ABSTRACT
Asthma is a complex airways disease with a wide spectrum which ranges from eosinophilic (Th2 driven) to mixed granulocytic (Th2/Th17 driven) phenotypes. Mixed granulocytic asthma is a cause of concern as corticosteroids often fail to control this phenotype. Different kinases such as Brutons's tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) play a pivotal role in shaping allergic airway inflammation. Ibrutinib is primarily a BTK inhibitor, however it is reported to be an ITK inhibitor as well. In this study, we sought to determine the effect of Ibrutinib on Th1, Th17 and Th2 immune responses in a cockroach allergen extract (CE)-induced mixed granulocytic (eosinophilic and neutrophilic) mouse model in preventative mode. Ibrutinib attenuated neutrophilic inflammation at a much lower doses (25-75⯵g/mouse) in CE-induced mixed granulocytic asthma whereas Th2/Th17 immune responses remained unaffected at these doses. However, at a much higher dose, i.e. 250⯵g/mouse, Ibrutinib remarkably suppressed both Th17/Th2 and lymphocytic/neutrophilic/eosinophilic airway inflammation. At molecular level, Ibrutinib suppressed phosphorylation of BTK in neutrophils at lower doses and ITK in CD4â¯+â¯T cells at higher doses in CE-treated mice. Further, effects of Ibrutinib were compared with dexamethasone on CE-induced mixed granulocytic asthma in therapeutic mode. Ibrutinib was able to control granulocytic inflammation along with Th2/Th17 immune response in therapeutic mode whereas dexamethasone limited only Th2/eosinophilic inflammation. Thus, Ibrutinib has the potential to suppress both Th17/Th2 and neutrophilic/eosinophilic inflammation during mixed granulocytic asthma and therefore may be pursued as alternative therapeutic option in difficult-to-treat asthma which is resistant to corticosteroids.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Anti-Asthmatic Agents/pharmacology , Asthma/drug therapy , Inflammation/drug therapy , Interleukin-2/antagonists & inhibitors , Neutrophils/drug effects , Protein-Tyrosine Kinases/metabolism , Agammaglobulinaemia Tyrosine Kinase/immunology , Allergens/immunology , Animals , Asthma/chemically induced , Asthma/immunology , Asthma/metabolism , Cockroaches/immunology , Cytokines/immunology , Cytokines/metabolism , Disease Models, Animal , Granulocytes/immunology , Granulocytes/metabolism , Inflammation/immunology , Inflammation/metabolism , Interleukin-2/immunology , Male , Mice , Mice, Inbred BALB C , Neutrophils/immunology , Neutrophils/metabolism , Plant Extracts/immunology , Protein-Tyrosine Kinases/immunology , Th17 Cells/drug effects , Th17 Cells/immunology , Th17 Cells/metabolism , Th2 Cells/drug effects , Th2 Cells/immunology , Th2 Cells/metabolismABSTRACT
The discovery of protein ligands, capable of forming a reversible covalent bond with amino acid residues on a protein target of interest, may represent a general strategy for the discovery of potent small-molecule inhibitors. We analyzed the ability of different aromatic aldehydes to form imines by reaction with lysine using 1 Hâ NMR techniques. 2-Hydroxybenzaldehyde derivatives were found to efficiently form imines in the millimolar concentration range. These benzaldehyde derivatives could increase the binding affinity of protein ligands towards the cognate protein target. Affinity maturation was achieved not only by displaying ligand and aldehyde moieties on two complementary locked nucleic acid strands but also by incorporating the binding fragments in a single small-molecule ligand. The affinity gain was only observed when lysine residues were accessible in the immediate surroundings of the ligand-binding site and could be abrogated by quenching with a molar excess of hydroxylamine.
