RESUMO
BACKGROUND: Felcisetrag (5-hydroxytryptamine-4 receptor [5-HT4] agonist) is under investigation as prophylaxis or active treatment for accelerating resolution of gastrointestinal function post-surgery. METHODS: Phase 2, randomized, placebo-controlled, parallel five-arm, double-blind, multicenter study (NCT03827655) in 209 adults undergoing open or laparoscopic-assisted bowel surgery. Patients received intravenous placebo, felcisetrag 0.1 mg/100 âmL or 0.5 mg/100 âmL pre-surgery only, or pre-surgery and daily post-surgery until return of gastrointestinal function or for up to 10 days. PRIMARY ENDPOINT: time to recovery of gastrointestinal function. RESULTS: Median time to recovery of gastrointestinal function was 2.6 days for both felcisetrag 0.5 âmg daily and 0.5 âmg pre-surgery versus 1.9 days for placebo (p â> â0.05). There were no notable differences in adverse events between treatment arms. CONCLUSIONS: Felcisetrag was well tolerated with no new safety concerns. However, no clinically meaningful difference in time to recovery of gastrointestinal function versus placebo was observed. Further investigation of the utility of 5-HT4 agonists in complicated, open abdominal surgeries may be warranted.
Assuntos
Complicações Pós-Operatórias , Agonistas do Receptor 5-HT4 de Serotonina , Humanos , Método Duplo-Cego , Masculino , Pessoa de Meia-Idade , Agonistas do Receptor 5-HT4 de Serotonina/uso terapêutico , Feminino , Complicações Pós-Operatórias/prevenção & controle , Adulto , Idoso , Gastroenteropatias/cirurgia , Procedimentos Cirúrgicos do Sistema Digestório/efeitos adversos , Laparoscopia/efeitos adversos , Recuperação de Função Fisiológica/efeitos dos fármacos , Resultado do TratamentoRESUMO
Palmoplantar pustular psoriasis (PPPP) and nonâpustular palmoplantar psoriasis (NPPP) are localized, debilitating forms of psoriasis. The inflammatory circuits involved in PPPP and NPPP are not well-understood. To compare the cellular and immunological features that differentiate PPPP and NPPP, skin biopsies were collected from a total of 30 participants with PPPP, NPPP, and psoriasis vulgaris (PV) and from 10 healthy participants. A subset consented to a second biopsy after 3 additional weeks off medication. Histologic staining of lesional and nonlesional skin showed higher neutrophil counts in PPPP than in NPPP and PV and higher CD8+ T-cell counts in NPPP. RNA sequencing and transcriptional analysis of skin biopsies showed enhanced IFN-γ pathway activation in NPPP lesions but stronger signatures of IL-17 pathway and neutrophil-related genes (e.g., IL36A) in PPPP lesional skin. Serum analysis on the Olink platform detected higher concentrations of T helper type 1, IFN-γâinducible chemokines in NPPP, and higher neutrophil-associated cytokines in PPPP. Taken together, this evidence suggests more pronounced T helper 1âmediated inflammation in NPPP than in PV and PPPP and stronger neutrophil-associated activity in PPPP than in NPPP and PV. These data support targeting inflammatory pathways associated with neutrophilic inflammation (e.g., IL-36 signaling) for therapeutic development in PPPP.
