ABSTRACT
Immune potentiators, termed adjuvants, trigger early innate immune responses to ensure the generation of robust and long-lasting adaptive immune responses of vaccines. Presented here is a study that takes advantage of a self-assembling small-molecule library for the development of a novel vaccine adjuvant. Cell-based screening of the library and subsequent structural optimization led to the discovery of a simple, chemically tractable deoxycholate derivative (molecule 6, also named cholicamide) whose well-defined nanoassembly potently elicits innate immune responses in macrophages and dendritic cells. Functional and mechanistic analyses indicate that the virus-like assembly enters the cells and stimulates the innate immune response through Toll-like receptor 7 (TLR7), an endosomal TLR that detects single-stranded viral RNA. As an influenza vaccine adjuvant in mice, molecule 6 was as potent as Alum, a clinically used adjuvant. The studies described here pave the way for a new approach to discovering and designing self-assembling small-molecule adjuvants against pathogens, including emerging viruses.
Subject(s)
Adjuvants, Immunologic/chemistry , Amides/chemistry , Amides/immunology , Amides/pharmacology , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Dendritic Cells/cytology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Deoxycholic Acid/chemistry , Drug Evaluation, Preclinical , Fluorescent Dyes/chemistry , Immunity, Innate , Immunoglobulin G/blood , Influenza Vaccines/chemistry , Influenza Vaccines/immunology , Interleukin-6/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nanostructures/chemistry , RAW 264.7 Cells , Structure-Activity Relationship , Toll-Like Receptor 7/metabolismABSTRACT
Nowadays, instrumental methodologies and rapid bioanalytical techniques complement each other for the analysis of toxic chemical compounds. Fluxapyroxad was commercialized a few years ago as a fungicide and today it is being used worldwide to control a variety of pests. In the present study, the development of monoclonal antibody-based immunochemical methods for the analysis of this chemical in food samples was evaluated for the first time. Novel haptens were synthesized and protein bioconjugates were prepared. High-affinity and specific monoclonal antibodies to fluxapyroxad were generated from two haptens with alternative linker tethering sites. Haptens with linker site heterology and a structurally heterologous hapten with a minor modification of the molecule conformation and volume but with a significant alteration of the electronic density of the pyrazole moiety were evaluated for immunoassay development. Direct and indirect competitive immunoassays were characterized and optimized, showing IC50 values for fluxapyroxad of 0.14 and 0.05 ng mL-1, respectively. The combination of two heterologies was particularly adequate in the indirect format. The two developed immunoassays showed excellent recoveries and coefficients of variation in fluxapyroxad-fortified plums and four varieties of grapes. Finally, a good correlation was found between the indirect immunoassay and UPLC-MS/MS when fruit samples with incurred residues of fluxapyroxad were analyzed. These monoclonal antibody-based immunochemical methods hold great promise for fluxapyroxad monitoring.
Subject(s)
Amides/analysis , Food Contamination/analysis , Fungicides, Industrial/analysis , Pyrazoles/analysis , Amides/immunology , Animals , Antibodies, Monoclonal/immunology , Enzyme-Linked Immunosorbent Assay/methods , Fruit/chemistry , Fungicides, Industrial/immunology , Haptens/chemistry , Limit of Detection , Mice , Pyrazoles/immunologyABSTRACT
Human cystatin C (hCC) is a small cysteine protease inhibitor whose oligomerization by propagated domain swapping is linked to certain neurological disorders. One of the ways to prevent hCC dimerization and fibrillogenesis is to enable its interaction with a proper antibody. Herein, the sites of interaction of hCC with dimer-preventing mouse monoclonal anti-hCC antibodies Cyst28 are studied and compared with the binding sites found for mAb Cyst10 that has almost no effect on hCC dimerization. In addition, hCC epitopes in complexes with native polyclonal antibodies extracted from human serum were studied. The results obtained with hydrogen-deuterium exchange mass spectrometry (HDX MS) were compared with the previous findings made using the excision/extraction MS approach. The main results from the two complementary MS-based approaches are found to be in agreement with each other, with some differences being attributed to the specificity of each method. The findings of the current studies may be important for future design of hCC dimerization inhibitors.