Subject(s)
Carbonic Anhydrase II/antagonists & inhibitors , Interleukin-2/antagonists & inhibitors , Lysine/pharmacology , Serum Albumin, Human/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Amines/chemistry , Amines/pharmacology , Animals , Benzaldehydes/chemistry , Benzaldehydes/pharmacology , Carbonic Anhydrase II/metabolism , Cattle , Humans , Ligands , Lysine/chemistry , Models, Molecular , Molecular Structure , Small Molecule Libraries/chemistryABSTRACT
The objective of the study was to isolate the effect of feeding a diet supplemented with docosahexaenoic acid (DHA) during the suckling and/or the weaning period on immune system development and function in offspring. Dams were randomized to one of two nutritionally adequate diets: control diet (N=12, 0% DHA) or DHA diet (N=8, 0.9% DHA). Diets were fed to dams throughout lactation, and then at weaning (21d), two pups per dam were randomly assigned to continue on the same diet as the dam or consume the other experimental diet for an additional 21d. At 6 weeks, splenocyte phenotypes and ex vivo cytokine production after stimulation with concanavalin A (ConA), lipopolysaccharide (LPS) or ovalbumin were assessed. Pups who received the control diet during both periods had the lowest production of IL-2 after ConA (P<.05 for interaction). Pups fed DHA during suckling had higher IL-10 production after all mitogens, regardless of the weaning diet (P<.05). Feeding DHA at weaning, regardless of the suckling diet, resulted in a lower production of IL-1ß and TNF-α in LPS-stimulated splenocytes and a higher proportion of total CD27+ cells (all P<.03). Our findings suggest that providing no DHA during critical periods of immune development resulted in a less efficient Th1 response upon challenge (IL-2 production). Feeding DHA during suckling had a programming effect on the ability of splenocytes to produce the regulatory cytokine IL-10. Feeding a DHA diet during weaning led to a lower TNF-α and IL-1ß response to a bacterial antigen.
Subject(s)
Dietary Supplements , Docosahexaenoic Acids/therapeutic use , Immune System Diseases/prevention & control , Immune System/immunology , Immunity, Innate , Lactation , Maternal Nutritional Physiological Phenomena , Animals , Cells, Cultured , Female , Immune System/cytology , Immune System/growth & development , Immune System/pathology , Immune System Diseases/immunology , Immune System Diseases/pathology , Interleukin-10/agonists , Interleukin-10/antagonists & inhibitors , Interleukin-10/metabolism , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/metabolism , Interleukin-2/antagonists & inhibitors , Interleukin-2/metabolism , Lymphocyte Activation/drug effects , Lymphocytes/cytology , Lymphocytes/drug effects , Lymphocytes/immunology , Lymphocytes/metabolism , Mitogens/toxicity , Random Allocation , Rats, Sprague-Dawley , Spleen/cytology , Spleen/growth & development , Spleen/immunology , Spleen/pathology , Th1 Cells/cytology , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , WeaningABSTRACT
Iron oxide nanoparticles have been used in a wide range of biomedical applications, including drug delivery, molecular imaging, and cellular imaging. Various surface modifications have been applied to the particles to stabilize their surface and to give them a moiety for anchoring tags and/or drug molecules. Conventional methods of delivering immunosuppressant drugs often require a high dose of drugs to ensure therapeutic effects, but this can lead to toxic side effects. In this study, we used silica-coated iron oxide nanoparticles (IOSs) for a drug delivery application in which the nanoparticles carry the minimum amount of drug required to be effective to the target cells. IOSs could be loaded with water-insoluble immunosuppressive drug molecules (MPA: mycophenolic acid) and be used as a contrast agent for MRI. We characterized the IOSs for their physicochemical properties and found their average hydrodynamic diameter and core size to be 40.5nm and 5nm, respectively. Following the introduction of MPA-loaded IOSs (IOS/M), we evaluated the secretion dynamics of cytokines from peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA). The results showed that IOS/M effectively inhibited the secretion of the cytokines interleukin-2 and tumor necrosis factor α, with a minimal concentration of MPA. In conclusion, IOS/M may have potential applications in both efficient drug delivery and MRI.
Subject(s)
Drug Carriers , Ferric Compounds/chemistry , Immunosuppressive Agents/pharmacology , Magnetite Nanoparticles/chemistry , Mycophenolic Acid/pharmacology , Silicon Dioxide/chemistry , Cell Survival/drug effects , Drug Compounding , Drug Liberation , Humans , Hydrophobic and Hydrophilic Interactions , Immunosuppressive Agents/chemistry , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Interleukin-2/metabolism , Kinetics , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Magnetite Nanoparticles/ultrastructure , Mycophenolic Acid/chemistry , Particle Size , Phytohemagglutinins/pharmacology , Primary Cell Culture , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Oral oil formulations have been reported to deliver drugs into the lymph. Lymphatic delivery of immunomodulatory drugs can more efficiently expose the drugs to T-cells in lymph, consequently induce higher efficacy and lower side effects. In this study, effects of tacrolimus oral oil formulations on drug blood exposure, and on inhibition of T-cell's interleukin-2 (IL-2) production were investigated in rats. Oil formulations (sunflower oil, cacao butter, medium chain triglyceride, and palm oil) dissolving tacrolimus showed lower drug blood concentration than a solid dispersion formulation (SDF). The sunflower oil, and cacao butter formulations suppressed drug blood exposure to 50% of the SDF, and inhibited T-cell's IL-2 production similar to the SDF. In vitro digestion tests indicated that slower digestion of the oils might reduce amount and rate of tacrolimus blood absorption. The cacao butter formulations showed 3.0 times more rapid tacrolimus absorption to lymphatic fluid than the SDF. Ratio of the rate constants of absorption into lymph to that into blood was higher in oil formulations (15 times in cacao butter, 15 times sunflower oil, and 3.5 times palm oil) than in the SDF. These results indicated that the oral oil formulations might be suitable for reduced tacrolimus blood concentration for low systemic side effects, and keep high lymph concentration for high efficacy in organ transplantation patients.
