RESUMO
The impact of PD-1 immune checkpoint therapy prompts exploration of other strategies to downregulate PD-1 for cancer therapy. We previously showed that the serine/threonine kinase, glycogen synthase kinase, GSK-3α/ß, is a central regulator of PD-1 transcription in CD8+ T cells. Here, we show that the use of small-molecule inhibitors of GSK-3α/ß (GSK-3i) to reduce pcdc1 (PD-1) transcription and expression was as effective as anti-PD-1 and PD-L1-blocking antibodies in the control of B16 melanoma, or EL4 lymphoma, in primary tumor and metastatic settings. Furthermore, the conditional genetic deletion of GSK-3α/ß reduced PD-1 expression on CD8+ T cells and limited B16 pulmonary metastasis to the same degree as PD-1 gene deficiency. In each model, GSK-3i inhibited PD-1 expression on tumor-infiltrating lymphocytes, while increasing Tbx21 (T-bet) transcription, and the expression of CD107a+ (LAMP1) and granzyme B (GZMB) on CD8+ T cells. Finally, the adoptive transfer of T cells treated ex vivo with a GSK-3 inhibitor delayed the onset of EL4 lymphoma growth to a similar extent as anti-PD-1 pretreatment. Overall, our findings show how GSK-3 inhibitors that downregulate PD-1 expression can enhance CD8+ T-cell function in cancer therapy to a similar degree as PD-1-blocking antibodies.Significance: These findings show how GSK-3 inhibitors that downregulate PD-1 expression can enhance CD8+ T-cell function in cancer therapy to a similar degree as PD-1 blocking antibodies, offering a next-generation approach in the design of immunotherapeutic approaches for cancer management. Cancer Res; 78(3); 706-17. ©2017 AACR.
Assuntos
Anticorpos Bloqueadores/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Linfócitos do Interstício Tumoral/imunologia , Linfoma/prevenção & controle , Melanoma Experimental/prevenção & controle , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfoma/imunologia , Linfoma/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Células Tumorais CultivadasRESUMO
A variable region fusion strategy was used to generate an immunosuppressive antibody based on a novel "stalk-knob" structural motif in the ultralong complementary-determining region (CDR) of a bovine antibody. The potent Kv1.3 channel inhibitory peptides Moka1-toxin and Vm24-toxin were grafted into different CDRs of the humanized antibodies BVK and Synagis (Syn) using both ß-sheet and coiled-coil linkers. Structure-activity relationship efforts led to generation of the fusion protein Syn-Vm24-CDR3L, which demonstrated excellent selectivity and potency against effector human memory T cells (subnanomolar to picomolar EC50 values). This fusion antibody also had significantly improved plasma half-life and serum stability in rodents compared with the parent Vm24 peptide. Finally, this fusion protein showed potent in vivo efficacy in the delayed type hypersensitivity in rats. These results illustrate the utility of antibody CDR fusions as a general and effective strategy to generate long-acting functional antibodies, and may lead to a selective immunosuppressive antibody for the treatment of autoimmune diseases.
Assuntos
Anticorpos Bloqueadores/farmacologia , Desenho de Fármacos , Imunossupressores/farmacologia , Canal de Potássio Kv1.3/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células CHO , Bovinos , Regiões Determinantes de Complementaridade/química , Cricetinae , Cricetulus , Células HEK293 , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Leukocyte transendothelial migration (TEM) is an essential component of the inflammatory response. In vitro studies with human cells have demonstrated that platelet/endothelial cell adhesion molecule (PECAM) functions upstream of CD99 during TEM; however, results in vivo with mice have been apparently contradictory. In this study we use four-dimensional (4D) intravital microscopy to demonstrate that the site and order of function of PECAM and CD99 in vivo are dependent on the strain of mice. In FVB/n mice, PECAM functions upstream of CD99, as in human cells in vitro, and blocking antibodies against either molecule arrest neutrophils before they traverse the endothelium. However, in C57BL/6 mice, PECAM and CD99 appear to function at a different step, as the same antibodies arrest leukocyte migration through the endothelial basement membrane. These results are the first direct comparison of PECAM and CD99 function in different murine strains as well as the first demonstration of the sequential function of PECAM and CD99 in vivo.