Subject(s)
Amides/immunology , Cystatin C/immunology , Epitope Mapping , Amides/chemistry , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Binding Sites , Cystatin C/antagonists & inhibitors , Cystatin C/chemistry , Deuterium Exchange Measurement , Humans , Mice , Protein Multimerization/drug effects , Protein Multimerization/immunologyABSTRACT
This study was undertaken to isolate active secondary metabolites from immunostimulatory Alcaligenes faecalis FY-3 and evaluate their activities using grass carp Ctenopharyngodon idella kidney (CIK) cells. By applying chromatography techniques and successive recrystallization, three purified metabolites were obtained and identified by spectral data (mass spectrometry and nuclear magnetic resonance) as: (1) phenylacetic acid, (2) p-hydroxyphenylacetylamide and (3) cyclo-(Gly-(L)-Pro). CIK cells were stimulated by different concentrations (1, 10 and 100 µg/ml) of the isolated compounds, and expression of MyD88, IL-1ß, TNF-α, type I-IFN and IL-8 genes at different time points (2, 8 and 24 h) post-stimulation was quantified by real-time PCR. The known immunostimulatory agent lipopolysaccharide (LPS) was used as a positive control. To analyse whether these compounds are toxic to the cells, the methyl tetrazolium assay was employed to measure changes in cell viability. The obtained results revealed that transcribing level of MyD88, an important adaptor molecule in toll-like receptor signalling pathway, was augmented remarkably by all the three isolated compounds and LPS as early as 2-h exposure. These compounds also induced gene expression of cytokines such as IL-1ß, TNF-α and type I-IFN. Under the experimental conditions, none of the test compounds is toxic to the CIK cells. These findings demonstrate that the immunostimulatory properties of the three metabolites [phenylacetic acid, p-hydroxyphenylacetylamide and cyclo-(Gly-(L)-Pro)] from A. faecalis FY-3 in CIK cells and highlight the potential of using these metabolites as immunostimulants in fish aquaculture.
Subject(s)
Adjuvants, Immunologic/metabolism , Alcaligenes faecalis/immunology , Carps/immunology , Gene Expression Regulation , Kidney/immunology , Adjuvants, Immunologic/chemistry , Amides/immunology , Amides/metabolism , Animals , Cells, Cultured , Cytokines/genetics , Cytokines/immunology , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/immunology , Peptides, Cyclic/immunology , Peptides, Cyclic/metabolism , Phenylacetates/immunology , Phenylacetates/metabolismABSTRACT
The interaction disorder between gut microbiota and its host has been documented in different non-communicable diseases (NCDs) such as metabolic syndrome, neurodegenerative disease, and autoimmune disease. The majority of these altered interactions arise through metabolic cross-talk between gut microbiota and host immune system, inducing a low-grade chronic inflammation that characterizes all NCDs. In this review, we discuss the contribution of bacterial metabolites to immune signaling pathways involved in NCDs. We then review recent advances that aid to rationally design microbial therapeutics. A deeper understanding of these intersections between host and gut microbiota metabolism using metabolomics-based system biology platform promises to reveal the fundamental mechanisms that drive metabolic predispositions to disease and suggest new avenues to use microbial therapeutic opportunities for NCDs treatment and prevention. Abbreviations: NCDs: non-communicable disease, IBD: inflammatory bowel disease, IL: interleukin, T2D: type 2 diabetes, SCFAs: short-chain fatty acids, HDAC: histone deacetylases, GPCR: G-protein coupled receptors, 5-HT: 5-hydroxytryptamine receptor signaling, DCs: dendritic cells, IECs: intestinal epithelial cells, T-reg: T regulatory cell, NF-κB: nuclear factor κB, TNF-α: tumor necrosis factor alpha, Th: T helper cell, CNS: central nervous system, ECs: enterochromaffin cells, NSAIDs: non-steroidal anti-inflammatory drugs, AhR: aryl hydrocarbon receptor, IDO: indoleamine 2,3-dioxygenase, QUIN: quinolinic acid, PC: phosphatidylcholine, TMA: trimethylamine, TMAO: trimethylamine N-oxide, CVD: cardiovascular disease, NASH: nonalcoholic steatohepatitis, BAs: bile acids, FXR: farnesoid X receptor, CDCA: chenodeoxycholic acid, DCA: deoxycholic acid, LCA: lithocholic acid, UDCA: ursodeoxycholic acid, CB: cannabinoid receptor, COBRA: constraint-based reconstruction and analysis.