Assuntos
Psoríase , Dermatopatias , Humanos , Pele/patologia , Dermatopatias/patologia , Inflamação/patologiaRESUMO
Biomarkers have the potential to accelerate drug development, as early indicators of improved clinical response, to improve patient safety, and for personalised medicine. However, few have been approved through the biomarker qualification pathways of the regulatory agencies. This paper outlines how biomarkers can accelerate drug development, and reviews the lessons learned by the EU IMI2-funded LITMUS consortium, which has had several interactions with regulatory agencies in both the US and EU regarding biomarker qualification in patients with non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Sharing knowledge of such interactions with the scientific community is of paramount importance to increase the chances of qualification of relevant biomarkers that may accelerate drug development, and thereby help patients, across disease indications. A qualified biomarker enables a decision to be made that all understand and support in a common framework.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Biomarcadores/metabolismo , Desenvolvimento de MedicamentosRESUMO
Background and Aims: Primary Biliary Cholangitis (PBC) is an organ-specific autoimmune liver disease. Mononuclear phagocytes (MNPs), comprise of monocyte, dendritic cells and monocyte-derived macrophages, constitute major arm of the innate immune system known to be involved in the pathogenesis of autoimmune disorders. MNPs were shown to accumulate around intra-hepatic bile ducts in livers of PBC patients. Interleukin 23 (IL-23) is a pro-inflammatory cytokine. IL-23-positive cells were detected in livers of patients with advanced stage PBC and IL-23 serum levels found to be in correlation with PBC disease severity. Our overall goal was to assess the importance of IL-23 derived from MNPs in PBC pathogenesis. Methods: We utilized an inducible murine model of PBC and took advantage of transgenic mice targeting expression of IL-23 by specific MNP populations. Analysis included liver histology assessment, flow cytometry of hepatic immune cells and hepatic cytokine profile evaluation. Specific MNPs sub-populations were sorted and assessed for IL-23 expression levels. Results: Flow cytometry analysis of non-parenchymal liver cells in autoimmune cholangitis revealed massive infiltration of the liver by MNPs and neutrophils and a decrease in Kupffer cells numbers. In addition, a 4-fold increase in the incidence of hepatic IL-17A producing CD4+ T cells was found to be associated with an increase in hepatic IL23-p19 and IL17A expression levels. Disease severity was significantly ameliorated in both CD11ccreP19flox/flox and CX3CR1creP19 flox/flox mice as assessed by reduced portal inflammation and decreased hepatic expression of various inflammatory cytokines. Amelioration of disease severity was associated with reduction in IL-17A producing CD4+ T cells percentages and decreased hepatic IL23-p19 and IL17A expression levels. qRT-PCR analysis of sorted hepatic MNPs demonstrated high expression levels of IL-23 mRNA specifically by CX3CR1hiCD11c+ monocyte-derived macrophages. Conclusion: Our results indicate a major role for IL-23 produced by hepatic monocyte-derived macrophages in the pathogenesis of PBC. These results may pave the road for the development of new immune-based and cell specific therapeutic modalities for PBC patients not responding to current therapies.
Assuntos
Suscetibilidade a Doenças , Interleucina-23/biossíntese , Cirrose Hepática Biliar/etiologia , Cirrose Hepática Biliar/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Autoimunidade , Biomarcadores , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Expressão Gênica , Imuno-Histoquímica , Imunofenotipagem , Interleucina-23/genética , Cirrose Hepática Biliar/patologia , Camundongos , Camundongos Transgênicos , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Cytokines maintain intestinal health, but precise intercellular communication networks remain poorly understood. Macrophages are immune sentinels of the intestinal tissue and are critical for gut homeostasis. Here, we show that in a murine inflammatory bowel disease (IBD) model based on macrophage-restricted interleukin-10 (IL-10) receptor deficiency (Cx3cr1Cre:Il10rafl/fl mice), proinflammatory mutant gut macrophages cause severe spontaneous colitis resembling the condition observed in children carrying IL-10R mutations. We establish macrophage-derived IL-23 as the driving factor of this pathology. Specifically, we report that Cx3cr1Cre:Il10rafl/fl:Il23afl/fl mice harboring macrophages deficient for both IL-10R and IL-23 are protected from colitis. By analyzing the epithelial response to proinflammatory macrophages, we provide evidence that T cells of colitic animals produce IL-22, which induces epithelial chemokine expression and detrimental neutrophil recruitment. Collectively, we define macrophage-specific contributions to the induction and pathogenesis of colitis, as manifested in mice harboring IL-10R deficiencies and human IBDs.