Subject(s)
Drug Delivery Systems/methods , Interleukin-2/antagonists & inhibitors , Lymph Nodes/drug effects , Plant Oils/administration & dosage , T-Lymphocytes/drug effects , Tacrolimus/administration & dosage , Animals , Chemistry, Pharmaceutical , Dietary Fats/administration & dosage , Dietary Fats/pharmacokinetics , Dose-Response Relationship, Drug , Interleukin-2/biosynthesis , Lymph Nodes/metabolism , Male , Palm Oil , Plant Oils/chemistry , Plant Oils/pharmacokinetics , Rats , Rats, Inbred Lew , Sunflower Oil , T-Lymphocytes/metabolism , Tacrolimus/chemistry , Tacrolimus/pharmacokineticsABSTRACT
The effect of vitamin C on T lymphocyte function is not clear. In-vivo supplementation of vitamin C is found to have a stimulatory effect on T cells while studies in which ascorbic acid was added to T cell cultures show an inhibition of cell function. The study aims to investigate the effect of ascorbic acid on interleukin-2 secretion by T cells in whole blood cultures which better resembles the physiological environment than pure T cell cultures. It was found that ascorbic acid, when added to whole blood cultures at a very high concentration of 1 mM, inhibits interleukin-2 secretion by T cells (p<0.05). However at lower concentrations of 0.25 mM and 0.5 mM significant inhibition was not seen which is contrary to earlier reports in pure T cell cultures. Hence it can be concluded that ascorbic acid inhibits T cell function in-vitro at high concentrations but the effect is relatively less in whole blood cultures compared to pure T cell cultures.
Subject(s)
Ascorbic Acid/pharmacology , Interleukin-2/metabolism , T-Lymphocytes/drug effects , Ascorbic Acid/administration & dosage , Blood Cells/cytology , Blood Cells/drug effects , Cell Culture Techniques , Cells, Cultured , Dose-Response Relationship, Drug , Healthy Volunteers , Humans , Interleukin-2/antagonists & inhibitors , T-Lymphocytes/cytology , T-Lymphocytes/metabolismABSTRACT
Icaritin is a native compound from Epimedium Genus, a traditional Chinese herbal medicine which is effective in treating asthma, autoimmune diseases and viral infections. In the present paper, the immunosuppressive effects of icaritin were found through in vitro and in vivo studies. Icaritin could dose-dependently inhibit murine CD4(+) T cells proliferation stimulated with mitogens or specific antigen ovabumin (OVA). Icaritin at 0.25-25µM could down-regulate T cell activation marker CD25 expression and inhibit IL-2 production. It could also reduce the Th1 cytokine IFN-γ production significantly if the T cells were activated by ConA or anti-CD3; while the inhibition of IL-4 secretion was only seen on anti-CD3 activated T cells treated with low concentrations of icaritin. In vivo study showed that treatment of icaritin at 10mg/kg/day on mice could suppress the immune response with prolonged allograft skin survival. Further study demonstrated that it reduced the alloantigen-induced splenocytes proliferation and Th1/Th2 cytokines. It could also increase NF-AT luciferase activity in Jurkat-NF-AT-luc T cells. The above results suggested that icaritin might be used to treat Th1 dominated immune diseases by interfering T cells activation with mechanism different to CsA.
Subject(s)
Drugs, Chinese Herbal/therapeutic use , Flavonoids/therapeutic use , Graft Survival/drug effects , Immunosuppressive Agents/therapeutic use , Lymphocyte Activation/drug effects , Skin Transplantation , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/administration & dosage , Drugs, Chinese Herbal/isolation & purification , Female , Flavonoids/administration & dosage , Flavonoids/isolation & purification , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/isolation & purification , Interleukin-2/antagonists & inhibitors , Interleukin-2 Receptor alpha Subunit/antagonists & inhibitors , Jurkat Cells , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Spleen/cytology , Spleen/drug effects , Spleen/immunology , Th1 Cells/drug effects , Th1 Cells/immunology , Transplantation, HomologousABSTRACT
A number of studies have implicated tumor-induced T(reg) cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting T(reg) cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-ß1-induced T(reg) activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-ß1 and T(reg) cell frequency as well as peripheral blood TGF-ß1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-ß1-mediated T(reg) activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in T(reg) culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3ß and ERK1/2 signaling by SocL and wogonin in T(reg) cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells' response to TGF-ß1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-ß1 secretion as well as via inhibition of T cells' response to TGF-ß1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.
