NOD-like receptors in autoimmune diseases.

Autoimmune diseases are chronic immune diseases characterized by dysregulation of immune system, which ultimately results in a disruption in self-antigen tolerance. Cumulative data show that nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) play essential roles in various autoimmune diseases, such as inflammatory bowel disease (IBD), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), psoriasis, multiple sclerosis (MS), etc. NLR proteins, consisting of a C-terminal leucine-rich repeat (LRR), a central nucleotide-binding domain, and an N-terminal effector domain, form a group of pattern recognition receptors (PRRs) that mediate the immune response by specifically recognizing cellular pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and triggering numerous signaling pathways, including RIP2 kinase, caspase-1, nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK) and so on. Based on their N-terminal domain, NLRs are divided into five subfamilies: NLRA, NLRB, NLRC, NLRP, and NLRX1. In this review, we briefly describe the structures and signaling pathways of NLRs, summarize the recent progress on NLR signaling in the occurrence and development of autoimmune diseases, as well as highlight numerous natural products and synthetic compounds targeting NLRs for the treatment of autoimmune diseases.

Lipid Mediators From Timothy Grass Pollen Contribute to the Effector Phase of Allergy and Prime Dendritic Cells for Glycolipid Presentation.

Plant pollen are an important source of antigens that evoke allergic responses. Protein antigens have been the focus of studies aiming to elucidate the mechanisms responsible for allergic reactions to pollen. However, proteins are not the sole active agent present in pollen. It is known that pollen grains contain lipids essential for its reproduction and bioactive lipid mediators. These small molecular compounds are co-delivered with the allergens and hence have the potential to modulate the immune response of subjects by activating their innate immune cells. Previous reports showed that pollen associated lipid mediators exhibited neutrophil- and eosinophil-chemotactic activity and induced polarization of dendritic cells (DCs) toward a Th2-inducing phenotype. In our study we performed chemical analyses of the pollen associated lipids, that are rapidly released upon hydration. As main components we have identified different types of phytoprostanes (PhytoPs), and for the first time phytofurans (PhytoFs), with predominating 16-F1t-PhytoPs (PPF1-I), 9-F1t-PhytoPs (PPF1-II), 16-E1t-PhytoPs (PPE1-I) and 9-D1t-PhytoPs (PPE1-II), and 16(RS)-9-epi-ST-Δ14-10-PhytoFs. Interestingly 16-E1t-PhytoP and 9-D1t-PhytoPs were found to be bound to glycerol. Lipid-containing samples (aqueous pollen extract, APE) induced murine mast cell chemotaxis and IL-6 release, and enhanced their IgE-dependent degranulation, demonstrating a role for these lipids in the immediate effector phase of allergic inflammation. Noteworthy, mast cell degranulation seems to be dependent on glycerol-bound, but not free phytoprostanes. On murine dendritic cells, APE selectively induced the upregulation of CD1d, likely preparing lipid-antigen presentation to iNKT cells. Our report contributes to the understanding of the activity of lipid mediators in the immediate effector phase of allergic reactions but identifies a yet undescribed pathway for the recognition of pollen-derived glycolipids by iNKT cells.

[Synthesis and identification of artificial antigen of forsythin].

The establishment of high specificity and sensitivity method of small molecule monoclonal antibody-based immunoassay has a great importance in the study of small molecule compounds in Chinese medicine, wherein synthesis of small molecule artificial antigen is a critical step in the preparation of small molecule antibodies. Oxidation method using sodium iodide was used to synthesize immunogenic antigen (FRn-BSA) and coating antigen (FRn-OVA) of forsythin. UV spectroscopy and thin layer chromatography showed that forsythin was successfully conjugated with BSA and OVA. After immuned FRn-BSA, the mice could specifically produce anti-forsythin antibodies with titer up to 1:8 000, and the linear range was from 1 mg x L(-1) to 100 mg x L(-1). In this paper, the artificial antigen of forsythin was successfully synthesized, which can be applied for preparation of monoclonal antibodies and establishment of appropriate immune method.

