Distribution and function of macrophage galactose-type C-type lectin 2 (MGL2/CD301b): efficient uptake and presentation of glycosylated antigens by dendritic cells.

Dendritic cells (DCs) express cell surface lectins that are potentially involved in the recognition, uptake, and presentation of glycosylated foreign substances. A unique calcium-type (C-type) lectin, the macrophage galactose (Gal)-type C-type lectin (MGL/CD301) expressed on DCs, is thought to participate in the recognition of molecules from both altered self and pathogens due to its monosaccharide specificity for Gal and N-acetylgalactosamine (GalNAc). Although mice have two MGL genes, Mgl1 and Mgl2, their distinct roles have not been previously explored. The present report characterizes the properties of MGL2 by examining its distribution and its role in antigen presentation by DCs. We generated an MGL2-specific monoclonal antibody and examined MGL2 expression in tissues by immunohistochemistry and in isolated cells by flow cytometry. The cells reactive with this antibody were shown to be a portion of MGL1-expressing cells, mostly conventional DCs. Internalization of soluble polyacrylamide polymers (PAA) with alpha-GalNAc residues (GalNAc-PAA) by bone marrow-derived DCs (BM-DCs) was mediated by MGL2, as revealed by a comparison of Mgl1(-/-) and Mgl2(-/-) BM-DCs with wild-type BM-DCs. Biotinylated GalNAc-PAA conjugated to streptavidin (SAv) was more efficiently presented to SAv-primed T cells by BM-DCs than beta-N-acetylglucosamine-PAA conjugated to SAv or SAv alone as shown by thymidine uptake and cytokine production. This is the first report that demonstrates the involvement of GalNAc residues in antigen uptake and presentation by DCs that lead to CD4(+) T cell activation.

Low susceptibility of NC/Nga mice to tumor necrosis factor-alpha-mediated lethality and hepatocellular damage with D-galactosamine sensitization.

The susceptibility of NC/Nga mice to tumor necrosis factor (TNF)-alpha was examined by using sensitization with d-galactosamine (d-GalN). Administration of TNF-alpha and d-GalN killed none of the NC/Nga mice, whereas it killed all of the BALB/c mice. Treatment with TNF-alpha and d-GalN caused few hepatic lesions in NC/Nga mice but massive hepatocellular apoptosis in BALB/c mice. Unlike BALB/c mice, there was no elevation in caspase 3 and 8 activities in the livers of NC/Nga mice receiving TNF-alpha and d-GalN. On the other hand, administration of anti-Fas antibody definitely killed both NC/Nga and BALB/c mice via activation of caspases 3 and 8. Treatment with TNF-alpha and d-GalN led to translocation of nuclear factor (NF)-kappaB in NC/Nga and BALB/c mice. However, NF-kappaB translocation was sustained in NC/Nga mice, although it disappeared in BALB/c mice 7 h after the treatment. NF-kappaB inhibitors activated caspases 3 and 8, and enhanced TNF-alpha-mediated lethality in NC/Nga. Taken together, the low susceptibility of NC/Nga mice to TNF-alpha-mediated lethality was suggested to be responsible for the sustained NF-kappaB activation.

Enantioselective visualization of D-alanine in rat anterior pituitary gland: localization to ACTH-secreting cells.

The cellular localization of D: -alanine (D: -Ala) in the rat pituitary gland, the tissue containing the highest amount of D: -Ala, has been clarified for the first time by enantioselective visualization of D: -Ala using our own established mouse monoclonal antibody against D: -Ala. D: -Ala immunopositive cells were present predominantly in the anterior lobe, while no intense staining was observed in the intermediate and posterior lobes. The anterior pituitary gland contains five types of cells secreting specific hormones (growth hormone, adrenocorticotropic hormone (ACTH), gonadotropic hormone, prolactin, and thyroid-stimulating hormone), and the double staining results indicated that D: -Ala is localized to the ACTH-secreting cells. The localization of D: -Ala is clearly different from that of D: -aspartic acid (D: -Asp), which is observed in the prolactin cells. Considered together with our previous findings that D: -Ala is localized to the insulin-secreting beta-cells in the pancreas, and both ACTH and insulin are typical regulatory hormones of blood glucose, D: -Ala is suggested to have some functional relationships to blood glucose level regulation in mammals.