Assuntos
Antígeno 12E7/metabolismo , Músculos Abdominais/metabolismo , Dermatite de Contato/metabolismo , Leucócitos/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Migração Transendotelial e Transepitelial , Antígeno 12E7/antagonistas & inibidores , Músculos Abdominais/patologia , Animais , Anticorpos Bloqueadores/farmacologia , Membrana Basal , Adesão Celular , Óleo de Cróton/efeitos adversos , Dermatite de Contato/etiologia , Dermatite de Contato/patologia , Fármacos Dermatológicos/efeitos adversos , Citometria de Fluxo , Microscopia Intravital , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , NeutrófilosRESUMO
Stimulation of feeding is necessary for treatment of pathological conditions of chronic malnutrition due to anorexia. Ghrelin, a hunger hormone, is one of the candidate for pharmacological treatments of anorexia, but because of its instability in plasma has limited efficacy. We previously showed that plasmatic IgG protect ghrelin from degradation and that IgG from obese subjects and mice may increase ghrelin׳s orexigenic effect. In this study we tested if ghrelin alone or combined with IgG may improve feeding in chronically food-restricted mice with or without physical activity-based anorexia (ABA) induced by free access to a running wheel. Mice received a single daily intraperitoneal injection of ghrelin (1nM) together or not with total IgG (1nM) from obese ob/ob or lean mice before access to food during 8 days of 3h/day feeding time. We found that both ghrelin and ghrelin combined with IgG from obese, but not lean mice, prevented ABA, however, they were not able to diminish body weight loss. Physical activity was lower during the feeding period and was increased shortly after feeding in mice receiving ghrelin together with IgG from obese mice. In food-restricted mice without ABA, ghrelin treatments did not have significant effects on food intake. Thus, this study supports pharmacological use of ghrelin or ghrelin combined with IgG from obese animals for treatment of anorexia accompanied by elevated physical activity. The utility of combining ghrelin with protective IgG should be further determined in animal models of anorexia with unrestricted access to food.
Assuntos
Anorexia/prevenção & controle , Grelina/uso terapêutico , Atividade Motora , Animais , Anorexia/psicologia , Anticorpos Bloqueadores/farmacologia , Peso Corporal/efeitos dos fármacos , Restrição Calórica , Ingestão de Alimentos/efeitos dos fármacos , Grelina/antagonistas & inibidores , Grelina/sangue , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Imunoglobulina G/imunologia , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/imunologiaRESUMO
Mycobacteria in complete Freund's adjuvant (CFA) are an essential component of immunization protocols in a number of autoimmune disease animal models including experimental autoimmune encephalomyelitis and uveoretinitis (EAE and EAU, respectively). We determined the role in EAU of two C-type lectin receptors on myeloid cells that recognize and respond to mycobacteria. Using receptor-specific antibodies and knockout mice, we demonstrated for the first time that the macrophage mannose receptor delays disease development but does not affect severity. In contrast, dectin-1 is critically involved in the development of CFA-mediated EAU. Disease severity is reduced in dectin-1 knockout mice and antibody blockade of dectin-1 during the induction, but not the effector phase, prevents EAU development. Significantly, similar blockade of dectin-1 in vivo has no effect in non-CFA-mediated, spontaneously induced or adoptive transfer models of EAU. Thus dectin-1 plays a critical role in the ability of complete Freund's adjuvant to induce EAU in mice.
Assuntos
Doenças Autoimunes/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Retinite/metabolismo , Uveíte/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Quimiocinas/metabolismo , Modelos Animais de Doenças , Adjuvante de Freund/imunologia , Humanos , Imunização , Mediadores da Inflamação/metabolismo , Lectinas Tipo C/deficiência , Lectinas Tipo C/imunologia , Linfonodos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Reconhecimento de Padrão/deficiência , Receptores de Reconhecimento de Padrão/imunologia , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Retinite/imunologia , Retinite/patologia , Proteínas de Ligação ao Retinol/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Fatores de Tempo , Uveíte/imunologia , Uveíte/patologiaRESUMO
Interleukin 6 (IL-6) has a broad effect on cells of the immune system and those not of the immune system and often displays hormone-like characteristics that affect homeostatic processes. IL-6 has context-dependent pro- and anti-inflammatory properties and is now regarded as a prominent target for clinical intervention. However, the signaling cassette that controls the activity of IL-6 is complicated, and distinct intervention strategies can inhibit this pathway. Clinical experience with antagonists of IL-6 has raised new questions about how and when to block this cytokine to improve disease outcome and patient wellbeing. Here we discuss the effect of IL-6 on innate and adaptive immunity and the possible advantages of various antagonists of IL-6 and consider how the immunobiology of IL-6 may inform clinical decisions.