Assuntos
Colite/imunologia , Células Epiteliais/imunologia , Interleucina-23/imunologia , Interleucinas/imunologia , Macrófagos/imunologia , Receptores de Interleucina-10/imunologia , Animais , Colite/patologia , Intestinos/imunologia , Intestinos/patologia , Masculino , Camundongos , Neutrófilos/imunologia , Receptores de Interleucina-10/genética , Interleucina 22RESUMO
BACKGROUND: Colonoscopy is the gold standard to diagnose and follow up the evolution of inflammatory bowel diseases. However, this technique can still present a risk of severe complications, a general discomfort in patients, and its diagnostic value is limited to the visualization of the colon mucosal changes. Magnetic resonance imaging (MRI) is emerging as a noninvasive imaging technique of choice to overcome these limitations. The aim of this work was to evaluate the potential of colon wall thickness measured using MRI as an in vivo imaging biomarker of inflammation for inflammatory bowel disease in an animal model of this disease. METHODS: On day 0, 2% or 3% Dextran sodium sulfate was added to the drinking water of mice (n = 10/group) for 5 days. Six mice were left as controls. Animals were imaged with colonoscopy and MRI on days 7, 11, and 21 to study the colitis progression. Histology was performed at the end of the protocol. RESULTS: The colon wall thickness measured in Dextran sodium sulfate-treated animals was shown to be significantly and dose dependently increased compared to controls. Colonoscopy showed similar results and excellently correlated with MRI measurements and histology. The proposed protocol showed high robustness, with negligible interoperator and intraoperator variability. CONCLUSIONS: The findings of this investigation suggest the feasibility of using MRI for the noninvasive assessment of colon wall thickness as a robust surrogate biomarker for colon inflammation detection and follow-up. The data presented show the potential of MRI in in vivo preclinical longitudinal studies, including testing of new drugs or investigation of inflammatory bowel disease development mechanisms.
Assuntos
Colite/diagnóstico por imagem , Colo/diagnóstico por imagem , Imageamento por Ressonância Magnética , Animais , Biomarcadores , Biópsia , Colite/induzido quimicamente , Colite/patologia , Colo/patologia , Colonoscopia , Sulfato de Dextrana , Modelos Animais de Doenças , Feminino , Estudos Longitudinais , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos TestesRESUMO
Interleukin-4 (IL-4) and IL-13 are critical drivers of immune activation and inflammation in ulcerative colitis, asthma and other diseases. Because these cytokines may have redundant function, dual targeting holds promise for achieving greater efficacy. We have recently described a bifunctional therapeutic targeting IL-4 and IL-13 developed on a novel protein scaffold, generated by combining specific binding domains in an optimal configuration using appropriate linker regions. In the current study, the bifunctional IL-4/IL-13 antagonist was evaluated in the murine oxazolone-induced colitis model, which produces disease with features of ulcerative colitis. The bifunctional IL-4/IL-13 antagonist reduced body weight loss throughout the 7-day course of the model, and ameliorated the increased colon weight and decreased colon length that accompany disease. Colon tissue gene expression was modulated in accordance with the treatment effect. Concentrations of serum amyloid P were elevated in proportion to disease severity, making it an effective biomarker. Serum concentrations of the bifunctional IL-4/IL-13 antagonist were inversely proportional to disease severity, colon tissue expression of pro-inflammatory genes, and serum amyloid P concentration. Taken together, these results define a panel of biomarkers signifying engagement of the IL-4/IL-13 pathway, confirm the T helper type 2 nature of disease in this model, and demonstrate the effectiveness of dual cytokine blockade.
