RESUMEN
Multiple studies have established associations between human gut bacteria and host physiology, but determining the molecular mechanisms underlying these associations has been challenging1-3. Akkermansia muciniphila has been robustly associated with positive systemic effects on host metabolism, favourable outcomes to checkpoint blockade in cancer immunotherapy and homeostatic immunity4-7. Here we report the identification of a lipid from A. muciniphila's cell membrane that recapitulates the immunomodulatory activity of A. muciniphila in cell-based assays8. The isolated immunogen, a diacyl phosphatidylethanolamine with two branched chains (a15:0-i15:0 PE), was characterized through both spectroscopic analysis and chemical synthesis. The immunogenic activity of a15:0-i15:0 PE has a highly restricted structure-activity relationship, and its immune signalling requires an unexpected toll-like receptor TLR2-TLR1 heterodimer9,10. Certain features of the phospholipid's activity are worth noting: it is significantly less potent than known natural and synthetic TLR2 agonists; it preferentially induces some inflammatory cytokines but not others; and, at low doses (1% of EC50) it resets activation thresholds and responses for immune signalling. Identifying both the molecule and an equipotent synthetic analogue, its non-canonical TLR2-TLR1 signalling pathway, its immunomodulatory selectivity and its low-dose immunoregulatory effects provide a molecular mechanism for a model of A. muciniphila's ability to set immunological tone and its varied roles in health and disease.
Asunto(s)
Akkermansia , Homeostasis , Inmunidad , Fosfatidiletanolaminas , Akkermansia/química , Akkermansia/citología , Akkermansia/inmunología , Membrana Celular/química , Membrana Celular/inmunología , Citocinas/inmunología , Homeostasis/inmunología , Humanos , Mediadores de Inflamación/síntesis química , Mediadores de Inflamación/química , Mediadores de Inflamación/inmunología , Fosfatidiletanolaminas/síntesis química , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/inmunología , Relación Estructura-Actividad , Receptor Toll-Like 1/inmunología , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/inmunologíaRESUMEN
Omega-3 polyunsaturated fatty acids (PUFAs) have been known to have beneficial effects in the prevention of various diseases. Recently, it was identified that the bioactivities of omega-3 are related to lipid mediators, called pro-resolving lipid mediators (SPMs), converted from PUFAs, so they have attracted much attention as potential pharmaceutical targets. Here, we aimed to build an efficient production system composed of enzymatic and chemical catalysis that converts docosahexaenoic acid (DHA) into lipid mediators. The cyanobacterial lipoxygenase, named Osc-LOX, was identified and characterized, and the binding poses of enzyme and substrates were predicted by ligand docking simulation. DHA was converted into three lipid mediators, a 17S-hydroxy-DHA, a 7S,17S-dihydroxy-DHA (RvD5), and a 7S,15R-dihydroxy-16S,17S-epoxy-DPA (new type), by an enzymatic reaction and deoxygenation. Also, two lipid mediators, 7S,15R,16S,17S-tetrahydroxy-DPA (new type) and 7S,16R,17S-trihydroxy-DHA (RvD2), were generated from 7S,15R-dihydroxy-16S,17S-epoxy-DPA by a chemical reaction. Our study suggests that discovering new enzymes that have not been functionally characterized would be a powerful strategy for producing various lipid mediators. Also, this combination catalysis approach including biological and chemical reactions could be an effective production system for the manufacturing lipid mediators.
