI genes. This is the first study to identify the LBP family genes from nonmammalian vertebrates.