Assuntos
Anticorpos Monoclonais/farmacologia , Colite Ulcerativa/metabolismo , Interleucina-13/antagonistas & inibidores , Interleucina-4/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/farmacologia , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Subunidade alfa2 de Receptor de Interleucina-13/antagonistas & inibidores , Camundongos , Oxazolona/efeitos adversos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteína Amiloide A Sérica/metabolismo , Componente Amiloide P Sérico/metabolismo , Índice de Gravidade de DoençaRESUMO
Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to PGs via the cyclooxygenase 1 and 2 pathways and to leukotrienes via the 5-lipoxygenase pathway. We used adoptive transfer and relapsing-remitting forms of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, in two different strains of mice (SJL or C57BL/6) to demonstrate that blockade of cPLA(2)α with a highly specific small-molecule inhibitor during the tissue-damage effector phase abrogates the clinical manifestation of disease. Using the adoptive transfer model in SJL mice, we demonstrated that the blockade of cPLA(2)α during the effector phase of disease was more efficacious in ameliorating the disease pathogenesis than the blockade of each of the downstream enzymes, cyclooxygenase-1/2 and 5-lipooxygenase. Similarly, blockade of cPLA(2)α was highly efficacious in ameliorating disease pathogenesis during the effector phase of EAE in the adoptive transfer model of EAE in C57BL/6 mice. Investigation of the mechanism of action indicates that cPLA(2)α inhibitors act on APCs to diminish their ability to induce Ag-specific effector T cell proliferation and proinflammatory cytokine production. Furthermore, cPLA(2)α inhibitors may prevent activation of CNS-resident microglia and may increase oligodendrocyte survival. Finally, in a relapsing-remitting model of EAE in SJL mice, therapeutic administration of a cPLA(2)α inhibitor, starting from the peak of disease or during remission, completely protected the mice from subsequent relapses.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Esclerose Múltipla/prevenção & controle , Transferência Adotiva , Animais , Células Apresentadoras de Antígenos/enzimologia , Células Apresentadoras de Antígenos/patologia , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/imunologia , Araquidonato 5-Lipoxigenase/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/imunologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2 , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/imunologia , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Microglia/enzimologia , Microglia/imunologia , Microglia/patologia , Esclerose Múltipla/enzimologia , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Oligodendroglia/enzimologia , Oligodendroglia/imunologia , Oligodendroglia/patologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
TBK1 is critical for immunity against microbial pathogens that activate TLR4- and TLR3-dependent signaling pathways. To address the role of TBK1 in inflammation, mice were generated that harbor two copies of a mutant Tbk1 allele. This Tbk1(Δ) allele encodes a truncated Tbk1(Δ) protein that is catalytically inactive and expressed at very low levels. Upon LPS stimulation, macrophages from Tbk1(Δ/Δ) mice produce normal levels of proinflammatory cytokines (e.g., TNF-α), but IFN-ß and RANTES expression and IRF3 DNA-binding activity are ablated. Three-month-old Tbk1(Δ/Δ) mice exhibit mononuclear and granulomatous cell infiltrates in multiple organs and inflammatory cell infiltrates in their skin, and they harbor a 2-fold greater amount of circulating monocytes than their Tbk1(+/+) and Tbk1(+/Δ) littermates. Skin from 2-week-old Tbk1(Δ/Δ) mice is characterized by reactive changes, including hyperkeratosis, hyperplasia, necrosis, inflammatory cell infiltrates, and edema. In response to LPS challenge, 3-month-old Tbk1(Δ/Δ) mice die more quickly and in greater numbers than their Tbk1(+/+) and Tbk1(+/Δ) counterparts. This lethality is accompanied by an overproduction of several proinflammatory cytokines in the serum of Tbk1(Δ/Δ) mice, including TNF-α, GM-CSF, IL-6, and KC. This overproduction of serum cytokines in Tbk1(Δ/Δ) mice following LPS challenge and their increased susceptibility to LPS-induced lethality may result from the reactions of their larger circulating monocyte compartment and their greater numbers of extravasated immune cells.