Asunto(s)
Ácidos Docosahexaenoicos/síntesis química , Mediadores de Inflamación/síntesis química , Inflamación/tratamiento farmacológico , Lípidos/síntesis química , Catálisis , Ácidos Docosahexaenoicos/química , Ácidos Docosahexaenoicos/farmacología , Ácidos Grasos Omega-3/síntesis química , Ácidos Grasos Omega-3/química , Ácidos Grasos Omega-3/farmacología , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/farmacología , Humanos , Inflamación/patología , Mediadores de Inflamación/química , Mediadores de Inflamación/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/química , Lípidos/farmacología , Lipooxigenasa/químicaRESUMEN
MYMD-1 is a synthetic derivative of tobacco alkaloids, compounds that possess immunoregulatory properties and have been linked to the epidemiological observation that smoking reduces the odds of developing thyroid Abs and hypothyroidism. To assess the effect and mechanism(s) of the action of MYMD-1, we chose the NOD.H-2h4 mouse model of spontaneous thyroiditis. We began in vitro using T cells isolated from NOD.H-2h4 spleens and found that MYMD-1 suppressed TNF-α production by CD4+ T cells in a dose-dependent manner. We then treated 58 NOD.H-2h4 mice for 12 wk with either unsupplemented water that contained (10 mice) or did not contain (16 mice) MYMD-1 (185 mg/l) or water supplemented with sodium iodide (500 mg/l) that contained (16 mice) or did not contain (16 mice) MYMD-1. Mice were bled at baseline and then every 2 wk until sacrifice. MYMD-1 decreased the incidence and severity (p < 0.001) of thyroiditis, as assessed by histopathology. Similarly, the number of CD3+ T cells and CD19+ B cells infiltrating the thyroid was dampened by MYMD-1, as assessed by flow cytometry. Interestingly, the subset of thyroidal CD3+CD4+Tbet+RORγT- effector Th1 cells and the systemic levels of TNF-α were decreased by MYMD-1. Serum thyroglobulin Abs decreased in the MYMD-1 group. Thyroid hormones did not differ among the four groups, whereas thyroid-stimulating hormone increased upon iodine supplementation but remained normal in MYMD-1-treated mice. Overall, the study suggests that MYMD-1 ameliorates thyroiditis acting on specific lymphoid subsets. Further studies, including other models of autoimmunity, will confirm the potential clinical use of MYMD-1 as a novel immunometabolic regulator.
Asunto(s)
Alcaloides/farmacología , Mediadores de Inflamación/farmacología , Tiroiditis Autoinmune/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Alcaloides/síntesis química , Alcaloides/química , Animales , Femenino , Mediadores de Inflamación/síntesis química , Mediadores de Inflamación/química , Masculino , Ratones , Ratones Endogámicos NOD , Células TH1/efectos de los fármacos , Células TH1/inmunología , Tiroiditis Autoinmune/inmunología , Nicotiana/química , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Resolvins, maresins, and protectins can be formed from fish oils. These specialized pro-resolving mediators (SPMs) have been implicated in the resolution of inflammation. Synthetic versions of such SPMs exert anti-inflammatory effects in vitro and when administered to animal models. However, their importance as endogenous products formed in sufficient amounts to exert anti-inflammatory actions in vivo remains speculative. We biased our ability to detect SPMs formed in healthy volunteers by supplementing fish oil in doses shown previously to influence blood pressure and platelet aggregation under placebo-controlled conditions. Additionally, we sought to determine the relative formation of SPMs during an acute inflammatory response and its resolution, evoked in healthy volunteers by bacterial lipopolysaccharide (LPS). Bioactive lipids, enzymatic epoxyeicosatrienoic acids (EETs), and free radical-catalyzed prostanoids [isoprostanes (iPs)] formed from arachidonic acid and the fish oils, served as comparators. Despite the clear shift from ω-6 to ω-3 EETs and iPs, we failed to detect a consistent signal, in most cases, of SPM formation in urine or plasma in response to fish oil, and in all cases in response to LPS on a background of fish oil. Our results question the relevance of these SPMs to the putative anti-inflammatory effects of fish oils in humans.