Biological activities of an engineered tautomycetin analogue via disruption of tmcR-encoding hydroxylase in Streptomyces sp. CK4412.

Tautomycetin (TMC), originally isolated from Streptomyces griseochromogenes, has been reported to possess biological functions including T cell-specific immunosuppressive and anticancer activities through a mechanism of differential inhibition of protein phosphatases such as PP1, PP2A, and SHP2. Independently isolated Streptomyces sp. CK4412 was also reported to produce a structurally identical TMC compound. Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. CK4412. In silico database comparison revealed a 1,359-bp tmcR as a putative bacterial Cytochrome P450 hydroxylase gene in the TMC biosynthetic gene cluster. Through targeted gene disruption and complementation, the tmcR mutant was confirmed to produce a C5-deoxy-TMC, the same analogue produced by the S. griseochromogenes ttnI mutant, implying that TmcR behaves as a regiospecific C5-oxygenase in the TMC biosynthetic pathway in Streptomyces sp. CK4412. In particular, the C5-deoxy-TMC from the tmcR mutant exhibited 3.2-fold higher inhibition activity toward SHP2 with significantly reduced inhibition activities toward PP1, and human Vero and lung cancer cells. These results suggested that C5 regiospecific modification of the TMC polyketide moiety may result in a drug development target for use in preferentially enhancing immunosuppressive activity while minimizing its undesirable biological activities.

Biological evaluation of isoegomaketone isolated from Perilla frutescens and its synthetic derivatives as anti-inflammatory agents.

The anti-inflammatory activities of a prepared isoegomaketone 3a and its derivatives 3b-3f were evaluated in RAW 264.7 cells. Among these, the compound 3d was displayed the most potent inhibitory activities against production of nitric oxide, monocyte chemoattractant protein-1 and interleukin-6. Based on these results, the abilities of compounds 3a-3f to modulate NF-κB and AP-1-mediated gene transcription using a luciferase reporter assay were investigated. The transcriptional activities of NF-κB and AP-1 decreased when pretreated with 3a-3f. Interestingly, at 10 µM, compound 3d markedly suppressed the lipopolysaccharide-induced NF-κB and activator protein-1 DNA binding activities. Some preliminary structure-activity relationships were proposed that may provide a direction for further study.

Isolation of the biosynthetic gene cluster for tautomycetin, a linear polyketide T cell-specific immunomodulator from Streptomyces sp. CK4412.

The bacterial genus Streptomyces has long been appreciated for its ability to produce various kinds of medically important secondary metabolites, such as antibiotics, anti-tumour agents, immunosuppressants and enzyme inhibitors. Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Using a Streptomyces polyketide methylmalonyl-CoA acyltransferase gene as a probe, three overlapping cosmids were isolated from the genomic library of TMC-producing Streptomyces sp. CK4412. Sequence information of an approximately 70 kb contiguous DNA region revealed two multi-modular type I polyketide synthases (PKSs), and 12 additional gene products presumably involved in TMC biosynthesis. The deduced roles for most of the TMC PKS catalytic domains were consistent with the expected functions necessary for TMC chain elongation and processing. In addition, disruption of a putative TMC acyl-CoA transferase gene, located upstream of the PKS gene locus, completely abolished TMC biosynthesis. Taken together, these data provide strong supporting evidence that the cloned gene cluster identified in this study is responsible for TMC biosynthesis in Streptomyces sp. CK4412, and set the stage for detailed genetic and biochemical studies of the biosynthesis of this important metabolite.

Reactivity of MEST-1 (antigalactofuranose) with Trypanosoma cruzi glycosylinositol phosphorylceramides (GIPCs): immunolocalization of GIPCs in acidic vesicles of epimastigotes.

Using confocal microscopy, MEST-1-positive immunofluorescence was observed within various Trypanosoma cruzi forms, except in cell-derived trypomastigotes. Glycosylinositol phosphorylceramides were identified by thin-layer chromatography immunostaining as the antigens recognized by MEST-1 in these parasites. In epimastigotes, labeling of MEST-1 coincided with acidic vesicles, indicating an internal localization of these glycoconjugates.