Subject(s)
Bacteria/metabolism , Gastrointestinal Microbiome/physiology , Noncommunicable Diseases , Signal Transduction/immunology , Amides/immunology , Amides/metabolism , Bacteria/classification , Bacteria/isolation & purification , Bile Acids and Salts/immunology , Bile Acids and Salts/metabolism , Choline/immunology , Choline/metabolism , Disease Susceptibility/immunology , Disease Susceptibility/microbiology , Fatty Acids, Volatile/immunology , Fatty Acids, Volatile/metabolism , Humans , Immune System/immunology , Indoles/immunology , Indoles/metabolism , Polyamines/immunology , Polyamines/metabolism , Vitamins/immunology , Vitamins/metabolismABSTRACT
The purpose of this multidisciplinary investigation was to characterize cytokine production by human blood mononuclear cells after 2 contrasting exercise bouts (a maximal graded oxygen consumption [VO(2)max] test and 90 min of cycling at 85% of ventilatory threshold [VT]) when stimulated in vitro with extracts from bloodroot (Sanguinaria canadensis), coneflower (Echinacea tennesseensis), or solvent vehicle controls. Blood was sampled pre- and post-exercise. Production of TNF, IL-1beta, and IL-10 were measured at 24, 48, and 72 h, respectively. In the VO(2)max test there was a main effect of exercise such that exercise increased cytokine synthesis and a main effect of stimulant such that bloodroot extracts significantly increased cytokine production compared to other stimulants or controls. In the 90-min bout, there was a main effect of exercise for TNF and IL-1beta (but not IL-10) such that exercise decreased cytokine synthesis and a main effect of stimulant such that bloodroot extracts significantly increased cytokine production compared to other stimulants or controls, with exercisexstimulant interactions for both IL-1beta and IL-10. A similar though weaker effect was seen with Echinacea extracts; subsequent biochemical analyses suggested this was related to alkamide decay during 3 years undisturbed storage at ultralow (-80 degrees C) temperature. In this study, the VO(2)max test was associated with enhanced cytokine production whereas the 90-min cycling at 85% VT was associated with suppressed cytokine production. Bloodroot extracts were able to increase cytokine production in both contexts. Herbal extracts purported to offset exercise-associated effects on immune activity warrant continued investigation.
Subject(s)
Benzophenanthridines/pharmacology , Cytokines/blood , Echinacea , Exercise , Leukocytes, Mononuclear/drug effects , Sanguinaria , Adult , Amides/analysis , Amides/immunology , Amides/metabolism , Humans , Interleukin-10/agonists , Interleukin-10/blood , Interleukin-1beta/agonists , Interleukin-1beta/blood , Leukocytes, Mononuclear/immunology , Plant Extracts/pharmacology , Tumor Necrosis Factor-alpha/agonists , Tumor Necrosis Factor-alpha/blood , Young AdultABSTRACT
Site-selective labelling of antibodies (Abs) can circumvent problems from heterogeneity of conventional conjugation. Here, we evaluate the industrially-applied chemoenzymatic 'Q-tag' strategy based on transglutaminase-mediated (TGase) amide-bond formation in the generation of 89Zr-radiolabelled antibody conjugates. We show that, despite previously suggested high regioselectivity of TGases, in the anti-Her2 Ab Herceptin™ more precise native MS indicates only 70-80% functionalization at the target site (Q298H), in competition with modification at other sites, such as Q3H critically close to the CDR1 region.