Subject(s)
Flavanones/pharmacology , Plant Extracts/pharmacology , Scutellaria/chemistry , T-Lymphocytes, Regulatory/drug effects , Transforming Growth Factor beta1/antagonists & inhibitors , Animals , Cell Line, Tumor , Flavanones/immunology , Glioma/drug therapy , Glioma/immunology , Glioma/metabolism , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/immunology , Glycogen Synthase Kinase 3 beta , Interleukin-10/antagonists & inhibitors , Interleukin-10/immunology , Interleukin-2/antagonists & inhibitors , Interleukin-2/immunology , MAP Kinase Signaling System/drug effects , Phosphorylation/drug effects , Plant Extracts/immunology , Plant Leaves/chemistry , Rats , Signal Transduction , Smad3 Protein/antagonists & inhibitors , Smad3 Protein/immunology , T-Lymphocytes, Regulatory/immunology , Transforming Growth Factor beta1/immunology , Transforming Growth Factor beta1/metabolism , p38 Mitogen-Activated Protein Kinases/immunologyABSTRACT
Owing to their high content of flavonoids and saponins, plantlets of Avena sativa L. (Poaceae) are likely to possess anti-inflammatory and immunoregulatory properties of value in the treatment of atopic dermatitis (AD). With a view to its potential use in atopic subjects at risk of developing sensitisation to dietary proteins, we prepared a plantlet extract without proteins and isolated 2 flavonoids, isoorientin-2''- O-arabinoside (1) and isovitexin-2''- O-arabinoside (2), and two saponins, avenacosides A (3) and B (4). The absence of protein in this extract was evidenced by electrophoresis and Western immunoblotting. Furthermore, Western immunoblotting demonstrated the absence of cross-reaction between grain and plantlet proteins. We evaluated the anti-inflammatory activity of the plantlet extract and its compounds IN VITRO in a model of keratinocyte inflammation: 6-keto prostaglandin F1 α production was inhibited by the plantlet extract (- 35â% and - 57â% at 10 and 30 µg/mL, respectively; p < 0.001) and isoorientin-2''- O-arabinoside (- 31â%, - 51â%, and - 56â% at 3, 10, and 30 µg/mL, respectively; p < 0.001). Intracellular interleukin-2 production in activated T lymphocytes was also inhibited by 16â%, 27â%, and 31â% with 3, 10, and 30 µg/mL plantlet extract, respectively, and by 23â% and 32â% with 3 and 10 µg/mL avenacoside A, respectively, (p < 0.001), demonstrating their immunoregulatory activity IN VITRO. The plantlet extract was also effective on the phenotype and function of dendritic cells (DC) differentiated from monocytes. It decreased the expression of major histocompatibility complex class II molecules on DC and significantly impaired their stimulatory activity on autologous T-cell proliferation (-25â%, p < 0.05). In conclusion, this protein-free oat plantlet extract exhibits anti-inflammatory and immunoregulatory activities in vitro.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Avena/chemistry , Flavonoids/pharmacology , Immunologic Factors/pharmacology , Plant Extracts/chemistry , Saponins/pharmacology , 6-Ketoprostaglandin F1 alpha/antagonists & inhibitors , 6-Ketoprostaglandin F1 alpha/metabolism , Anti-Inflammatory Agents/isolation & purification , Cell Proliferation/drug effects , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Epoprostenol/metabolism , Humans , Interleukin-2/antagonists & inhibitors , Interleukin-2/metabolism , Keratinocytes/drug effects , Keratinocytes/metabolism , Phenotype , Plant Components, Aerial/chemistry , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
CONTEXT: Calcineurin (CN), a unique protein phosphatase, plays an important role in immune regulation. Our laboratory has established an effective molecular drug-screening model based on CN activity. OBJECTIVE: Our aim is to search for an effective immunosuppressant from Glycyrrhiza uralensis (Leguminosae). MATERIALS AND METHODS: As guided by CN inhibitory test, an active compound was purified and identified as glycyrol. Immunosuppressive activity of glycyrol in vitro was assayed by T lymphocytes proliferation and mixed lymphocyte reaction (MLR). In addition, delayed-type hypersensitivity reaction (DTH) and skin allograft test in vivo were also carried out. Further, we have investigated the effect of glycyrol on phorbol 12-myristate 13-acetate (PMA)/ionomycin (Io)-stimulated IL-2 expression in Jurkat cells. RESULTS: The enzymatic assay showed glycyrol (IC(50) = 84.6 µM) inhibited calcineurin activity in a dose-dependent manner. Glycyrol, at the non-cytotoxic concentration, significantly inhibited proliferation of murine spleen T lymphocytes induced by Concanavalin A (Con A) and mixed lymphocyte reaction (MLR) in vitro. In addition, mice treated with glycyrol had shown the dose-dependent decrease in delayed type hypersensitivity (DTH) and prolonged the graft survival by 59% compared to the control group (*p < 0.05). RT-PCR showed glycyrol suppressed IL-2 production in a concentration-dependent manner. DISCUSSION AND CONCLUSION: Our results show the immunosuppressive activity of glycyrol and this activity should be due to its inhibitory effect on CN activity, thereby suppressing IL-2 production and regulating T lymphocytes. Thus, glycyrol could be a candidate for development as a novel immunomodulatory drug.