Asunto(s)
Suplementos Dietéticos , Aceites de Pescado/administración & dosificación , Mediadores de Inflamación/sangre , Inflamación/dietoterapia , Metabolismo de los Lípidos/efectos de los fármacos , Adulto , Antiinflamatorios/administración & dosificación , Antígenos CD59/administración & dosificación , Ácidos Grasos Omega-3/sangre , Femenino , Voluntarios Sanos , Humanos , Inflamación/sangre , Inflamación/inducido químicamente , Mediadores de Inflamación/síntesis química , Lipopolisacáridos/administración & dosificación , MasculinoRESUMEN
Acid-sensing ion channels (ASICs) are activated by acidic pH and may play a significant role in the development of hyperalgesia. Earlier studies show ASIC3 is important for induction of hyperalgesia after muscle insult using ASIC3-/- mice. ASIC3-/- mice lack ASIC3 throughout the body, and the distribution and composition of ASICs could be different from wild-type mice. We therefore tested whether knockdown of ASIC3 in primary afferents innervating muscle of adult wild-type mice prevented development of hyperalgesia to muscle inflammation. We cloned and characterized artificial miRNAs (miR-ASIC3) directed against mouse ASIC3 (mASIC3) to downregulate ASIC3 expression in vitro and in vivo. In CHO-K1 cells transfected with mASIC3 cDNA in culture, the miR-ASIC3 constructs inhibited protein expression of mASIC3 and acidic pH-evoked currents and had no effect on protein expression or acidic pH-evoked currents of ASIC1a. When miR-ASIC3 was used in vivo, delivered into the muscle of mice using a herpes simplex viral vector, both muscle and paw mechanical hyperalgesia were reduced after carrageenan-induced muscle inflammation. ASIC3 mRNA in DRG and protein levels in muscle were decreased in vivo by miR-ASIC3. In CHO-K1 cells co-transfected with ASIC1a and ASIC3, miR-ASIC3 reduced the amplitude of acidic pH-evoked currents, suggesting an overall inhibition in the surface expression of heteromeric ASIC3-containing channels. Our results show, for the first time, that reducing ASIC3 in vivo in primary afferent fibers innervating muscle prevents the development of inflammatory hyperalgesia in wild-type mice, and thus, may have applications in the treatment of musculoskeletal pain in humans.
Asunto(s)
Marcación de Gen/métodos , Hiperalgesia/prevención & control , MicroARNs/farmacología , Dolor Musculoesquelético/terapia , Miositis/genética , Canales de Sodio/genética , Canales Iónicos Sensibles al Ácido , Animales , Células CHO , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Terapia Genética/métodos , Hiperalgesia/genética , Hiperalgesia/patología , Mediadores de Inflamación/síntesis química , Mediadores de Inflamación/fisiología , Mediadores de Inflamación/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/síntesis química , MicroARNs/uso terapéutico , Dolor Musculoesquelético/genética , Dolor Musculoesquelético/patología , Miositis/complicaciones , Miositis/patología , Canales de Sodio/deficiencia , Canales de Sodio/fisiologíaRESUMEN
Amyloid-ß (Aß40/42) aggregates containing the cross-ß-sheet structure are associated with the pathogenesis of Alzheimer's disease (AD). It is generally accepted that the N-terminal peptide of Aß40/42, Aß1-16, does not aggregate, and is not cytotoxic. However, we here show that Aß1-16 can aggregate, and form cytotoxic aggregates containing ß-turns and regular non-amyloid ß-sheet structures. Factors such as pH, ionic strength, and agitation were found to influence Aß1-16 aggregation, and the amino acid residues Asp1, His6, Ser8, and Val12 in Aß1-16 may play a role in this aggregation. Our MTT results showed that Aß1-16 monomers or oligomers were toxic to SH-SY5Y cells, but Aß1-16 fibrils exhibited less cytotoxicity. Our studies also indicate that Aß1-16 aggregates can increase the formation of reactive oxygen species and nitric oxide, induce the loss of calcium homeostasis, and incur the microglial production of TNF-α and IL-4. Thus, our findings suggest that Aß1-16 may contribute to AD pathogenesis.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Citocinas/biosíntesis , Óxido Nítrico/biosíntesis , Fragmentos de Péptidos/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Péptidos beta-Amiloides/síntesis química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Humanos , Mediadores de Inflamación/síntesis química , Mediadores de Inflamación/metabolismo , Fragmentos de Péptidos/síntesis química , Multimerización de Proteína , Estructura Secundaria de Proteína , Especies Reactivas de Oxígeno/síntesis químicaRESUMEN
Resolution of inflammation, an actively coordinated program, is essential to maintain host health. It involves effective removal of inflammatory stimuli and the spatio-temporal control of leukocyte trafficking as well as chemical mediator generation. During the active resolution process, new classes of small, local acting endogenous autacoids, namely the lipoxins, D and E series resolvins, (neuro)protectins, and maresins have been identified. These specialized pro-resolving lipid mediators (SPM) prevent excessive inflammation and promote removal of microbes and apoptotic cells, thereby expediting resolution and return to tissue homeostasis. As part of their molecular mechanism, SPM exert their potent actions via activating specific pro-resolving G-protein coupled receptors. Together these SPM and their receptors provide new concepts and opportunities for therapeutics, namely promoting active resolution as opposed to the conventionally used enzyme inhibitors and receptor antagonists. This approach may offer new targets suitable for drug design for treating inflammation related diseases, for the new terrain of resolution pharmacology.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Mediadores de Inflamación/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Diseño de Fármacos , Humanos , Mediadores de Inflamación/síntesis química , Mediadores de Inflamación/química , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-ActividadRESUMEN
Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.