Subject(s)
Antibodies/chemistry , Immunoconjugates/chemistry , Radioisotopes/chemistry , Zirconium/chemistry , Amides/chemistry , Amides/immunology , Amides/metabolism , Antibodies/immunology , Immunoconjugates/immunology , Molecular Structure , Transglutaminases/chemistry , Transglutaminases/immunology , Transglutaminases/metabolism , Zirconium/immunologyABSTRACT
Potato plants treated with the pathogen-associated molecular pattern Pep-13 mount salicylic acid- and jasmonic acid-dependent defense responses, leading to enhanced resistance against Phytophthora infestans, the causal agent of late blight disease. Recognition of Pep-13 is assumed to occur by binding to a yet unknown plasma membrane-localized receptor kinase. The potato genes annotated to encode the co-receptor BAK1, StSERK3A and StSERK3B, are activated in response to Pep-13 treatment. Transgenic RNAi-potato plants with reduced expression of both SERK3A and SERK3B were generated. In response to Pep-13 treatment, the formation of reactive oxygen species and MAP kinase activation, observed in wild type plants, is highly reduced in StSERK3A/B-RNAi plants, suggesting that StSERK3A/B are required for perception of Pep-13 in potato. In contrast, defense gene expression is induced by Pep-13 in both control and StSERK3A/B-depleted plants. Altered morphology of StSERK3A/B-RNAi plants correlates with major shifts in metabolism, as determined by untargeted metabolite profiling. Enhanced levels of hydroxycinnamic acid amides, typical phytoalexins of potato, in StSERK3A/B-RNAi plants are accompanied by significantly decreased levels of flavonoids and steroidal glycoalkaloids. Thus, altered metabolism in StSERK3A/B-RNAi plants correlates with the ability of StSERK3A/B-depleted plants to mount defense, despite highly decreased early immune responses.
Subject(s)
Gene Expression Regulation, Plant/immunology , Phytophthora infestans/pathogenicity , Plant Diseases/immunology , Plant Proteins/immunology , Protein Serine-Threonine Kinases/immunology , Receptors, Pattern Recognition/immunology , Solanum tuberosum/immunology , Alkaloids/immunology , Alkaloids/metabolism , Amides/immunology , Amides/metabolism , Coumaric Acids/immunology , Coumaric Acids/metabolism , Cyclopentanes/immunology , Cyclopentanes/metabolism , Disease Resistance/genetics , Flavonoids/immunology , Flavonoids/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/immunology , Metabolome/genetics , Metabolome/immunology , Oxylipins/immunology , Oxylipins/metabolism , Pathogen-Associated Molecular Pattern Molecules/immunology , Phytophthora infestans/physiology , Plant Diseases/genetics , Plant Proteins/antagonists & inhibitors , Plant Proteins/genetics , Plants, Genetically Modified , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reactive Oxygen Species/immunology , Reactive Oxygen Species/metabolism , Receptors, Pattern Recognition/antagonists & inhibitors , Receptors, Pattern Recognition/genetics , Salicylic Acid/immunology , Salicylic Acid/metabolism , Sesquiterpenes/immunology , Sesquiterpenes/metabolism , Solanum tuberosum/genetics , Solanum tuberosum/parasitology , PhytoalexinsABSTRACT
BACKGROUND: An effective HIV-1 vaccine or microbicide must block the transmitted virus variants that initially establish a new infection; consequently, it is critical that such viruses be isolated and characterized. OBJECTIVE: To evaluate HIV-1 envelope variants from early in infection from individuals infected heterosexually with subtype A HIV-1 for their sensitivity to antibody-mediated neutralization and to inhibitors of viral entry. METHODS: Full-length subtype A HIV-1 envelope clones from 28-75 days postinfection were used to generate pseudoviruses for infection studies. The susceptibility of these pseudoviruses to neutralization by autologous and heterologous plasma and by monoclonal antibodies was examined. The sensitivity of these pseudoviruses to PSC-RANTES and TAK-779, inhibitors of CCR5, and to soluble CD4 (sCD4) was also evaluated. RESULTS: Pseudoviruses with subtype A HIV-1 envelopes from early in infection demonstrated a broad range of neutralization sensitivities to both autologous and heterologous plasma. However, neutralization by the monoclonal antibodies b12, 2G12, 4E10 and 2F5 was generally poor; notably, none of the 14 early virus variants were neutralized by 2G12 and only one was neutralized by b12. Viruses bearing these early CCR5-using envelopes were generally sensitive to the CCR5 inhibitors PSC-RANTES and TAK-779, but they demonstrated more variable sensitivity to sCD4. CONCLUSIONS: These subtype A HIV-1 variants, representing the viruses that must be blocked by antibody-based prevention strategies, vary in their susceptibility to neutralization. A subset of these HIV-1 variants from early in infection will be useful for screening candidate vaccines and microbicides.