Immunologic findings in workers formerly exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and its congeners.

One hundred ninety-two workers in a German pesticide factory who were exposed to polychlorinated dibenzodioxins and -furans (PCDD/PCDF) were investigated for former and present diseases and laboratory changes of the immune system. Moreover, in a subgroup of 29 highly exposed and 28 control persons, proliferation studies were performed. In addition to assays such as blood count, immunoglobulins, serum electrophoresis, monoclonal bands, surface markers, autoantibodies, and lymphocyte proliferation, two new methods, the rise of tetanus antibody concentration after vaccination and the in vitro resistance of lymphocytes to chromate, were used to diagnose the morphologic and functional state of the immune system. There was no stringent correlation of actual PCDD/PCDF concentrations with the occurrence of infections or with one of the immune parameters. In addition, outcomes of the tetanus vaccination and the chromate resistance test were not correlated with PCDD/PCDF. However, the chromate resistance of lymphocytes stimulated by phytohemagglutinin of highly exposed persons was significantly lower than that for the control group. These findings indicate that the function of lymphocytes can be stressed and possibly impaired by high exposure to PCDD/PCDF.

Identification by flow cytometry of seiridin, one of the main phytotoxins produced by three Seiridium species pathogenic to cypress.

Seiridin (SE), one of the main phytotoxins produced in vitro by Seiridium species pathogenic to cypress, was oxidized and the corresponding ketone derivative covalently linked to bovine serum albumin (BSA). The conjugate (SE-BSA) was used to prepare an antiserum to SE. The antibodies were absorbed with BSA and their specificity was assayed by ELISA and flow cytometry against SE, iso-seiridin (ISE), a structural isomer of SE, and some derivatives of these two metabolites. The antibodies tested in a competitive indirect ELISA did not show any binding activity to SE, ISE and their derivatives. The cytometry test, instead, was successful. SE-BSA and SE showed the highest binding activity with the antibodies. SE derivatives having a shift on the adjacent carbon, oxidation, or acetylation of the hydroxy group of the heptyl side chain at C-4 or conversion of the gamma-lactone in the corresponding planar furane ring reacted less than SE. The 2'-dansylhydrazoneSE and the 3,4-dihydroSE having a bulky group attached to the heptyl side chain and a saturated lactone ring, respectively, showed a weak reactivity. SE derivatives in which the gamma-lactone ring was destroyed and ISE derivatives presenting the shift of the hydroxy group at C-3' and another structural modification had no binding activity.

Sylvaticin: a new cytotoxic and insecticidal acetogenin from Rollinia sylvatica (Annonaceae).

Sylvaticin (I), a new tetrahydroxy annonaceous acetogenin with nonadjacent tetrahydrofuran rings, has been isolated from the dried fruits of Rollinia sylvatica St. Hil. (Annonaceae). This compound is extremely cytotoxic to human tumor cells and shows promising insect control properties.

Interferon induction by SMANCS: a polymer-conjugated derivative of neocarzinostatin.

Poly (styrene-co-maleic acid)-conjugated neocarzinostatin, SMANCS, induced antiviral activities in the circulation of mice. The maximum titer of the activity (240 U/ml) was observed 20 h after administration an 8 mg/kg iv dose of SMANCS. Various experiments showed that this antiviral substance induced by SMANCS was an interferon (IFN). Sixty percent of the IFN response stimulated by SMANCS was impaired in mice pretreated with anti-IFN gamma antiserum. This suggests that the serum IFN induced by the agent contained about 60% of IFN gamma. When Thy 1+ or Lyt 2+ T-cells were depleted from mice by administration of anti-Thy 1.2 or anti-Lyt 2.2 monoclonal antibody (mAb), this IFN response was eliminated. However, when natural killer cells were depleted from mice by treatment with antiasialo GM1 antiserum, no alteration in the IFN response was observed. The SMANCS-stimulated IFN response was also abrogated in mice treated with macrophage blockers (carrageenan and trypan blue), whereas administration of anti-Lyt 1.2 mAb had no effect on the IFN response. These results suggest that macrophage and T-cells w Lyt 1-2+ phenotype may have an important role in the IFN response stimulated by SMANCS.