Asunto(s)
Inmunidad Adaptativa/efectos de los fármacos , Enfermedades Autoinmunes/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Inmunidad Innata/efectos de los fármacos , Inmunosupresores/farmacología , Mediadores de Inflamación/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Línea Celular , Línea Celular Transformada , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunosupresores/química , Inmunosupresores/uso terapéutico , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/síntesis química , Mediadores de Inflamación/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Pirimidinas/síntesis química , Pirimidinas/uso terapéutico , Pirroles/síntesis química , Pirroles/uso terapéutico , RatasRESUMEN
BACKGROUND AND OBJECTIVES: The non-allelic variant of CXCL4/PF4, CXCL4L1/PF4alt, differs from CXCL4 in three amino acids of the C-terminal α-helix and has been characterized as a potent anti-angiogenic regulator. Although CXCL4 structurally belongs to the chemokine family, it does not behave like a 'classical' chemokine, lacking significant chemotactic properties. Specific hallmarks are its angiostatic, anti-proliferative activities, and proinflammatory functions, which can be conferred by heteromer-formation with CCL5/RANTES enhancing monocyte recruitment. METHODS AND RESULTS: Here we show that tube formation of endothelial cells was inhibited by CXCL4L1 and CXCL4, while only CXCL4L1 triggered chemokinesis of endothelial cells. The chemotactic response towards VEGF and bFGF was attenuated by both variants and CXCL4L1-induced chemokinesis was blocked by bFGF or VEGF. Endothelial cell proliferation was inhibited by CXCL4 (IC(50) 6.9 µg mL(-1)) but not by CXCL4L1, while both chemokines bound directly to VEGF and bFGF. Moreover, CXCL4 enhanced CCL5-induced monocyte arrest in flow adhesion experiments and monocyte recruitment into the mouse peritoneal cavity in vivo, whereas CXCL4L1 had no effect. CXCL4L1 revealed lower affinity to CCL5 than CXCL4, as quantified by isothermal fluorescence titration. As evidenced by the reduction of the activated partial thromboplastin time, CXCL4L1 showed a tendency towards less heparin-neutralizing activity than CXCL4 (IC(50) 2.45 vs 0.98 µg mL(-1)). CONCLUSIONS: CXCL4L1 may act angiostatically by causing random endothelial cell locomotion, disturbing directed migration towards angiogenic chemokines, serving as a homeostatic chemokine with a moderate structural distinction yet different functional profile from CXCL4.