Concanavalin A induces formation of osteoclast-like cells in RAW 264.7 mouse macrophage cells.

The effect of lectins on the formation of osteoclasts in RAW 264.7 mouse macrophage cells was examined. Concanavalin A (Con A) induced the formation of multinucleated giant cells (MGC) whereas pokeweed mitogen and phytohemagglutinin did not do it. Con A-induced MGC were positive for tartrate- resistant acid phosphatase (TRAP) activity, a histochemical marker of osteoclasts. Murine splenic macrophages differentiated into TRAP-positive and multinucleated cells in response to Con A whereas peritoneal macrophages did not. The culture supernatant from Con A-stimulated RAW 264.7 cells did not cause the MGC formation. The relationship between Con A-induced GMC formation and osteoclastgenesis is discussed.

Circadian changes of D-alanine and related compounds in rats and the effect of restricted feeding on their amounts.

The circadian changes of D-alanine (D-Ala), an intrinsic D-amino acid found in mammals, were investigated in rats with diurnal and nocturnal habits, and the profiles were compared to those of L-Ala, other D-amino acids and several hormones. Determination of D-Ala in the rat plasma, pancreas and anterior pituitary gland was carried out using a sensitive and selective two-dimensional HPLC system combining a micro-ODS column and an enantioselective column after fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The amount of D-Ala was high during the sleeping period and low during the active period in rats with both diurnal and nocturnal habits, indicating for the first time that the D-Ala is closely related to the activity rhythm of animals. In contrast, L-Ala and other D-amino acids did not show any clear circadian changes. The circadian change of D-Ala inversely correlated with that of the plasma insulin level in rats with both diurnal and nocturnal habits. Considered together with our previous findings that D-Ala is localized in the insulin secreting beta-cells in the rat pancreas, it is strongly suggested that D-Ala has some functional relationships to insulin in mammals.

Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals.

A fully automated 2D-HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column has been developed for the simultaneous enantiomer determination of proline, trans-4-hydroxyproline and cis-4-hydroxyproline in mammals. As a first dimension, a monolithic ODS column of 0.53 mm i.d. showed 3-6 times larger theoretical plate numbers than those of particle-packed ODS columns, and the best enantioseparations were obtained by a Chiralpak QN-2-AX column of 1.5 mm i.d. in the second dimension (separation factors: 1.44-1.83). The R.S.D. values for within-day and dayto-day precisions were less than 5.8%, and the lower limits of quantitation for the D-enantiomers were 1 fmol. The present method was successfully applied to the determination of proline and hydroxyproline enantiomers in the serum and collagen-rich skin tissue. Small amounts of D-proline were found both in the serum (1.57 +/- 0.19 nmol/mL) and in the skin (0.093 +/- 0.015 nmol/mg protein), while the amounts of D-hydroxyproline were smaller than the lower limit of quantitation.

A genetic variant of the serine racemase gene is associated with schizophrenia.

BACKGROUND: Serine racemase (SRR) is a brain-enriched enzyme that converts L-serine to D-serine, which acts as an endogenous ligand of N-methyl D-aspartate (NMDA) receptors. Dysfunction of SRR may reduce the function of NMDA receptors and susceptibility to schizophrenia. METHODS: We genotyped three single-nucleotide polymorphisms (SNPs) of the 5' region of the SRR gene in 525 patients with schizophrenia and 524 healthy controls. Effects of SNPs on the promoter activity and on serum levels of total and D-serine were examined. RESULTS: We found a significant excess of the IVS1a+465C allele of the SRR gene in schizophrenia, especially in the paranoid subtype (p = .0028). A reporter assay showed that the IVS1a+465C allele had 60% lower promoter activity than did the IVS1a+465G allele. CONCLUSIONS: The IVS1a+465C allele of the SRR gene, which reduces expression of the gene, is a risk factor for schizophrenia, especially the paranoid subtype.

Comprehensive analysis of branched aliphatic D-amino acids in mammals using an integrated multi-loop two-dimensional column-switching high-performance liquid chromatographic system combining reversed-phase and enantioselective columns.