Subject(s)
Antibodies, Viral/immunology , CCR5 Receptor Antagonists , HIV Infections/virology , HIV-1/classification , Viral Envelope Proteins/immunology , Amides/immunology , Antibodies, Monoclonal/immunology , CD4 Antigens/immunology , Chemokine CCL5/immunology , Female , HIV Antigens/immunology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp160/immunology , HIV Fusion Inhibitors/immunology , HIV Infections/immunology , HIV-1/immunology , Humans , Kenya , Neutralization Tests , Phylogeny , Quaternary Ammonium Compounds/immunology , Receptors, CCR5/immunologySubject(s)
Amides/immunology , Anesthetics, Local/immunology , Esters/immunology , Hypersensitivity, Immediate/diagnosis , Immunoglobulin E/blood , Adult , Amides/adverse effects , Anesthetics, Local/adverse effects , Anesthetics, Local/chemistry , Esters/adverse effects , Female , Humans , Hypersensitivity, Immediate/etiology , Hypersensitivity, Immediate/immunologyABSTRACT
The influence of the presence of a terminal COOH or CONH2 on the antigenic characters of synthetic immunogenic peptides has been studied on a streptococcal synthetic vaccine model. The obtained results show that when a peptide amide is used, the antibodies raised specifically against the amide group recognize neither free COOH nor the parent protein. The carboxamide group is thus unsuitable as was postulated for raising antibodies which recognize the peptide bond.
Subject(s)
Amides/immunology , Antigens, Bacterial/immunology , Carbon Dioxide/immunology , Peptide Fragments/immunology , Streptococcus pyogenes/immunology , Animals , Antibodies, Bacterial/biosynthesis , Binding, Competitive , Epitopes/analysis , Female , Mice , Mice, Inbred BALB CABSTRACT
The analysis of T-cell responses to peptides has recently become a busy area of immunologic research. Peptides may be used as single stimulants, pools or libraries, or as part of peptide/major histocompatibility complexes (MHC) for direct T-cell receptor staining. For stimulating T cells, peptides must be bound to MHC molecules. In this study we have used 9- or 10-amino acid peptides, 15-amino acid peptides containing stimulating shorter sequences, and peptides with modified C-terminal function. On average 67% of the T cells from healthy cytomegalovirus-positive donors that bound a frequently used cytomegalovirus pp65/HLA-A*0201 tetramer were able to produce interferon-gamma on stimulation with the respective 9-amino acid peptide. Peptides of 15 amino acids length used at the same concentration (in microg/ml) stimulated CD8 T cells somewhat less efficiently (on average 77% of the frequencies induced with the respective shorter peptides). Modifications of 9-amino acid peptides such as addition of amino acids or functional groups often resulted in a decreased ability to stimulate. However, based on our own results, published data, and theoretic considerations, we conclude that sets of peptides of 15 amino acids length with 11 amino acids overlap represent a good compromise for stimulating both CD8 and CD4 T cells in a number of applications. These parameters may be modified subject to the purpose of a study.
Subject(s)
Epitopes, T-Lymphocyte/analysis , Glycine/analogs & derivatives , Lymphocyte Activation/immunology , Peptide Fragments/immunology , T-Lymphocytes/immunology , Amides/chemistry , Amides/immunology , Amino Acid Sequence , Amino Acids/immunology , Brefeldin A/pharmacology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Dose-Response Relationship, Immunologic , Epitopes, T-Lymphocyte/immunology , Flow Cytometry , Glycine/chemistry , Glycine/immunology , HLA-A Antigens/immunology , Humans , Immediate-Early Proteins/immunology , Interferon-gamma/metabolism , Leukocytes, Mononuclear/immunology , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Phosphoproteins/immunology , Viral Matrix Proteins/immunology , Viral Proteins/immunologyABSTRACT
Syntheses and cross-reactivities with progesterone toward the same specific antibody are reported for a series of amides of 11 alpha-hydroxyprogesterone 11-hemisuccinate. Some hypotheses are made regarding the effects of the chemical structure of the substituents on the immunological properties of derivatives.