Assuntos
Lipopolissacarídeos/toxicidade , Monócitos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Quimiocina CCL2/biossíntese , Feminino , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Cytosolic phospholipase A2 alpha (cPLA2 alpha) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to prostaglandins via the cyclooxygenase (COX) 1/2 pathways, and leukotrienes via the 5-lipoxygenase (LO) pathway. We utilized inhibitors of cPLA2 alpha, COX-1/2 and 5-LO to determine the potential roles of these enzymes in development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Blocking cPLA2 alpha prevented EAE development and greatly reduced antigen-induced production of Th1-type cytokines and IL-17. Blocking COX-1/2 delayed onset and reduced severity of EAE, and reduced production of Th1-type cytokines, but not IL-17. Blocking 5-LO delayed onset and reduced cumulative severity of EAE, but did not reduce production of Th1-type cytokines or IL-17. Finally, blockade of cPLA2 alpha from the onset of clinical EAE reduced duration of EAE relapses. Therefore, cPLA2 alpha represents a potential therapeutic target for treatment of MS.
Assuntos
Encefalomielite Autoimune Experimental/prevenção & controle , Encefalomielite Autoimune Experimental/fisiopatologia , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Células Th1/fisiologia , Análise de Variância , Animais , Benzoatos/farmacologia , Benzoatos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/enzimologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Glicoproteínas , Hidroxiureia/análogos & derivados , Hidroxiureia/farmacologia , Hidroxiureia/uso terapêutico , Inibidores de Lipoxigenase/farmacologia , Inibidores de Lipoxigenase/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Glicoproteína Mielina-Oligodendrócito , Naproxeno/farmacologia , Naproxeno/uso terapêutico , Fragmentos de Peptídeos , Índice de Gravidade de Doença , Vazamento Acidental em Seveso , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Células Th1/efeitos dos fármacos , Fatores de TempoRESUMO
Cytosolic phospholipase A(2) alpha (cPLA(2)alpha, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA(2)alpha inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA(2)alpha and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA(2)alpha inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 degrees C and pH 7.4. The compounds tested bound cPLA(2)alpha with a 1:1 stoichiometry, and the dissociation constant K(d) of the inhibitors calculated from the ITC experiments correlated well with the IC(50) values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA(2)alpha was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA(2)alpha. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA(2)alpha was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (DeltaH), entropy (DeltaS), and free energy (DeltaG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics.
Assuntos
Citosol/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Animais , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologia , Benzofenonas/química , Benzofenonas/metabolismo , Benzofenonas/farmacologia , Células CHO , Domínio Catalítico , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Indóis/química , Indóis/metabolismo , Indóis/farmacologia , Organofosfonatos/metabolismo , Organofosfonatos/farmacologia , Ligação Proteica , Pirrolidinas/química , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Solubilidade , TermodinâmicaRESUMO
The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Benzoatos/síntese química , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Sulfonamidas/síntese química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/tratamento farmacológico , Benzoatos/química , Benzoatos/farmacologia , Disponibilidade Biológica , Broncoconstrição/efeitos dos fármacos , Calorimetria , Carragenina , Linhagem Celular , Inibidores de Ciclo-Oxigenase 2/síntese química , Inibidores de Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Edema/induzido quimicamente , Edema/tratamento farmacológico , Humanos , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Masculino , Camundongos , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Ovinos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Experimental autoimmune encephalomyelitis (EAE), a T cell-mediated inflammatory disease of the CNS, is a rodent model of human multiple sclerosis. IL-23 is one of the critical cytokines in EAE development and is currently believed to be involved in the maintenance of encephalitogenic responses during the tissue damage effector phase of the disease. In this study, we show that encephalitogenic T cells from myelin oligodendrocyte glycopeptide (MOG)-immunized wild-type (WT) mice caused indistinguishable disease when adoptively transferred to WT or IL-23-deficient (p19 knockout (KO)) recipient mice, demonstrating that once encephalitogenic cells have been generated, EAE can develop in the complete absence of IL-23. Furthermore, IL-12/23 double-deficient (p35/p19 double KO) recipient mice developed EAE that was indistinguishable from WT recipients, indicating that IL-12 did not compensate for IL-23 deficiency during the effector phase of EAE. In contrast, MOG-specific T cells from p19KO mice induced EAE with delayed onset and much lower severity when transferred to WT recipient mice as compared with the EAE that was induced by cells from WT controls. MOG-specific T cells from p19KO mice were highly deficient in the production of IFN-gamma, IL-17A, and TNF, indicating that IL-23 plays a critical role in development of encephalitogenic T cells and facilitates the development of T cells toward both Th1 and Th17 pathways.