A validated two-dimensional HPLC method for the comprehensive analysis of small quantities of branched aliphatic D-amino acids in the presence of large amounts of their L-congeners in mammalian tissues and physiological fluids is described. The quantitative analysis of these aliphatic amino acids (Val, allo-Ile, Ile, and Leu) is important for the diagnosis of various inherent metabolic disorders of amino acids, and the D-enantiomers are expected to be of particular interest from a pharmacological point of view. Target analytes were determined as their fluorescent derivatives, pre-column labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), using an automated two-dimensional column-switching high-performance liquid chromatographic system combining a narrow bore reversed-phase column and an enantioselective column connected with an integrated multi-loop peak fraction storage device. The described two-dimensional analysis concept proved to be successful for the given task in biological samples taken from mammals. Total analysis time for the reversed-phase separation of the four target NBD-amino acids is 60 min, and the integrated enantiomer separation of each of the four analytes is completed in approximately 5 min. In the rat, significant amounts of D-Leu were found in all tissues and physiological fluids tested (trace-1.3 nmol/g tissue), and in the urine, the presence of high amounts of D-allo-Ile (D-isomer of a non-proteinogenic amino acid, 22.2 nmol/ml) was demonstrated. D-allo-Ile was also found in the urine of dog and mouse, which indicates the ubiquitous presence of this unusual D-amino acid and the potential need to clarify its unique metabolism in mammals.

Defective responsiveness of CD5+ B1 cells to lipopolysaccharide in cytokine production.

Previously, we found that mouse TH2.52 cells possess the characteristic of CD5(+) B1 cells and proliferate in response to lipopolysaccharide (LPS). The effect of LPS on cytokine production by TH2.52 B1 cells was studied. TH2.52 cells constitutively produced a small amount of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and TNF-alpha and IL-6 production was markedly enhanced by LPS stimulation. Although interferon (IFN)-gamma caused the production of various cytokines, such as IL-2, IL-4, IL-6 and TNF-alpha in TH2.52 cells, LPS did not cause the production of such cytokines. LPS did not induce IFN-beta production in TH2.52 cells and TH2.52 cells lacked the expression of several molecules participating in the MyD88-independent pathway in LPS signaling. Defective responsiveness of TH2.52 B1 cells to LPS in cytokine production might be responsible for the failure of IFN-beta production due to the lack of molecules participating in the MyD88-independent pathway.

Sensitive high-performance liquid chromatographic assay for D-amino-acid oxidase activity in mammalian tissues using a fluorescent non-natural substrate, 5-fluoro-D-tryptophan.

A sensitive assay for D-amino-acid oxidase (DAO) activity in mammalian tissues has been established. D-Tryptophan (D-Trp) analogs were tested as substrates for DAO, and 5-fluoro-D-tryptophan (D-FTP) was found to be the best substrate. By the enzymatic reaction, D-FTP was converted to 5-fluoroindole-3-acetic acid (FIAA), a highly fluorescent product, and the product was determined by an RP-HPLC system with a fluorescence detector. The detection limit for purified DAO (from hog kidney) was 0.25 microU, and the within-day and day-to-day precisions of the assays were 4.6% (RSD, n=5), and 13.8% (RSD, 5 days), respectively. By the present method, the detailed distribution of DAO activity in the mouse brain was determined using individual animals for the first time, and significant activities were observed in the cerebellum, medulla oblongata and midbrain. Because sensitive DAO assay is frequently required in small tissues or in limited-tissue regions, the present method is useful for various research studies concerning DAO and the related D-amino acids.

Streptococcal Toxic Shock Syndrome: Life Saving Role of Peritoneal Lavage and Drainage.

PURPOSE: We encountered a case where an infection with group A streptococcus (GAS; ie, Streptococcus pyogenes) initially caused primary peritonitis and then subsequently caused streptococcal toxic shock syndrome. The patient's life was likely saved by an emergency laparotomy followed by extensive peritoneal lavage and drainage. CASE PRESENTATION: A 40-year-old woman was admitted to the Emergency Department for lower abdominal pain and numbness in the extremities. She presented with systemic inflammatory response syndrome. An emergency laparotomy was performed, and ascites that resembled pus and general peritonitis were noted. Peritoneal lavage and drainage were performed, and GAS was isolated from peritoneal fluid. Gram staining of cervical polyp specimens revealed Gram-positive bacteria. CONCLUSIONS: The patient was diagnosed with streptococcal toxic shock syndrome due to an ascending GAS infection originating from vagina.