Subject(s)
Amides/immunology , Antibody Affinity , Hydroxyprogesterones/immunology , Progesterone/analogs & derivatives , Amides/chemical synthesis , Antibody Specificity , Chemical Phenomena , Chemistry , Chromatography/methods , Cross Reactions , Hydroxyprogesterones/chemical synthesis , Progesterone/immunology , Radioimmunoassay , Structure-Activity RelationshipSubject(s)
Antihypertensive Agents/adverse effects , Dermatitis, Allergic Contact/etiology , Drug Eruptions/etiology , Eyelid Diseases/chemically induced , Prostaglandins F, Synthetic/adverse effects , Amides/administration & dosage , Amides/immunology , Antihypertensive Agents/administration & dosage , Antihypertensive Agents/immunology , Bimatoprost , Cloprostenol/administration & dosage , Cloprostenol/analogs & derivatives , Cloprostenol/immunology , Cross Reactions , Dermatitis, Allergic Contact/diagnosis , Drug Eruptions/diagnosis , Eyelid Diseases/diagnosis , Female , Glaucoma, Open-Angle/drug therapy , Humans , Instillation, Drug , Latanoprost , Middle Aged , Patch Tests , Prostaglandins F, Synthetic/administration & dosage , Prostaglandins F, Synthetic/immunologySubject(s)
Adjuvants, Immunologic , Cancer Vaccines/immunology , Melanoma, Experimental/immunology , Saponins/chemistry , Saponins/immunology , Skin Neoplasms/immunology , Adjuvants, Immunologic/chemical synthesis , Adjuvants, Immunologic/chemistry , Amides/immunology , Animals , Antibody Formation , Female , Gangliosides/immunology , Mice , Mice, Inbred C57BL , Saponins/chemical synthesisSubject(s)
Allergens/immunology , Anesthetics, Local/immunology , Bupivacaine/immunology , Carticaine/immunology , Dentistry , Drug Hypersensitivity/diagnosis , Lidocaine/immunology , Adult , Amides/chemistry , Amides/immunology , Anesthetics, Local/therapeutic use , Angioedema , Bupivacaine/therapeutic use , Carticaine/therapeutic use , Cross Reactions , Drug Tolerance , Erythema , Female , Humans , Hypersensitivity, Delayed , Lidocaine/therapeutic use , Patch TestsABSTRACT
Complete, life-long exclusion of gluten containing foods from the diet is the only available treatment for celiac sprue, a widespread immune disease of the small intestine. Investigations into the molecular pathogenesis of celiac sprue have identified the major histocompatibility complex protein HLA-DQ2 and the multi-functional enzyme transglutaminase 2 as potential pharmacological targets. Based upon the crystal structure of HLA-DQ2, we rationally designed an aldehyde-functionalized, gluten peptide analogue as a tight-binding HLA-DQ2 ligand. Aldehyde-bearing gluten peptide analogues were also designed as high-affinity, reversible inhibitors of transglutaminase 2. By varying the side-chain length of the aldehyde-functionalized amino acid, we found that the optimal transglutaminase 2 inhibitor was 5 methylene units in length, 2 carbon atoms longer than its natural glutamine substrate.
Subject(s)
Aldehydes/chemistry , Amides/chemistry , Enzyme Inhibitors/pharmacology , GTP-Binding Proteins/antagonists & inhibitors , Glutens/chemistry , HLA-DQ Antigens/chemistry , Peptide Fragments/chemistry , Transglutaminases/antagonists & inhibitors , Aldehydes/immunology , Amides/immunology , Antigen Presentation , Binding Sites , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Drug Design , Enzyme Inhibitors/chemistry , GTP-Binding Proteins/metabolism , Gamma Rays , Glutens/immunology , HLA-DQ Antigens/metabolism , Humans , Ligands , Models, Molecular , Peptide Fragments/immunology , Protein Glutamine gamma Glutamyltransferase 2 , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transglutaminases/metabolismABSTRACT
Several new amides 4 of 5-substituted 3-methyl-4-isothiazolecarboxylic acid were obtained. These compounds have acetylamino or benzoylamino groups in position 5 of the isothiazole ring. In position 4, the carboxylic group was transformed in the amides using amino-acid esters. Activities of the obtained derivatives were checked in the humoral immune response and delayed type hypersensitivity reaction to sheep red blood cells (SRBCs) in vivo.