Subject(s)
Calcineurin Inhibitors , Flavonoids/pharmacology , Glycyrrhiza uralensis/chemistry , Immunosuppressive Agents/pharmacology , Plant Extracts/pharmacology , Animals , Cell Proliferation/drug effects , Concanavalin A/immunology , Concanavalin A/pharmacology , Dose-Response Relationship, Drug , Early Growth Response Protein 3/immunology , Flavonoids/chemistry , Graft Survival/drug effects , Graft Survival/immunology , Humans , Hypersensitivity, Delayed/immunology , Immunosuppressive Agents/chemistry , In Vitro Techniques , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Jurkat Cells , Lymphocyte Activation/drug effects , Lymphocyte Culture Test, Mixed , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , NFATC Transcription Factors/metabolism , Plant Extracts/chemistry , Plant Roots/chemistry , Signal Transduction/drug effects , Signal Transduction/physiology , Spleen/drug effects , Spleen/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
In order to explore the anti-inflammatory effects of Nodosin from Isodon serra, a traditional Chinese herb medicine, mouse T lymphocytes were incubated with Nodosin. In the current study, Nodosin suppressed the overproduction of the T lymphocytes; moreover, cell mitosis cycle was modulated by interfering with DNA replication in G1 stages via inhibition of IL-2 cytokine secretion at the mRNA level by Nodosin. Interestingly, Xylene-induced mouse tumescence model results suggested Nodosin depressed the murine ear-swelling extent and the level of IL-2 in the blood serum. Finally, Nodosin possessed significant anti-inflammatory effects and is a potential candidate for further clinical trial.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Diterpenes/therapeutic use , Drugs, Chinese Herbal/therapeutic use , Interleukin-2/antagonists & inhibitors , Isodon/chemistry , T-Lymphocytes/drug effects , Animals , Anti-Inflammatory Agents/isolation & purification , Anti-Inflammatory Agents/pharmacology , Cell Cycle/drug effects , DNA Replication/drug effects , Diterpenes/isolation & purification , Diterpenes/pharmacology , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Ear , Edema/chemically induced , Edema/drug therapy , Edema/immunology , Female , Interleukin-2/blood , Male , Mice , Mice, Inbred BALB C , Phytotherapy , Plant Components, Aerial , RNA, Messenger/metabolism , T-Lymphocytes/metabolism , XylenesABSTRACT
In the course of our studies on the isolation of bioactive compounds from the roots of Moringa oleifera, a traditional herb in southeast Asia, rare aurantiamide acetate 4 and 1,3-dibenzyl urea 5 have been isolated and characterized. And also, this is the first report of isolation from this genus. Isolated compound inhibited the production of TNF-alpha and IL-2; further compound 5 showed significant analgesic activities in a dose dependant manner. These findings may help in understanding the mechanism of action of this traditional plant leading to control of activated mast cells on inflammatory conditions like arthritis, for which the crude extract has been used.