Assuntos
Linfócitos B/imunologia , Imunoterapia/tendências , Doenças Inflamatórias Intestinais/terapia , Interleucina-6/fisiologia , Psoríase/terapia , Receptores de Interleucina-6/metabolismo , Espondilite Anquilosante/terapia , Imunidade Adaptativa , Animais , Anticorpos Bloqueadores/farmacologia , Avaliação Pré-Clínica de Medicamentos , Homeostase , Humanos , Imunidade Inata , Doenças Inflamatórias Intestinais/imunologia , Interleucina-6/antagonistas & inibidores , Psoríase/imunologia , Transdução de Sinais , Espondilite Anquilosante/imunologiaRESUMO
Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Predisposição Genética para Doença , Antígenos de Histocompatibilidade/genética , Sequência de Aminoácidos , Aminoácidos/genética , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Sítios de Ligação/genética , Peso Corporal/efeitos dos fármacos , Peso Corporal/imunologia , Modelos Animais de Doenças , Genótipo , Haplótipos/imunologia , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/imunologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Polimorfismo Genético/imunologia , Estrutura Terciária de Proteína , Ratos , Índice de Gravidade de Doença , Terpenos/imunologiaRESUMO
The mammalian neocortex is composed of various types of neurons that reflect its laminar and area structures. It has been suggested that not only intrinsic but also afferent-derived extrinsic factors are involved in neuronal differentiation during development. However, the role and molecular mechanism of such extrinsic factors are almost unknown. Here, we attempted to identify molecules that are expressed in the thalamus and affect cortical cell development. First, thalamus-specific molecules were sought by comparing gene expression profiles of the developing rat thalamus and cortex using microarrays, and by constructing a thalamus-enriched subtraction cDNA library. A systematic screening by in situ hybridization showed that several genes encoding extracellular molecules were strongly expressed in sensory thalamic nuclei. Exogenous and endogenous protein localization further demonstrated that two extracellular molecules, Neuritin-1 (NRN1) and VGF, were transported to thalamic axon terminals. Application of NRN1 and VGF to dissociated cell culture promoted the dendritic growth. An organotypic slice culture experiment further showed that the number of primary dendrites in multipolar stellate neurons increased in response to NRN1 and VGF, whereas dendritic growth of pyramidal neurons was not promoted. These molecules also increased neuronal survival of multipolar neurons. Taken together, these results suggest that the thalamus-specific molecules NRN1 and VGF play an important role in the dendritic growth and survival of cortical neurons in a cell type-specific manner.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Dendritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Tálamo/química , Tálamo/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , Primers do DNA , DNA Complementar/biossíntese , DNA Complementar/genética , Eletroporação , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/farmacologia , Vetores Genéticos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Análise em Microsséries , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Plasmídeos/genética , Gravidez , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
BACKGROUND: Among birch pollen allergic patients up to 70% develop allergic reactions to Bet v 1-homologue food allergens such as Api g 1 (celery) or Dau c 1 (carrot), termed as birch pollen-related food allergy. In most cases, specific immunotherapy with birch pollen extracts does not reduce allergic symptoms to the homologue food allergens. We therefore genetically engineered a multi-allergen chimer and tested if mucosal treatment with this construct could represent a novel approach for prevention of birch pollen-related food allergy. METHODOLOGY: BALB/c mice were poly-sensitized with a mixture of Bet v 1, Api g 1 and Dau c 1 followed by a sublingual challenge with carrot, celery and birch pollen extracts. For prevention of allergy sensitization an allergen chimer composed of immunodominant T cell epitopes of Api g 1 and Dau c 1 linked to the whole Bet v 1 allergen, was intranasally applied prior to sensitization. RESULTS: Intranasal pretreatment with the allergen chimer led to significantly decreased antigen-specific IgE-dependent ß-hexosaminidase release, but enhanced allergen-specific IgG2a and IgA antibodies. Accordingly, IL-4 levels in spleen cell cultures and IL-5 levels in restimulated spleen and cervical lymph node cell cultures were markedly reduced, while IFN-γ levels were increased. Immunomodulation was associated with increased IL-10, TGF-ß and Foxp3 mRNA levels in NALT and Foxp3 in oral mucosal tissues. Treatment with anti-TGF-ß, anti-IL10R or anti-CD25 antibodies abrogated the suppression of allergic responses induced by the chimer. CONCLUSION: Our results indicate that mucosal application of the allergen chimer led to decreased Th2 immune responses against Bet v 1 and its homologue food allergens Api g 1 and Dau c 1 by regulatory and Th1-biased immune responses. These data suggest that mucosal treatment with a multi-allergen vaccine could be a promising treatment strategy to prevent birch pollen-related food allergy.
Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Betula/imunologia , Hipersensibilidade Alimentar/prevenção & controle , Mucosa Nasal/efeitos dos fármacos , Pólen/imunologia , Proteínas Recombinantes/farmacologia , Administração Intranasal , Alérgenos/farmacologia , Animais , Anticorpos Bloqueadores/farmacologia , Basófilos/efeitos dos fármacos , Basófilos/imunologia , Basófilos/fisiologia , Degranulação Celular/efeitos dos fármacos , Citocinas/biossíntese , Epitopos/imunologia , Feminino , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Imunidade Humoral/efeitos dos fármacos , Imunidade Humoral/imunologia , Imunização , Imunoglobulina E/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
Food allergy is an increasing health problem in Western countries. Previously, it has been shown that the intensity of food allergic reactions can be regulated by regulatory T (T(reg)) cells. In addition, it has been shown that activation of the aryl hydrocarbon receptor (AhR) regulates T-cell responses by induction of T(reg) cells. Therefore, we hypothesized that activation of the AhR pathway can suppress development of food allergic responses through the induction of T(reg) cells. This was investigated by using a mouse model for peanut allergy. C3H/HeOuJ mice (AhR(b)(-2)) were sensitized to peanut by administering peanut extract (PE) by gavage in the presence of cholera toxin and were treated with the prototypical AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.6, 1.7, 5, and 15 µg/kg body weight) on days 3 and 11 orally. The functional role of CD4(+)CD25(+)Foxp3(+) T(reg) cells was investigated by depleting these cells with anti-CD25 mAb during sensitization to PE. TCDD treatment dose dependently suppressed sensitization to peanut (PE-specific IgE, IgG1, and IgG2a and PE-induced IL-5, IL-10, and IL-13, respectively). The percentage, but not the number, of CD4(+)CD25(+)Foxp3(+) T(reg) cells dose dependently increased by AhR activation in both spleen and mesenteric lymph nodes. Depletion of CD4(+)CD25(+)Foxp3(+) T(reg) cells markedly reversed the suppressive effect of TCDD on PE-specific antibody levels and PE-induced IL-5, IL-10, and IL-13 cytokine production. Present data demonstrate for the first time that activation of the AhR by TCDD suppressed the development of Th2-mediated food allergic responses. A functional shift within the CD4(+) cell population toward CD4(+)CD25(+)Foxp3(+) T(reg) cells appeared to underlie this effect. This suggests that the AhR pathway might provide potential therapeutic targets to treat food allergic diseases.