Asunto(s)
Proteínas Angiostáticas/metabolismo , Movimiento Celular , Proliferación Celular , Quimiotaxis de Leucocito , Células Endoteliales/inmunología , Mediadores de Inflamación/metabolismo , Monocitos/inmunología , Neovascularización Fisiológica , Factor Plaquetario 4/metabolismo , Proteínas Angiostáticas/síntesis química , Proteínas Angiostáticas/genética , Animales , Coagulación Sanguínea , Células Cultivadas , Quimiocina CCL5/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Mediadores de Inflamación/síntesis química , Ratones , Ratones Endogámicos C57BL , Tiempo de Tromboplastina Parcial , Factor Plaquetario 4/síntesis química , Factor Plaquetario 4/genética , Multimerización de Proteína , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The metalloproteinases ADAM10 and ADAM17 are involved in various diseases: neurodegeneration, cancer and inflammation. OBJECTIVE: The inhibition of these proteases is a promising target in the treatment of inflammation and cancer. METHODS AND RESULTS: In this study, we present an improved synthesis of the ADAM10 reference inhibitor GI254023X with a higher overall yield, enhanced detection ability and increased acid stability, providing easier handling. CONCLUSION: This upscaled synthesis, free of diastereomeric intermediates, ensures single-batch identity, thus warranting its reproducibility in further biological investigations.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Dipéptidos/síntesis química , Dipéptidos/farmacología , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacología , Mediadores de Inflamación/síntesis química , Proteínas de la Membrana/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Proteínas ADAM/metabolismo , Proteína ADAM10 , Amidas/síntesis química , Amidas/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Línea Celular , Humanos , Inflamación/tratamiento farmacológico , Inflamación/enzimología , Mediadores de Inflamación/farmacología , Proteínas de la Membrana/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Inhibidores de Proteasas/farmacología , Reproducibilidad de los ResultadosRESUMEN
Novel therapies are needed to address the vascular endothelial cell (EC) barrier disruption that occurs in inflammatory diseases such as acute lung injury (ALI). We previously demonstrated the potent barrier-enhancing effects of both sphingosine 1-phosphate (S1P) and the structurally similar compound FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] in inflammatory lung injury. In this study, we examined the therapeutic potential of several novel FTY720 analogs to reduce vascular leak. Similar to S1P and FTY720, the (R)- and (S)-enantiomers of FTY720 phosphonate and enephosphonate analogs produce sustained EC barrier enhancement in vitro, as seen by increases in transendothelial electrical resistance (TER). In contrast, the (R)- and (S)-enantiomers of FTY720-regioisomeric analogs disrupt EC barrier integrity in a dose-dependent manner. Barrier-enhancing FTY720 analogs demonstrate a wider protective concentration range in vitro (1-50 microM) and greater potency than either S1P or FTY720. In contrast to FTY720-induced EC barrier enhancement, S1P and the FTY720 analogs dramatically increase TER within minutes in association with cortical actin ring formation. Unlike S1P, these FTY720 analogs exhibit differential phosphorylation effects without altering the intracellular calcium level. Inhibitor studies indicate that barrier enhancement by these analogs involves signaling via G(i)-coupled receptors, tyrosine kinases, and lipid rafts. Consistent with these in vitro responses, the (S)-phosphonate analog of FTY720 significantly reduces multiple indices of alveolar and vascular permeability in a lipopolysaccharide-mediated murine model of ALI (without significant alterations in leukocyte counts). These results demonstrate the capacity for FTY720 analogs to significantly decrease pulmonary vascular leakage and inflammation in vitro and in vivo.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/fisiología , Clorhidrato de Fingolimod/análogos & derivados , Mediadores de Inflamación/síntesis química , Mediadores de Inflamación/farmacología , Organofosfonatos/síntesis química , Organofosfonatos/farmacología , Glicoles de Propileno/síntesis química , Glicoles de Propileno/farmacología , Arteria Pulmonar/efectos de los fármacos , Esfingosina/análogos & derivados , Animales , Línea Celular , Clorhidrato de Fingolimod/síntesis química , Clorhidrato de Fingolimod/farmacología , Humanos , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Arteria Pulmonar/patología , Esfingosina/síntesis química , Esfingosina/farmacologíaRESUMEN