Subject(s)
Analgesics/chemical synthesis , Anti-Inflammatory Agents/chemical synthesis , Dipeptides/chemical synthesis , Moringa oleifera/chemistry , Urea/chemical synthesis , Analgesics/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Dipeptides/pharmacology , Interleukin-2/antagonists & inhibitors , Mast Cells/drug effects , Plant Extracts/chemistry , Plant Extracts/pharmacology , Plant Roots/chemistry , Rats , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Urea/pharmacologyABSTRACT
The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular processes. The PKC theta (PKCtheta) isoform is involved in TCR signal transduction and T cell activation and regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus inhibition of PKCtheta enzyme activity by a small molecule represents an attractive strategy for the treatment of asthma. A PKCtheta high-throughput screening (HTS) campaign led to the identification of 4-(3-bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low microM ATP competitive PKCtheta inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCtheta inhibitor. Compound 4p was selective for inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the conventional and atypical PKCs, PKCbeta, and PKCzeta, respectively. Compound 4p also inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.
Subject(s)
Indoles/pharmacology , Isoenzymes/antagonists & inhibitors , Nitriles/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Animals , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , Indoles/chemical synthesis , Indoles/chemistry , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Isoenzymes/deficiency , Isoenzymes/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Molecular Structure , Nitriles/chemical synthesis , Nitriles/chemistry , Protein Kinase C/deficiency , Protein Kinase C/drug effects , Protein Kinase C-theta , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyridines/chemical synthesis , Pyridines/chemistry , Spleen/cytology , Spleen/drug effects , Spleen/immunology , Stereoisomerism , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
The plant sterols campesterol, beta-sitosterol and beta-sitostanol were investigated for potential immunomodulatory effects in Jurkat T cells. Treatments involved supplementing cells with or without concanavalin A (ConA) or phorbol-12-myristate-13-acetate plus ionomycin (PMA+IoM) in the presence or absence of increasing concentrations (10-100 microM) of each plant sterol for 24 h. None of the plant sterols significantly affected mitogen-stimulated IL-4, IL-10 or IFN-gamma production. However, campesterol, beta-sitosterol and beta-sitostanol significantly suppressed mitogen-induced IL-2 production in a dose-dependent manner. Both bisindolylmaleimide-I (BIM-I), a specific protein kinase C (PKC) inhibitor, and the immunosuppressant drug known as Tacrolimus (FK506), an IL-2 inhibitor, prevented mitogen-stimulated IL-2 production in Jurkat cells. Treatment with PMA+IoM alone significantly increased PKC activity and the presence of BIM-I prevented PKC activation by PMA+IoM. Following 24 h treatments, the plant sterols did not affect PMA+IoM-enhanced PKC activity, cellular calcium content or calcineurin activity. Intracellular cyclic 3',5'-adenosine monophosphate (cAMP) levels were significantly reduced by PMA+IoM. The presence of FK506 prevented a PMA+IoM-induced reduction of intracellular cAMP. Likewise the plant sterols behaved in a similar manner as FK506. Our findings suggest that the suppression of IL-2 by the plant sterols was not mediated via PKC inhibition and that their effects occurred possibly via cAMP modulation and/or a calcium/calcineurin-independent pathway.
Subject(s)
Cytokines/biosynthesis , Immunologic Factors/pharmacology , Phytosterols/pharmacology , T-Lymphocytes/metabolism , Cell Division , Cell Survival , Cholesterol/analogs & derivatives , Cholesterol/pharmacology , Concanavalin A/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Immunosuppressive Agents/pharmacology , Indoles/pharmacology , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Jurkat Cells , Lymphocyte Activation , Maleimides/pharmacology , Protein Kinase C/antagonists & inhibitors , Sitosterols/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tacrolimus/pharmacology , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
En los últimos años han aparecido una serie de nuevos fármacos desarrollados por biología molecular. Estos medicamentos actúan bloqueando moléculas específicas del sistema inmunológico y se desarrollan para actuar sobre dianas específicas que tienen un papel importante en la fisiopatología de determinadas enfermedades para cuyo tratamiento son aprobadas. Con el tiempo se ha ido adquiriendo experiencia con estos medicamentos en el tratamiento de dermatosis para las que no han sido diseñados, pero para las que, por compartir un mismo mecanismo fisiopatológico, pueden ser útiles. El empleo de estos medicamentos en el tratamiento de casos difíciles de numerosas enfermedades dermatológicas para las cuales no están aprobados es creciente. Esta segunda parte de la revisión analiza el uso, fuera de indicación, en el tratamiento de la dermatosis de los siguientes fármacos biológicos: etanercept, efalizumab, alefacept, rituximab, daclizumab, basiliximab, omalizumab y cetuximab