Augmentation of lipopolysaccharide-induced nitric oxide production by alpha-galactosylceramide in mouse peritoneal cells.

The effect of alpha-galactosylceramide (alpha-GalCer) on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal cells was studied. alpha-GalCer augmented LPS-induced NO production in mouse peritoneal cells, but not in RAW 264.7 macrophage cells. alpha-GalCer augmented NO production, but not tumor necrosis factor (TNF)-alpha production in LPS-stimulated peritoneal cells. Peritoneal cells produced a significant level of interferon (IFN)-gamma in response to alpha-GalCer and anti-IFN-gamma antibody abolished the augmentation of LPS-induced NO production by alpha-GalCer. Moreover, anti-IFN-gamma antibody prevented the enhanced expression of an inducible type of NO synthase mRNA by alpha-GalCer. alpha-GalCer did not augment LPS-induced NO production in peritoneal cells from natural killer T (NKT)-deficient mice. Therefore, it was suggested that alpha-GalCer might augment LPS-induced NO production in peritoneal cells through release of IFN-gamma from NKT cells.

Secular trends and geographical variations in the dietary intake of polybrominated diphenyl ethers (PBDEs) using archived samples from the early 1980s and mid 1990s in Japan.

A retrospective exposure assessment among the general population for polybrominated diphenyl ethers (PBDEs) was conducted using dietary surveys. We analyzed samples of food duplicate portions collected in the early 1980s (1980 survey: N=40) and the mid 1990s (1995 survey: N=39) from female subjects (5 participants from each of 8 sites per survey except for one site) living throughout Japan, from the north (Hokkaido) to the south (Okinawa). The study populations in the 1980 and 1995 surveys were different, but lived in the same communities. We measured four PBDE congeners [2,2',4,4'-tetrabrominated diphenyl ether (tetraBDE): #47; 2,2',4,4',5-pentaBDE: #99; 2,2',4,4',6-pentaBDE: #100; and 2,2',4,4',5,5'-hexaBDE: #153] in the diet. #99 was the most abundant congener in the diet (49% of the total PBDEs), followed by #47 (33%), #100 (12%) and #153 (6%). Regional variations found in the 1980 survey decreased in the 1995 survey. The total daily intake of PBDEs (ng/d) [GM (GSD)] in the 1980 survey [91.4 (4.1)] was not significantly different from that in the 1995 survey [93.8 (3.4)] for the total population, nor did it differ among the sites including Shimane, in which a 20-fold increase in serum concentrations was observed in the same population1). In consideration of the significant increases in the serum concentration, inhalation may be more important than food ingestion as the route of human exposure to PBDEs.

Differences in the mechanism of nitric oxide production between mouse vascular endothelial cells and macrophages.

The detailed mechanism of NO production in mouse vascular endothelial cells, END-D, was studied. The NO production in END-D cells was triggered by gamma interferon (IFN-gamma), but not LPS. However, LPS augmented the NO production in IFN-gamma-stimulated END-D cells. A high level of NO production was due to the expression of an inducible type of NO synthase (iNOS) in those cells. A significant amount of NO was detected 18 h after IFN-gamma stimulation, accompanied by the delayed iNOS expression. The JAK/STAT signal pathway mediated IFN-gamma-induced NO production, but did not participate in the LPS-induced augmentation. Further, no activation of nuclear factor (NF)-kappaB was involved in the NO production in END-D cells stimulated with either IFN-gamma and/or LPS. The mechanism of NO production in END-D cells was suggested to be different from that in mouse macrophages. The differential regulation of NO production in mouse vascular endothelial cells and macrophages is discussed.

Butyrate enhances the production of nitric oxide in mouse vascular endothelial cells in response to gamma interferon.

The effect of butyrate, a natural bacterial product of colonic bacterial flora, on nitric oxide (NO) production in murine vascular endothelial cell line END-D in response to IFN-gamma and/or LPS was studied. Butyrate significantly augmented NO production in END-D cells in response to IFN-gamma or IFN-gamma + LPS, but not LPS alone. The NO production was augmented by the addition of butyrate until 6 h after the stimulation with IFN-gamma or IFN-gamma + LPS. The augmentation was abolished by the removal of butyrate from the cultures. Butyrate enhanced the expression of inducible type NO synthase (iNOS) in the stimulated END-D cells. Furthermore, butyrate-enhanced NO production in the presence of various signal inhibitors down-regulating the signal pathways using nuclear factor (NF)-kappaB, mitogen-activated protein (MAP) kinases and Janus tyrosine kinase. The putative mechanism of butyrate-induced augmentation of NO production in response to IFN-gamma or IFN-gamma + LPS is discussed.