Prostate cancer is the most common cancer in men and one of the leading causes of cancer-related deaths in Western countries. The extraordinary biological heterogeneity, the increasing incidence of this disease, and the presence of putative premalignant conditions make prostate cancer a crucial pathology to study and test pharmacological or nutritional chemopreventive strategies. It has been demonstrated that the incidence of prostate cancer is lower in Asian people, and that it increases in Asian men living in Western countries; these data point to a pivotal role of diet in the onset of prostate cancer. A large amount of work has been done in investigating chemopreventive properties of dietary compounds widely used in Asian countries (i.e. soy, soybeans, green tea, fish) in respect of the oxidants- and meat-rich diet typical of Western people, particularly of central and northern Europe. Some dietary products appear promising as chemo-preventive agents for prostate cancer, because they display both anti-oxidant and anti-inflammatory activity - and inflammation is crucial for the aetiology of adeno-carcinoma of the prostate. There is increasing evidence for close correlation between inflammation, the microenvironment and tumour-associated neo-angiogenesis causing the adverse outcomes of prostate cancer. It may thus be useful to develop new strategies to couple the treatment of inflammation-related prostate cancer and the generation of angiopreventive or antiinflammatory molecules to prevent this disease. The search for compounds with few or no adverse effects - particularly cardiovascular - as compared with the agents currently in use is therefore of greatest relevance.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Mediadores de Inflamación/fisiología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/prevención & control , Animales , Antineoplásicos Fitogénicos/síntesis química , Productos Biológicos/administración & dosificación , Productos Biológicos/química , Humanos , Mediadores de Inflamación/síntesis química , Mediadores de Inflamación/farmacología , MasculinoRESUMEN
Targeted silencing of disease-associated genes by synthetic short interfering RNA (siRNA) holds considerable promise as a novel therapeutic strategy. However, unmodified siRNA can be potent triggers of the innate immune response, particularly when associated with delivery vehicles that facilitate intracellular uptake. This represents a significant barrier to the therapeutic development of siRNA due to toxicity and off-target gene effects associated with this inflammatory response. Here we show that immune stimulation by synthetic siRNA can be completely abrogated by selective incorporation of 2'-O-methyl (2'OMe) uridine or guanosine nucleosides into one strand of the siRNA duplex. These noninflammatory siRNA, containing less than 20% modified nucleotides, can be readily generated without disrupting their gene-silencing activity. We show that, coupled with an effective systemic delivery vehicle, 2'OMe-modified siRNA targeting apolipoprotein B (apoB) can mediate potent silencing of its target mRNA, causing significant decreases in serum apoB and cholesterol. This is achieved at therapeutically viable siRNA doses without cytokine induction, toxicity, or off-target effects associated with the use of unmodified siRNA. This approach to siRNA design and delivery should prove widely applicable and represents an important step in advancing synthetic siRNA into a broad range of therapeutic areas.
Asunto(s)
Silenciador del Gen , Inmunosupresores/síntesis química , Mediadores de Inflamación/síntesis química , Leucocitos Mononucleares/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/síntesis química , Animales , Células Cultivadas , Metilación de ADN , Silenciador del Gen/inmunología , Humanos , Inmunosupresores/administración & dosificación , Mediadores de Inflamación/administración & dosificación , Mediadores de Inflamación/fisiología , Leucocitos Mononucleares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Interferencia de ARN/inmunología , ARN Interferente Pequeño/fisiologíaRESUMEN