In recent years, a series of new drugs have been developed through the application of molecular biology. These drugs act by blocking specific molecules of the immune system and have been developed to act on specific targets that play an important role in the pathophysiology of the diseases in which their therapeutic use has now been approved. Over time, experience has been accumulated in the use of these drugs in the treatment of skin diseases for which they have not been approved but in which the pathophysiology suggests that they could also be effective. The use of these drugs is increasing in difficult-to-treat cases of skin diseases for which the drugs are not approved. The second part of this review of off-label use of biologic agents in dermatology considers the use of etanercept, efalizumab, alefacept, rituximab, basiliximab, omalizumab, and cetuximab
Subject(s)
Humans , Biological Therapy/methods , Skin Diseases/drug therapy , Drug Approval , Lymphocyte Function-Associated Antigen-1 , CD58 Antigens , Antigens, CD20 , Immunoglobulin E , Interleukin-2/antagonists & inhibitors , ErbB Receptors/antagonists & inhibitors , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
In order to identify new, immune modulating compounds, aqueous extracts of plants pre-selected on ethno-pharmacological knowledge were screened for inhibitory effects in an anti-CD3 driven lymphocyte proliferation assay (MTT-assay). We found for the extract of the inner bark of Tabebuia avellanedae (Tabebuia) dose dependent and reproducible inhibitory effects on lymphocyte proliferation. We further analyzed Tabebuia in flow cytometry based whole blood T-cell function assays. We found that Tabebuia inhibited dose dependent ConA stimulated T-cell proliferation. Decreased T-lymphocyte proliferation was associated with dose dependent reduction of CD25 and CD71 expression on T-lymphocytes. In contrast Tabebuia exerted no effects on cytokine expression (Il-2 and TNF-alpha) by PMA/Ionomycin stimulated T-lymphocytes. Concentrations of Tabebuia used were not toxic for lymphocytes as verified by trypan blue exclusion assay. Further experiments showed that the immune inhibitory effects by Tabebuia were not mediated by its pharmacological lead compound beta-lapachone and only observed in aqueous but not in ethanol plant extracts.
Subject(s)
Cell Proliferation , Immune Tolerance/drug effects , Immunosuppressive Agents/pharmacology , Interleukin-2/physiology , Lymphocyte Activation/immunology , Plant Extracts/pharmacology , T-Lymphocytes/immunology , Tabebuia/immunology , Cell Proliferation/drug effects , Cells, Cultured , Growth Inhibitors/pharmacology , Humans , Immune Tolerance/immunology , Interleukin-2/antagonists & inhibitors , Lymphocyte Activation/drug effects , T-Lymphocytes/cytology , T-Lymphocytes/drug effectsABSTRACT
Myricetin is a naturally occurring flavonoid that is commonly found in tea, berries, fruits, vegetables, and medicinal herbs. This study examined the effects of myricetin on the production of interlukin-2 (IL-2), a potent T cell growth factor. Treatment with myricetin significantly inhibited the secretion of the IL-2 protein from mouse EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io) in a dose-dependent manner. Flow cytometric analysis showed that myricetin suppressed the intracellular production of the IL-2 protein. Furthermore, the effects of myricetin on mRNA expression were analyzed by reverse transcription-polymerase chain reaction and it showed that myricetin reduced the expression of IL-2 mRNA induced by PMA plus Io. This suggests that myricetin has potential immunosuppressive effects by inhibiting the production of IL-2.
Subject(s)
Flavonoids/pharmacology , Interleukin-2/antagonists & inhibitors , Animals , Cells, Cultured , Interleukin-2/biosynthesis , Interleukin-2/genetics , Mice , NF-kappa B/physiology , RNA, Messenger/analysis , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Areca quid chewing is a major risk factor associated with oral submucous fibrosis (OSF) and oral cancer. Experimental evidence indicates that immune deterioration is associated with the pathophysiology of OSF and oral cancer. In addition, reactive oxygen species (ROS) is shown to play a role in the cytotoxic and genotoxic effect induced by areca nut extracts (ANE) in oral cells. The present studies investigated the effects of ANE on T-cell reactivity and the role of ROS in ANE effects. Treatment of splenocytes with ANE induced a marked cytotoxic effect, and suppressed the production of IL-2 and IFN-gamma, whereas the production of IL-4 was unaffected. The ANE-mediated cytotoxicity, and suppression of IFN-gamma and IL-2 production were attenuated by the presence of antioxidant N-acetyl-l-cysteine (NAC). Moreover, flow cytometric analysis demonstrated an increase in cellular ROS levels in splenic T-cells treated with ANE, which was also attenuated by the presence of NAC. Concordantly, the cellular level of glutathione was diminished by ANE in splenic T-cells pretreated with NAC. Collectively, these results demonstrated that ANE markedly suppressed T-cell activation and Th1 cytokine production, which was mediated, at least in part, by the induction of oxidative stress.