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells.

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.

D-Amino acids in mammals and their diagnostic value.

Substantial amounts of D-amino acids are present in mammalian tissues; their function, origin and relationship between pathophysiological processes have been of great interest over the last two decades. In the present article, analytical methods including chromatographic, electrophoretic and enzymatic methods to determine D-amino acids in mammalian tissues are reviewed, and the distribution of these D-amino acids in mammals is discussed. An overview of the function, origin and relationship between the amino acids and pathophysiological processes is also given.

Determination of d- and l-enantiomers of threonine and allo-threonine in mammals using two-step high-performance liquid chromatography.

A sensitive and selective method for the determination of four threonine (Thr) isomers (L-Thr, D-Thr, L-allo-Thr and D-allo-Thr) in mammalian tissues has been established using two-step high-performance liquid chromatography. This method includes the precolumn fluorescence derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the separation using a combination of a reversed-phase column and a chiral column. The calibration ranges of D-Thr, D-allo-Thr and L-allo-Thr spiked in the rat cerebellum sample are 2.5 fmol-5 pmol per injection, and that of L-Thr is 50 fmol-50 pmol. Within-day and day-to-day precisions of the determination of the four Thr isomers are approximately 5% in the rat cerebellum. By using this method, the tissue distributions of D-Thr, D-allo-Thr and L-allo-Thr in mammals have been demonstrated for the first time in rats, and found that significant amounts of D-Thr and D-allo-Thr are present in the frontal brain areas and urine. Among the 12 tissues tested, the highest amounts of D-Thr (0.85 +/- 0.05 nmol/g wet tissue) and D-allo-Thr (5.01 +/- 0.32 nmol/g wet tissue) were found in the corpus striatum. L-allo-Thr was not present in any of the tested tissues and physiological fluids.

Immunological role of prostaglandin E2 production in mouse auditory cells in response to LPS.

The effect of LPS on the production of prostaglandin E2 (PGE2) in mouse HEI-OC1 auditory cells was examined. HEI-OC1 auditory cells constitutively produce a small amount of PGE2. LPS augmented the PGE2 production via enhanced cyclooxygenase 2 (COX2) expression. LPS-induced augmentation of COX2 expression was dependent on up-regulation of COX2 mRNA expression. LPS induced the production of TNF-α, but not IL-1ß· An anti-TNF-α neutralizing Ab significantly inhibited PGE2 production and COX2 mRNA expression in response to LPS. LPS-induced PGE2 production was prevented by a series of pharmacological signaling inhibitors to NF-κB and MAPKs. Pam3CSK4 as a TLR2 ligand, as well as LPS as a TLR4 ligand, augmented the PGE2 production. However, poly I:C as a TLR3 ligand, imiquimod as a TLR7 ligand and CpG DNA as a TLR9 ligand did not augment it. HEI-OC1 cells expressed TLR2, TLR4 and TLR9, but not TLR3 or TLR7. The putative role of LPS-induced PGE2 production in auditory cells is discussed.

Auditory cells produce nitric oxide in response to bacterial lipopolysaccharide.

The NO productivity of auditory cells in response to LPS was examined by using conditionally immortalized murine HEI-OC1 auditory cells. HEI-OC1 cells produced NO in response to LPS ranging from 0.1 µg/ml to 100 µg/ml in a concentration-dependent manner. LPS at 100 µg/ml exhibited no cytotoxic action against HEI-OC1 cells and led to the highest level of NO production. The NO output in LPS-treated HEI-OC1 cells gradually increased up to 72 h. LPS-induced NO production was mediated by the expression of an inducible NO synthase (iNOS) protein. TLR4 and CD14 was expressed on the cell surface of HEI-OC1 cells. LPS augmented the production of IFN-ß in the MyD88-independent pathway of LPS signalling. HEI-OC1 cells produced NO in response to a TLR2 ligand but not TLR3 ligand. LPS was suggested to lead to NO production in auditory cells via iNOS expression. The immunological significance of NO production in auditory cells is discussed.