Assuntos
Alérgenos/imunologia , Arachis/imunologia , Tolerância Imunológica/imunologia , Hipersensibilidade a Amendoim/imunologia , Receptores de Hidrocarboneto Arílico/biossíntese , Animais , Anticorpos Bloqueadores/farmacologia , Arachis/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Antagonismo de Drogas , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Hipersensibilidade a Amendoim/metabolismo , Extratos Vegetais/imunologia , Extratos Vegetais/toxicidade , Dibenzodioxinas Policloradas/farmacologia , Linfócitos T Reguladores/imunologiaRESUMO
In this study neural (N)-cadherin, neural cell adhesion molecule (N-CAM) and L1 proteins and their antibody equivalents were covalently immobilized on a polyethylene-imine (PEI)-coated glass surface to form neuron-adhesive coatings. Impedance sensing and (supplementary) image analysis were used to monitor the effects of these CAMs. Immobilization of high concentrations of both N-cadherin protein and antibody led to good adhesion of neurons to the modified surface, better than surfaces treated with 30.0 and 100.0 µg ml(-1) N-CAM protein and antibody. L1 antibody and protein coating revealed no significant effect on neuronal cell-substrate adhesion. In a second series of combinatorial experiments, we used the same antibodies and proteins as medium-additives to inhibit cell-cell adhesion between neurons. Adhesion of neurons cultured on N-cadherin protein or antibody-modified surfaces was lowered by the addition of a soluble N-cadherin protein and antibody to the culturing medium, accelerating neuronal aggregation. The presence of a soluble N-CAM antibody or protein had no effect on the adhesion of neuronal cells on a N-cadherin protein-modified surface. On a N-cadherin antibody-coated surface, the addition of a soluble N-CAM protein led to cell death of neurons after 48 h, while a N-CAM antibody had no effect. In the presence of a soluble N-cadherin protein and antibody the aggregation of neurons was inhibited, both on N-CAM protein and N-CAM antibody-modified surfaces. Neurons cultured on immobilized antibodies were less affected by the addition of soluble CAM blockers than neurons cultured on immobilized proteins, indicating that antibody-protein bonds are more stable compared to protein-protein bonds.
Assuntos
Caderinas/fisiologia , Adesão Celular/fisiologia , Complexo Antígeno L1 Leucocitário/fisiologia , Moléculas de Adesão de Célula Nervosa/fisiologia , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Anticorpos Bloqueadores/farmacologia , Caderinas/antagonistas & inibidores , Caderinas/imunologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Impedância Elétrica , Eletrodos , Complexo Antígeno L1 Leucocitário/imunologia , Moléculas de Adesão de Célula Nervosa/antagonistas & inibidores , Moléculas de Adesão de Célula Nervosa/imunologia , Neurônios/efeitos dos fármacos , Ratos , Propriedades de SuperfícieRESUMO
The aim of this study was to evaluate the effect and elucidate the potential mechanism of the extract of rhizomes from Dioscorea alata L. cv. Phyto, Dispo85E, on accelerating the elimination of advanced glycation end products (AGEs) in vitro and in vivo. Primary mouse nonparenchymal cells (NPCs) were used to evaluate the drug effect on AGEs clearance and autophagic-lysosomal activity. In an animal study, we used AGEs-induced diabetic mice to evaluate the drug effect on AGEs-induced vascular complications. Our results indicated that Dispo85E enhanced the endocytosis and degradation activity of AGEs in hepatic NPCs. Furthermore, the hepatocyte growth factor (HGF) expression level was positively correlated with the clearance capacity of the AGEs in NPCs after Dispo85E treatment. In addition, the effects of Dispo85E on the degradation and uptake capability of (14)C-AGEs were abolished in the presence of an anti-HGF neutralizing antibody. We further demonstrated that recombinant mouse HGF could enhance the endocytosis and autophagic clearance of AGEs in NPCs. The in vivo data indicated that Dispo85E increased hepatic HGF messenger RNA expression levels and decreased serum AGEs level in diabetic mice. Moreover, the function of retina and kidneys was improved by Dispo85E treatment in AGEs-induced diabetic mice. These results suggest that HGF may have an important role in the elimination of AGEs. This study suggests that Dispo85E is a botanical drug with a novel mechanism that enhances the clearance of AGEs through HGF-induced autophagic-lysosomal pathway and is a candidate drug for the treatment of diabetic vascular complications.
Assuntos
Autofagia/efeitos dos fármacos , Angiopatias Diabéticas/tratamento farmacológico , Angiopatias Diabéticas/metabolismo , Dioscorea/química , Produtos Finais de Glicação Avançada/metabolismo , Fator de Crescimento de Hepatócito/fisiologia , Hipoglicemiantes/uso terapêutico , Extratos Vegetais/uso terapêutico , Animais , Anticorpos Bloqueadores/farmacologia , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/biossíntese , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Lisossomos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fitoterapia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Rizoma/químicaRESUMO
Urease plays a crucial role in the survival and pathogenesis of Helicobacter pylori (H. pylori), and antibody neutralizing the urease activity may be implicated for the protection against H. pylori infection. Previously, a neutralizing monoclonal antibody (MAb) 6E6 against UreB of H. pylori was developed. In this work, we try to identify the B-cell epitope recognized by neutralizing MAb 6E6. Following screening a series of truncated proteins of UreB, an epitope was primarily localized in the aa 200-230 of UreB. Subsequently, we screened the overlapping synthetic peptides covering the aa 200-230 and identified a novel B-cell epitope (U(211-225), IEAGAIGFKIHEDWG) that was recognized by specific MAb 6E6. The newly identified epitope may help understanding of the protective immunity against H. pylori and be implicated for vaccine development.