The unusual amino acid diaminopimelic acid (DAP) was prepared by cross metathesis of appropriately protected vinyl glycine and allyl glycine derivatives. Catalytic hydrogenation of the cross-coupling product resulted in reduction of the double bond and the removal of protecting groups. The resulting compounds were appropriately protected for the polymer-supported and solution-phase synthesis of muramyl tripeptides 2 and 3, which differ in the amidation of the alpha-carboxylic acids of the isoglutamine and DAP moieties. Muramyl dipeptide (1, MDP), the DAP-containing muramyl tripeptide 3, and the lysine-containing muramyl tripeptides 4 and 5 induced TNF-alpha gene expression without TNF-alpha protein production in a human monocytic cell line. The observed block in translation could be removed by co-incubation with LPS, resulting in an apparent synergistic effect. Compound 2 did not induce TNF-alpha gene expression, neither did it exhibit a synergistic effect with LPS; this indicates that amidation of the alpha-carboxylic acids of the isoglutamine and DAP moieties results in a loss of biological activity. It is proposed that amidation of alpha-carboxylic acids is a strategy that may be used by pathogens to avoid detection by the innate immune system. Furthermore, the pattern recognition receptors Nod1 and Nod2 have been implicated in the possible induction of a synergistic effect of muropeptides with LPS.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Ácido Diaminopimélico/química , Mediadores de Inflamación/síntesis química , Mediadores de Inflamación/farmacología , Lisina/química , Acetilmuramil-Alanil-Isoglutamina/síntesis química , Acetilmuramil-Alanil-Isoglutamina/farmacología , Línea Celular , Humanos , ARN Mensajero/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
TLRs recognize and respond to conserved motifs termed pathogen-associated molecular patterns. TLRs are characterized by an extracellular leucine-rich repeat motif and an intracellular Toll/IL-1R domain. Triggering of TLRs by pathogen-associated molecular patterns initiates a series of intracellular signaling events resulting in an inflammatory immune response designed to contain and eliminate the pathogen. Vaccinia virus encodes immunoregulatory proteins, such as A52R, that can effectively inhibit intracellular Toll/IL-1R signaling, resulting in a diminished host immune response and enhancing viral survival. In this study, we report the identification and characterization of a peptide derived from the A52R protein (sequence DIVKLTVYDCI) that, when linked to the nine-arginine cell transduction sequence, effectively inhibits cytokine secretion in response to TLR activation. The peptide had no effect on cytokine secretion resulting from cell activation that was initiated independent of TLR stimulation. Using a mouse model of otitis media with effusion, administration of heat-inactivated Streptococcus pneumoniae into the middle ears of BALB/c mice resulted in a significant inflammatory response that was dramatically reduced with peptide treatment. The identification of this peptide that selectively targets TLR-dependent signaling may have application in the treatment of chronic inflammation initiated by bacterial or viral infections.
Asunto(s)
Citocinas/antagonistas & inhibidores , Mediadores de Inflamación/fisiología , Glicoproteínas de Membrana/fisiología , Otitis Media con Derrame/prevención & control , Fragmentos de Péptidos/fisiología , Infecciones Neumocócicas/prevención & control , Receptores de Superficie Celular/fisiología , Virus Vaccinia/inmunología , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Citocinas/metabolismo , Proteínas I-kappa B/antagonistas & inhibidores , Proteínas I-kappa B/metabolismo , Mediadores de Inflamación/síntesis química , Mediadores de Inflamación/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , Otitis Media con Derrame/inmunología , Otitis Media con Derrame/patología , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Fosforilación , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/patología , Receptores de Superficie Celular/antagonistas & inhibidores , Transducción de Señal/inmunología , Acetato de Tetradecanoilforbol/farmacología , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Virales/síntesis química , Proteínas Virales/metabolismoRESUMEN
We have identified a synthetic peptide derived from the secreted portion of HSV type 2 glycoprotein G, denoted gG-2p20, which has proinflammatory properties in vitro. The gG-2p20 peptide, corresponding to aa 190-205 of glycoprotein G-2, was a chemoattractant for both monocytes and neutrophils in a dose-dependent fashion, and also induced the release of reactive oxygen from these cells. The receptor mediating the responses was identified as the formyl peptide receptor. The gG-2p20-induced activation of phagocytes had a profound impact on NK cell functions. The reactive oxygen species produced by gG-2p20-activated phagocytes both inhibited NK cell cytotoxicity and accelerated the apoptotic cell death in NK cell-enriched lymphocyte populations. Hence, we have for the first time been able to identify a potential function of the secreted portion of HSV-2 glycoprotein G. We propose that the proinflammatory gG-2p20 peptide identified could contribute to a reduced function and viability of NK cells during HSV-2 infection due to its ability to recruit and activate phagocytic cells.