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Subunidade p35 da Interleucina-12/imunologia , Subunidade p19 da Interleucina-23/imunologia , Esclerose Múltipla/imunologia , Células Th1/imunologia , Animais , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Subunidade p35 da Interleucina-12/deficiência , Subunidade p19 da Interleucina-23/deficiência , Masculino , Camundongos , Camundongos Knockout , Esclerose Múltipla/patologia , Proteínas da Mielina , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Associada a Mielina/toxicidade , Glicoproteína Mielina-Oligodendrócito , Células Th1/patologia , Células Th1/transplanteRESUMO
Compound 1 was previously reported to be a potent inhibitor of cPLA(2)alpha in both artificial monomeric substrate and cell-based assays. However, 1 was inactive in whole blood assays previously used to characterize cyclooxygenase and lipoxygenase inhibitors. The IC(50) of 1 increased dramatically with cell number or lipid/detergent concentration. In an attempt to insert an electrophilic ketone between the indole and benzoic acid moieties, we discovered that increasing the distance between the two moieties gave a compound with activity in the GLU (7-hydroxycoumarinyl-gamma-linolenate) micelle assay, which contains lipid and detergent. Extensive structure-activity relationship work around this lead identified a potent pharmacophore for cPLA(2)alpha inhibition. The IC(50)s between the GLU micelle and rat whole blood assays correlated highly. No correlation was found for other parameters, including lipophilicity or acidity of the required acid functionality. Compounds 25, 39, and 94 emerged as potent, selective inhibitors of cPLA(2)alpha and represent well-validated starting points for further optimization.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Indóis/síntese química , Indóis/farmacologia , Fosfolipases A/antagonistas & inibidores , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citosol/enzimologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Fosfolipases A2 do Grupo IV , Humanos , Indóis/química , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2alpha (cPLA2alpha), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2alpha-/- mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2alpha+/- mice, whereas the lesions in cPLA2alpha-/- mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2alpha-/- mice compared with cPLA2alpha+/- mice, which indicates that cPLA2alpha plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2alpha-/- mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2alpha also plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2alpha-/- mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2alpha-/- mice susceptible to EAE. Our data indicate that cPLA2alpha plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype.
Assuntos
Diferenciação Celular/imunologia , Citosol/enzimologia , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Fosfolipases A/deficiência , Células Th1/imunologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Feminino , Fosfolipases A2 do Grupo IV , Imunidade Inata/genética , Imunofenotipagem , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas da Mielina , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Fosfolipases A/genética , Fosfolipases A/metabolismo , Fosfolipases A2 , Medula Espinal/imunologia , Medula Espinal/patologia , Células Th1/citologiaRESUMO
A search for noncarbohydrate sLe(x) mimics led to the development of quinic acid derivatives as selectin inhibitors. At Wyeth we solved the first cocrystal structure of a small molecule, quinic acid, with E-selectin. In the cocomplex two hydroxyls of quinic acid mimic the calcium-bound fucose of the tetrasaccharide sLe(x). The X-ray structure, together with structure based computational methods, was used to design quinic acid based libraries that were synthesized and evaluated for their ability to block the interaction of sLex with P-selectin. A large number of analogues were prepared using solution-phase parallel synthesis. Selected compounds showed decrease in leukocyte rolling in the IVM mouse model. Compound 2 inhibited neutrophil influx in the murine TIP model and demonstrated good plasma exposure.