Assuntos
Linfócitos B/imunologia , Vacinas Bacterianas/imunologia , Epitopos/imunologia , Helicobacter pylori/imunologia , Urease/imunologia , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/imunologia , Western Blotting , Avaliação Pré-Clínica de Medicamentos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Feminino , Infecções por Helicobacter/imunologia , Helicobacter pylori/genética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Molecular , Testes de Neutralização , Conformação Proteica , Urease/antagonistas & inibidoresRESUMO
In the present study, we aimed to investigate the neuromodulatory role played by hypothalamic brain-derived neurotrophic factor (BDNF) in the regulation of acute cardiovascular and feeding responses to melanocortin-4 receptor (MC4R) activation. In vitro, a selective MC4R agonist, MK1, stimulated BDNF release from isolated rat hypothalami and this effect was blocked by preincubation with the MC3/4R antagonist SHU-9119. In vivo, peripheral administration of MK1 decreased food intake in rats and this effect was blocked by pretreatment with an anti-BDNF antibody administered into the third ventricle. When anorexia was induced with the cannabinoid-1 receptor (CB1R) antagonist AM251, the anti-BDNF antibody did not prevent the reduction in food intake. Peripheral administration of MK1 also increased mean arterial pressure, heart rate and body temperature. These effects were prevented by pretreatment with the anti-BDNF antibody whereas the intracerebroventricular administration of BDNF caused changes similar to those of MK1. These findings demonstrate for the first time that activation of MC4R leads to an acute release of BDNF in the hypothalamus. This release is a prerequisite for MC4R-induced effects on appetite, body temperature and cardiovascular function. By contrast, CB1R antagonist-mediated anorexia is independent of the MC4R/BDNF pathway. Overall, these results show that BDNF is an important downstream mediator of the MC4R pathway.
Assuntos
Temperatura Corporal/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Sistema Cardiovascular/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Hipotálamo/metabolismo , Receptor Tipo 4 de Melanocortina/agonistas , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais , Depressores do Apetite/farmacologia , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Interpretação Estatística de Dados , Hipotálamo/efeitos dos fármacos , Técnicas In Vitro , Injeções Intraventriculares , Masculino , Hormônios Estimuladores de Melanócitos/administração & dosagem , Hormônios Estimuladores de Melanócitos/farmacologia , Piperidinas/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/agonistas , Transdução de Sinais/efeitos dos fármacos , Técnicas Estereotáxicas , TelemetriaRESUMO
The biological effects of rIgG(1) 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-kappaB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG(1) 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG(1) 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37 degrees C, together with recruitment of TCR, CD3zeta, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Ctheta, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cgamma1, and p95(vav). Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr(292) and Tyr(319) phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10-30 s following rIgG(1) 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-kappaB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG(1) 13B8.2 acts to induce its biological effects.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Antígenos CD4/metabolismo , Microdomínios da Membrana/imunologia , Fosfolipase C gama/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-vav/antagonistas & inibidores , Proteína-Tirosina Quinase ZAP-70/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anticorpos Bloqueadores/genética , Anticorpos Monoclonais/genética , Baculoviridae/genética , Baculoviridae/imunologia , Antígenos CD4/imunologia , Reagentes de Ligações Cruzadas/metabolismo , Detergentes , Humanos , Imunoglobulina G/genética , Imunoglobulina G/farmacologia , Células Jurkat , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/metabolismo , Fosfolipase C gama/metabolismo , Fosfoproteínas/metabolismo , Óleos de Plantas , Polietilenoglicóis , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
The leukocyte-specific integrin CD11b/CD18 plays a key role in the biological function of these cells and represents a validated therapeutic target for inflammatory diseases. Currently, the low affinity interaction between CD11b/CD18 integrin and its respective ligand poses a challenge in the development of cell-based adhesion assays for the high-throughput screening (HTS) environment. Here the authors describe a simple cell-based adhesion assay that can be readily used for HTS for the discovery of functional regulators of CD11b/CD18. The assay consistently produces acceptable Z' values (> 0.5) for HTS. After testing the assay using 2 established blocking antibodies as reference biologicals, the authors performed a proof-of-concept primary screen using a library of 6612 compounds and identified both agonist and antagonist hits.