Stereospecificity in allergic contact dermatitis to simple substituted methylene lactone derivatives.

The enantiomers of beta,gamma-dimethyl- and beta-methyl-alpha-methylene-gamma-butyrolactones have been synthesized stereospecifically from glutamic acid and beta-hydroxy isobutyric acid, respectively. Guinea pigs have been sensitized (Freund complete adjuvant technique) and tested to them. Both enantiomers of beta-methyl lactone as well as (+)-beta,gamma-dimethyl lactone induced enantiospecific allergic contact dermatitis (ACD); in turn, (-)-beta,gamma-dimethyl lactone showed no specificity. An interpretation is proposed.

Poly-maleic acid anhydride (PMA) coupled cortisol antibody in routine determination of urinary free cortisol.

Antibodies against cortisol-21-hemisuccinate conjugated to bovine serum albumin was raised in rabbits and conjugated with poly-maleic acid anhydride. The conjugate was used in a solid phase radioimmunoassay to measure the urinary excretion of unconjugated cortisol. Cortisol was extracted with methylene chloride, the extract purified by chromatography on Sephadex LH-20, and the cortisol content measured with the radioimmunoassay. The intra-assay variation of the method was 6 per cent and the interassay variation 10 per cent. In 95 normal adult persons the excretion of free cortisol was 11--70, mean 30 micrograms/24 h. No sex or age difference was found. In 8 children aged 7--14 years the excretion was 8--46 micrograms/24 h.

Laboratory studies on the immune effects of halogenated aromatics.

The effects of TCDD exposure on the developing immune system were investigated in Wistar/Fischer hybrid or Fischer rats. Fetal and neonatal rats were exposed to TCDD through maternal dosing (5 muk/kg) on day 18 of gestation and on days 0, 7, and 14 of postnatal life (group 1). Another group of neonatal rats was exposed to TCDD through maternal dosing on days 0, 7, and 14 of postnatal life only (group 2). Variable but significant effects on body weights and thymus/body weight ratios were found up to 133 days of age. Cell mediated immune functions were depressed up to 133 days of age in both groups but less severely in animals exposed only postnatally. Furthermore, TCDD suppressed cell-mediated immune functions without affecting humoral immune function. Adoptive cell transfer studies indicated suppression of T-cell functions was selective in that "helper" cell function was not suppressed. In other studies, the effects on lymphocyte function following brief exposure of spleens from B6C3F1 mice to TCDD in dimethylsulfoxide (DMSO) were investigated. DNA, RNA and protein synthesis were inhibited at concentrations less than 2 X 10(-7) M TCDD in DMSO. This concentration accounted for approximately 0.2 ng TCDD uptake per spleen. The structurally related chemicals 3,4,3',4'-tetrachlorobiphenyl and 1-amino, 3,7,8-trichlorodibenzop-p-dioxin did not show significant lymphocyte effects even at two-fold higher concentrations. The ability of lymphocyte mitogens to bind to their cell surface receptors was not affected by TCDD treatment. TCDD was slightly cytolytic to lymphocytes after 48 hours of culture. DMSO treatment alone was also slightly toxic to lymphoid cells as indicated by a 10--20% loss of cell viability, although this occurred within 4 hours after DMSO exposure. Studies were performed to investigate the effects of 2,3,7,8-tetrachlorodibenzofuran (TCDF) on immune function in adult Hartley guinea pigs. Animals received 6 weekly doses of either 0, 0.05, 0.17, 0.5 or 1.0 microgram TCDF/kg body weight. TCDF slightly depressed cell-mediated immune functions, particularly at the higher dose levels as indicated by decreased lymphocyte blastogenesis, delayed hypersensitivity reactions, and production of macrophage inhibitor factor. Additionally, thymus-to-body-weight ratios were slightly reduced in the 0.5 and 1.0 microgram dosage groups. Serum IgG levels and antibody titer to BGG did not differ from controls. These results indicate that TCDF-induced immunosuppression is similar to that of TCDD.