Subject(s)
Amides/chemical synthesis , Thiazoles/chemical synthesis , Amides/immunology , Amides/pharmacology , Animals , Antibody Formation/drug effects , Antibody Formation/immunology , Cell Proliferation/drug effects , Chemistry, Pharmaceutical/methods , Chemistry, Pharmaceutical/trends , Erythrocytes/drug effects , Erythrocytes/immunology , Hypersensitivity, Delayed/drug therapy , Hypersensitivity, Delayed/immunology , Male , Mice , Mice, Inbred CBA , Sheep , Spleen/cytology , Thiazoles/immunology , Thiazoles/pharmacologyABSTRACT
RATIONALE: Nuclear factor (NF)-kappaB is a transcription factor known to regulate the expression of many inflammatory genes, including cytokines, chemokines, and adhesion molecules. NF-kappaB is held inactive in the cytoplasm, bound to I-kappaB. The removal of I-kappaB, via the actions of inhibitor of kappaB (I-kappaB) kinase-2 (IKK-2), allows NF-kappaB to enter the nucleus. OBJECTIVES: To determine the impact of inhibiting IKK-2 on in vitro and in vivo models of airway inflammation. METHODS: The effect of inhibiting IKK-2 was assessed in stimulated, cultured, primary human airway smooth muscle cells and an antigen-driven rat model of lung inflammation. MEASUREMENTS: The release of cytokines from cultured cells and inflammatory cytokine expression and cellular burden in the lung were determined. MAIN RESULTS: Two structurally distinct molecules and dominant negative technology demonstrated that inhibition of IKK-2 activity completely blocked cytokine release from cultured cells, whereas the two glucocorticoid comparators had limited impact on granulocyte colony-stimulating factor, interleukin 8, and eotaxin release. In addition, in an in vivo antigen-driven model of airway inflammation, the IKK-2 inhibitor blocked NF-kappaB nuclear translocation, which was associated with a reduction in inflammatory cytokine gene and protein expression, airway eosinophilia, and late asthmatic reaction, similar in magnitude to that obtained with budesonide. CONCLUSION: This study demonstrates that inhibiting IKK-2 results in a general reduction of the inflammatory response in vitro and in vivo. Compounds of this class could have therapeutic utility in the treatment of asthma and may, in certain respects, possess a beneficial efficacy profile compared with that of a steroid.
Subject(s)
Amides/therapeutic use , Asthma/drug therapy , Disease Models, Animal , I-kappa B Kinase/antagonists & inhibitors , Muscle, Smooth/drug effects , Respiratory System/drug effects , Thiophenes/therapeutic use , Amides/immunology , Animals , Anti-Inflammatory Agents/immunology , Anti-Inflammatory Agents/therapeutic use , Asthma/immunology , Asthma/physiopathology , Budesonide/immunology , Budesonide/therapeutic use , Cells, Cultured/drug effects , Cells, Cultured/immunology , Chemokine CCL11 , Chemokines, CC/immunology , Dexamethasone/immunology , Dexamethasone/therapeutic use , Drug Evaluation, Preclinical , Gene Expression/drug effects , Gene Expression/immunology , Granulocyte Colony-Stimulating Factor/drug effects , Granulocyte Colony-Stimulating Factor/immunology , Humans , I-kappa B Kinase/immunology , Inflammation , Interleukin-8/immunology , Muscle, Smooth/cytology , Muscle, Smooth/immunology , Muscle, Smooth/physiopathology , NF-kappa B/drug effects , NF-kappa B/immunology , Rats , Respiratory System/cytology , Respiratory System/immunology , Respiratory System/physiopathology , Thiophenes/immunologyABSTRACT
A monoclonal antibody (clone AD4G2) was generated against a common part of microcystins and nodularins, the unusual amino acid Adda [(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4E,6E-dienoic acid]. A direct competitive ELISA based on this antibody was developed and the cross-reactivity pattern was measured. Different toxins showed a very similar response. The assay provides therefore a sum parameter of microcystins, nodularins and peptide fragments containing Adda. The IC50 for microcystin-LR was 0.33 microg L(-1) which leads to a detection limit of 0.07 microg L(-1). This is well below the concentration of 1 microg L(-1) proposed by the World Health Organisation (WHO) as the limit for drinking water. Microcystin-LR spiked water samples in the concentration range between 0.1 and 1 microg L(-1) were measured and a mean recovery of 113+/-23% was found. The antibody is well suited for the determination of microcystins in drinking as well as surface water.