Assuntos
Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Anticorpos Bloqueadores/farmacologia , Adesão Celular/efeitos dos fármacos , Células Clonais , Dimerização , Humanos , Células K562 , Peso MolecularRESUMO
NK cell-mediated cytotoxicity of target cells is the result of a balance between the activating and inhibitory signals provided by their respective ligand-receptor interactions. In our current study, we have investigated the significance of CD59 on human target cells in modulating this process. A range of CD59 site-specific Abs were used in NK cytotoxicity blocking studies against the CD59-expressing K562 target cell line. Significantly reduced cytotoxicity was observed in the presence of Abs previously shown to lack blocking capacity for C-mediated lysis. We investigated the consequences for alternative membrane attachment modalities, namely bis-myristoylated-peptidyl (BiMP) and GPI anchoring, on CD59-negative U937 cells. Expression of GPI-anchored CD59 either via transfection or incorporation rendered U937 targets more susceptible to NK cytotoxicity, whereas incorporation of CD59 via a BiMP anchor to similar levels did not alter susceptibility to NK cytotoxicity. Localization of both BiMP- and GPI-anchored CD59 proteins was shown to be within the lipid raft microdomain. A role for the GPI anchor and independence from glycosylation status was confirmed by expression of transmembrane-anchored CD59 or unglycosylated CD59 and by testing in NK cytotoxicity assays. To investigate mechanisms, we compared the signaling capacity of the various forms of expressed and incorporated CD59 following Ab cross-linking in calcium flux assays. GPI-anchored CD59, with or without glycosylation, mediated activation events, whereas CD59 forms lacking the GPI anchor did not. The data show that the increased susceptibility of target cells expressing CD59 to NK cytotoxicity requires GPI anchor-mediating signaling events, likely mediated by interactions between GPI-anchored CD59 on targets and NK receptors.
Assuntos
Adjuvantes Imunológicos/fisiologia , Antígenos CD59/biossíntese , Citotoxicidade Imunológica , Glicosilfosfatidilinositóis/metabolismo , Células Matadoras Naturais/imunologia , Anticorpos Bloqueadores/farmacologia , Antígenos CD59/imunologia , Antígenos CD59/metabolismo , Antígenos CD59/fisiologia , Linhagem Celular , Células Cultivadas , Glicosilação , Glicosilfosfatidilinositóis/deficiência , Glicosilfosfatidilinositóis/genética , Humanos , Células K562 , Transdução de Sinais/fisiologia , Células U937RESUMO
PURPOSE: To investigate the roles of interaction between OX40 and OX40 ligand (OX40L) in the development of experimental allergic conjunctivitis (EC) in mice. METHODS: BALB/c mice actively immunized with short ragweed pollen (RW) were intraperitoneally injected on days 0, 2, 4, 6, and 8 with agonistic anti-OX40 Ab, blocking anti-OX40L Ab, or normal rat (nr)IgG. On day 10, the mice were challenged with RW in eye drops, and 24 hours later their conjunctivas, spleens, and blood were harvested for analyses. For examination of the effects of the Abs during the late induction (or effector) phase, actively immunized mice were treated with the Abs just before or at the same time as the challenge. In addition, splenocytes from RW-primed mice were transferred into syngeneic naïve mice, and the recipients were treated with Abs twice (on days 2 and 4). On day 4, the mice were challenged with RW and evaluated. RESULTS: When the treatments were performed during the induction phase, anti-OX40 Ab treatment significantly increased clinical EC and eosinophil infiltration into the conjunctiva, whereas anti-OX40L Ab treatment significantly reduced eosinophil infiltration. Compared with splenocytes from nrIgG-treated mice, splenocytes from anti-OX40 Ab-treated mice proliferated vigorously against RW and produced significantly higher amounts of IL-2, -4, and -5 by RW stimulation but a significantly lesser amount of IFN-gamma after Con A stimulation. In contrast, splenocytes from anti-OX40L Ab-treated mice produced significantly less IL-5 with RW stimulation and IL-2 and IL-5 with Con A stimulation, whereas significantly more IFN-gamma was induced by Con A stimulation. Treatment with anti-OX40 and anti-OX40L Abs during the late induction or effector phase of EC did not affect eosinophil infiltration. CONCLUSIONS: Blocking of the interaction between OX40 and OX40L in vivo inhibits the development of EC. In contrast, forced stimulation of OX40 in vivo significantly exacerbates EC by activating T cells, especially Th2 cells. These effects were noted only in the induction phase of EC, suggesting that the interaction between OX40 and OX40L is important in the generation of Th2 immune responses in the development of EC.