Asunto(s)
Herpesvirus Humano 2/inmunología , Mediadores de Inflamación/fisiología , Células Asesinas Naturales/inmunología , Monocitos/inmunología , Neutrófilos/inmunología , Fragmentos de Péptidos/fisiología , Proteínas del Envoltorio Viral/fisiología , Secuencia de Aminoácidos , Apoptosis/inmunología , Quimiotaxis de Leucocito/inmunología , Citotoxicidad Inmunológica/inmunología , Inducción Enzimática/inmunología , Humanos , Inmunosupresores/síntesis química , Inmunosupresores/inmunología , Mediadores de Inflamación/síntesis química , Células Asesinas Naturales/patología , Células Asesinas Naturales/virología , Activación de Macrófagos/inmunología , Datos de Secuencia Molecular , Monocitos/patología , Monocitos/virología , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/metabolismo , Activación Neutrófila/inmunología , Neutrófilos/enzimología , Neutrófilos/patología , Neutrófilos/virología , Fragmentos de Péptidos/síntesis química , Toxina del Pertussis/farmacología , Fagocitos/enzimología , Fagocitos/inmunología , Fagocitos/patología , Fagocitos/virología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Formil Péptido/fisiología , Proteínas del Envoltorio Viral/síntesis químicaRESUMEN
Con la finalidad de investigar la síntesis de Proteína C Reactiva (PCR) en niños con diferentes grados de desnutrición y entre desnutridos graves infectados y eutróficos infectados; así como en un grupo control de niños eutróficos, se determinó las concentraciones de esta proteína por el método turbidimétrico en 109 niños venezolanos en edades comprendidas entre 6 meses y 6 años. Los resultados encontrados mostraron que el desnutrido grave infectado aumenta su valor de PCR de manera significativa (80,80 ± 38,39 mg/L) en relación con el no infectado (8,17 ± 3,06 mg/L) (p < 0,001). Igualmente hubo diferencias estadísticamente significativas entre los desnutridos graves infectados y el grupo de eutróficos infectados (p < 0,001) quienes presentaron valores más elevados. En relación con los grupos de desnutridos no infectados se encontró diferencia significativa entre los desnutridos graves con el resto de los grupos y el grupo control eutrófico (p < 0,05), no obstante, sus concentraciones permanecieron dentro del valor normal. Estos resultados permiten concluir que el desnutrido infectado es capaz de sintetizar PCR en respuesta a procesos infecciosos graves pero que difiere significativamente cuando sus valores se comparan con los del eutrófico infectado. Por otra parte el desnutrido libre de infección cualquiera que sea su grado de desnutrición mantiene sus valores de PCR en límites normales
Asunto(s)
Humanos , Masculino , Femenino , Niño , Desnutrición Proteico-Calórica , Infecciones/metabolismo , Mediadores de Inflamación/síntesis química , Proteína C-Reactiva/síntesis química , Medicina , VenezuelaRESUMEN
Site-directed mutagenesis of tumor necrosis factor (TNF) based on prediction of the interaction of specific residues with TNF receptors generated dominant-negative constructs, in which single- or double-amino acid changes result in decreased receptor binding and cellular activation. These dominant-negatives not only provide a novel manner to block the proinflammatory effects of TNF, but also can be used as a tool to examine ligand-receptor interactions and their importance in signaling. Because these TNF mutant molecules are smaller than those used for conventional anti-TNF therapies, such as etanercept or infliximab, they are likely to achieve greater tissue concentrations and may provide enhanced therapeutic effect. However, the immunogenicity, as well as efficacy, of the dominant-negative TNF constructs must be more completely examined.
Asunto(s)
Biología Computacional/métodos , Diseño de Fármacos , Variación Genética/genética , Mediadores de Inflamación/síntesis química , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/síntesis química , Humanos , Inflamación/prevención & control , Mutagénesis Sitio-Dirigida/genética , Isoformas de Proteínas/síntesis química , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Condensations of chiral diamines 11a-c with benzotriazole and formaldehyde gave benzotriazolyl intermediates 12a-c; similar condensations of alpha-amino-amides 10a-c with benzotriazole and paraformaldehyde gave 14a-c. Subsequent treatment of 12a-c and 14a-c with AlCl(3) led to enantiopure tricyclic 1,2,3,5,10,10a-hexahydroimidazo[1,5-b]isoquinolines 1a-c and 2,3,10,10a-tetrahydroimidazo[1,5-b]isoquinolin-1(5H)-ones 15a-c, respectively, via Lewis acid promoted iminium cation cyclizations.
Asunto(s)
Isoquinolinas/síntesis química , Ciclización , Iminas , Mediadores de Inflamación/síntesis química , EstereoisomerismoRESUMEN
Acylated beta-amino acids are described as potent, specific and orally bioavailable antagonists of VLA-4. The initial lead was identified from a combinatorial library. Subsequent optimization using a traditional medicinal chemistry approach led to significant improvement in potency (up to 8-fold) while maintaining good pharmacokinetic properties.