Subject(s)
Areca/chemistry , Interferon-gamma/biosynthesis , Oxidative Stress/drug effects , Plant Extracts/toxicity , T-Lymphocytes/drug effects , Animals , Antioxidants/pharmacology , Cell Survival/drug effects , Cell Survival/immunology , Drug Interactions , Glutathione/immunology , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/immunology , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Interleukin-2/immunology , Interleukin-4/antagonists & inhibitors , Interleukin-4/biosynthesis , Interleukin-4/immunology , Lymphocyte Activation/drug effects , Male , Mice , Mice, Inbred BALB C , Nuts/chemistry , Oxidative Stress/immunology , Random Allocation , Reactive Oxygen Species/metabolism , Spleen/cytology , Spleen/drug effects , Spleen/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Conjugated linoleic acid (CLA) refers to geometric and positional isomers of linoleic acid. Animal studies have shown that CLA modulates the immune system and suggest that it may have a therapeutic role in inflammatory disorders. This double-blind placebo-controlled intervention trial investigated the effects of CLA supplementation on indices of immunity relating to cardiovascular disease (CVD) in a cohort of healthy middle-aged male volunteers. Subjects were randomly assigned to supplement their diet with 2.2 g 50:50 isomeric blend of cis 9, trans 11 (c9, t11)-CLA and trans 10, cis 12 (t10, c12)-CLA or placebo daily for 8 weeks. Interleukin (IL) 2, IL-10 and tumour necrosis factor (TNF) alpha were measured in the supernatant of cultured unstimulated and concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) by ELISA. Serum IL-6 and plasma CRP were measured by ELISA and plasma fibrinogen by automated clotting assay. Gene expression was investigated by real-time RT-PCR. CLA supplementation significantly reduced Con A-stimulated PBMC IL-2 secretion (37.1%; P=.02). CLA supplementation had no significant effect on transcription of IL-2. CLA supplementation had no direct significant effects on PBMC TNFalpha or IL-10 secretion. Other inflammatory markers associated with CVD, including IL-6, CRP and fibrinogen, were not affected by CLA supplementation. This study showed that CLA supplementation reduced PBMC IL-2 secretion from Con A-stimulated PBMC but lacked effect on other markers of the human inflammatory response.
Subject(s)
Interleukin-2/biosynthesis , Leukocytes, Mononuclear/drug effects , Linoleic Acids, Conjugated/pharmacology , Adult , Concanavalin A/pharmacology , Double-Blind Method , Humans , Interleukin-10/biosynthesis , Interleukin-2/antagonists & inhibitors , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
Chinese herbs are useful edible and medicinal plants for their immune modulatory functions. We have proven that (S)-armepavine (C19H23O3N; MW313) from Nelumbo nucifera inhibits the proliferation of human PBMCs activated with PHA and improves autoimmune diseases in MRL/MpJ-lpr/lpr mice. In the present study, the pharmacological activities of (S)-armepavine were evaluated in PHA-activated PBMCs. The results showed that (S)-armepavine suppressed PHA-induced PBMC proliferation and genes expression of IL-2 and IFN-gamma without direct cytotoxicity. Inhibition of NF-AT and NF-kappaB activation suggested phospholipase Cgamma (PLCgamma)-mediated Ca2+ mobilization and protein kinase C activation were blocked by (S)-armepavine. Phosphorylation of PLCgamma is regulated by lymphocyte-specific kinase (Lck), ZAP-70, and IL-2-inducible T cell kinase (Itk). We found (S)-armepavine inhibited PHA-induced phosphorylation of Itk and PLCgamma efficiently but did not influence Lck or ZAP-70 phosphorylation. In addition, ZAP-70-mediated pathways, such as the association of linker for activation of T cells with PLCgamma and activation of ERK, were also intact in the presence of (S)-armepavine. Finally, reduction of phosphoinositide 3,4,5-trisphosphate formation and Akt phosphorylation suggested that (S)-armepavine inhibited Itk, and PLCgamma phosphorylation might be a result of the influence of PI-3K activation. Addition of exogenous IL-2 or PMA/A23187 rescued PBMC proliferation in the presence of (S)-armepavine. Therefore, we concluded that (S)-armepavine inhibited PHA-induced cell proliferation and cytokine production in a major way by blocking membrane-proximal effectors such as Itk and PLCgamma in a PI-3K-dependent manner.