Assuntos
Conjuntivite Alérgica/imunologia , Glicoproteínas de Membrana/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Fatores de Necrose Tumoral/fisiologia , Transferência Adotiva , Alérgenos/imunologia , Animais , Anticorpos Bloqueadores/farmacologia , Conjuntivite Alérgica/etiologia , Modelos Animais de Doenças , Eosinófilos/fisiologia , Feminino , Imunização , Imunoglobulina E/sangue , Interferon gama/metabolismo , Interleucinas/metabolismo , Ativação Linfocitária/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ligante OX40 , Pólen/imunologia , Receptores OX40 , Células Th2/imunologiaRESUMO
PECAM is a molecule used specifically during the diapedesis step when neutrophils and monocytes leave the blood compartment. Anti-PECAM reagents, such as Abs and soluble fusion proteins, block diapedesis both in vivo and in vitro. However, the PECAM knockout mouse in C57BL/6 strain has no serious defects in most models of inflammation. We show in this study that the same PECAM knockout backcrossed into the FVB/n strain clearly has reduced leukocyte emigration in two models of inflammation. Furthermore, we show that anti-PECAM reagents can block leukocyte emigration in several other wild-type strains of mice like FVB/n, SJL, and the outbred strain Swiss Webster. This clearly shows that the C57BL/6 strain is uniquely able to compensate for the loss of PECAM function. Murine models of inflammatory disease that have been studied using C57BL/6 mice should be re-evaluated using FVB/n or other mouse strains to determine whether PECAM plays a role in those models.
Assuntos
Inibição de Migração Celular , Movimento Celular/genética , Movimento Celular/imunologia , Regulação para Baixo/imunologia , Leucócitos/imunologia , Leucócitos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Animais , Anticorpos Bloqueadores/farmacologia , Cruzamentos Genéticos , Óleo de Cróton/administração & dosagem , Dermatite de Contato/genética , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Modelos Animais de Doenças , Regulação para Baixo/genética , Inativação Gênica , Leucócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peritonite/genética , Peritonite/imunologia , Peritonite/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Especificidade da Espécie , Tioglicolatos/administração & dosagemRESUMO
CD8 glycoproteins are expressed as either alphaalpha homodimers or alphabeta heterodimers on the surface of T cells. CD8alphabeta is a more efficient coreceptor than the CD8alphaalpha for peptide Ag recognition by TCR. Each CD8 subunit is composed of four structural domains, namely, Ig-like domain, stalk region, transmembrane region, and cytoplasmic domain. In an attempt to understand why CD8alphabeta is a better coreceptor than CD8alphaalpha, we engineered, expressed, and functionally tested a chimeric CD8alpha protein whose stalk region is replaced with that of CD8beta. We found that the beta stalk region enhances the coreceptor function of chimeric CD8alphaalpha to a level similar to that of CD8alphabeta. Surprisingly, the beta stalk region also restored functional activity to an inactive CD8alpha variant, carrying an Ala mutation at Arg(8) (R8A), to a level similar to that of wild-type CD8alphabeta. Using the R8A variant of CD8alpha, a panel of anti-CD8alpha Abs, and three MHC class I (MHCI) variants differing in key residues known to be involved in CD8alpha interaction, we show that the introduction of the CD8beta stalk leads to a different topology of the CD8alpha-MHCI complex without altering the overall structure of the Ig-like domain of CD8alpha or causing the MHCI to employ different residues to interact with the CD8alpha Ig domain. Our results show that the stalk region of CD8beta is capable of fine-tuning the coreceptor function of CD8 proteins as a coreceptor, possibly due to its distinct protein structure, smaller physical size and the unique glycan adducts associated with this region.