Assuntos
Selectina E/metabolismo , Oligossacarídeos/química , Ácido Quínico/análogos & derivados , Ácido Quínico/farmacologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Fucose , Veias Jugulares/efeitos dos fármacos , Veias Jugulares/fisiologia , Cinética , Antígenos do Grupo Sanguíneo de Lewis , Espectroscopia de Ressonância Magnética , Masculino , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Oligossacarídeos/síntese química , Oligossacarídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Antígeno Sialil Lewis X , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: T(H)2-mediated allergic asthma is characterized by eosinophilia, mucus overproduction, and airway hyperresponsiveness (AHR). Although it is clear that T(H)2 cells and their cytokines play an important role in AHR, the roles of T(H)1 cells and neutrophils in AHR are controversial. OBJECTIVE: We sought to determine the roles of T(H)1 cells and neutrophils in AHR. METHODS: Ovalbumin-specific CD4(+) T cells were purified from DO11.10 mice, differentiated into T(H)1 cells, and injected into naive BALB/c, IL-4RalphaKO, or IL-8RKO mice. After ovalbumin antigen challenge, cytokine mRNA levels in lung samples, as well as inflammatory cell types and numbers in bronchoalveolar lavage fluid (BALF), were determined. AHR was assessed by measuring resistance in tracheostomized mice and enhanced pause in freely moving mice. RESULTS: T(H)1 cells induced AHR as robust as T(H)2 cells. They also induced lung inflammation dominated by neutrophils. Neither AHR nor inflammation were reduced when T(H)1 cells were transferred into IL-4RalphaKO mice. When IL-8RKO mice were used as recipients of T(H)1 cells, neutrophilia was greatly reduced, but the AHR was as strong as that seen in wild-type mice. On the other hand, dexamethasone treatment had no effect on neutrophilia but has significantly reduced AHR. Reduction in AHR was accompanied by a reduction in the numbers of lymphocytes and macrophages in BALF. CONCLUSIONS: T(H)1 cells can induce strong AHR independent of IL-4 and IL-13. The AHR is associated with the presence of lymphocytes and macrophages, but not neutrophils, in BALF. Our results point to a pathway whereby T(H)1 cells mediate AHR independent of neutrophilic inflammation.
Assuntos
Hiper-Reatividade Brônquica/fisiopatologia , Bronquite/patologia , Neutrófilos/patologia , Células Th1/imunologia , Transferência Adotiva , Animais , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Citocinas/genética , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Interleucina-13/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Muco/metabolismo , Ovalbumina/farmacologia , RNA Mensageiro/metabolismo , Receptores de Interleucina-8A/deficiência , Índice de Gravidade de Doença , Células Th2/imunologiaRESUMO
Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins, which may be responsible for enlargement of alveoli in chronic obstructive pulmonary disease (COPD) and remodeling of pulmonary tissue associated with chronic asthma. Here, we provide novel evidence that MMP-12 is expressed and secreted by normal human bronchial epithelial cell cultures (NHBECs) and reveal the regulation of MMP-12 gene expression by tumor necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF), and interferon gamma (IFN-gamma). Reverse transcription-polymerase chain reaction analyses demonstrated MMP-12 mRNA presence in unstimulated differentiated NHBEC cultures. Cultures stimulated independently with EGF or IFN-gamma failed to alter MMP-12 mRNA abundance, while TNF-alpha, TNF-alpha+EGF, or TNF-alpha+IFN-gamma elicited relatively early (6 h) peak increases in MMP-12 mRNA levels. Western blot analyses specifically indicated the presence of MMP-12 in differentiated NHBEC-conditioned media. These findings indicate that the bronchial epithelium may be an important source of elastolytic activity in COPD and tissue remodeling in chronic asthma.