RESUMO
AIM: Peroxisome proliferator activated receptors (PPARs) are nuclear receptors involved in glucose and lipid metabolism. Three isoforms of PPARs have been identified with different tissue distribution and biological functions. Although the pharmacology of each receptor is well studied, the physiological effect of simultaneous activation of PPARalpha, gamma and delta is only starting to emerge. We sought to determine the biological effects of a novel PPAR pan activator and elucidate the physiological mechanisms involved. METHODS: Ob/ob, diet-induced obese (DIO) or PPARalpha knockout mice were administered a novel agonist that activates all PPARs to various degrees to determine the effect on body weight, body composition, food intake and energy expenditure. In addition, serum parameters including glucose, insulin, triglycerides and ketone bodies as well as tissue acylcarnitine were evaluated. The effect of the novel agonist on liver and skeletal muscle histopathology was also studied. RESULTS: We report that simultaneous activation of all PPARs resulted in substantial weight loss in ob/ob and DIO mice. Consistent with known PPAR pharmacology, we observed that agonist treatment increased lipid oxidation, although appetite suppression was mainly responsible for the weight loss. Agonist-induced weight loss was completely absent in PPARalpha knockout mice suggesting that PPARalpha pharmacology was the major contributor to weight regulation in mice. CONCLUSIONS: Our work provides evidence that simultaneous activation of PPARalpha, gamma and delta decreases body weight by regulating appetite. These effects of the pan agonist were completely absent in PPARalpha knockout mice, suggesting that PPARalpha pharmacology was the major contributor to weight loss.
Assuntos
Depressores do Apetite/farmacologia , Obesidade/tratamento farmacológico , PPAR alfa/agonistas , PPAR delta/agonistas , PPAR gama/agonistas , Redução de Peso/fisiologia , Animais , Regulação do Apetite/fisiologia , Metabolismo Energético/fisiologia , Resistência à Insulina/fisiologia , Peroxidação de Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/fisiopatologia , PPAR alfa/genética , PPAR gama/genética , Rosiglitazona , Tiazóis/farmacologia , Tiazolidinedionas/farmacologiaRESUMO
The selectins are a family of three calcium-dependent lectins that mediate adhesive interactions between leukocytes and the endothelium during normal and abnormal inflammatory episodes. Previous work has implicated the carbohydrate sialyl Lewis(x) (sLe(x); sialic acid alpha 2-3 galactose beta 1-4 [Fucose alpha 1-3] N-acetyl glucosamine) as a component of the ligand recognized by E- and P-selectin. In the case of P-selectin, other components of the cell surface, including 2'6-linked sialic acid and sulfatide (galactose-4-sulfate ceramide), have also been proposed for adhesion mediated by this selectin. We have recently defined a region of the E-selectin lectin domain that appears to be directly involved with carbohydrate recognition and cell adhesion (Erbe, D. V., B. A. Wolitzky, L. G. Presta, C. R. Norton, R. J. Ramos, D. K. Burns, R. M. Rumberger, B. N. N. Rao, C. Foxall, B. K. Brandley, and L. A. Lasky. 1992. J. Cell Biol. 119:215-227). Here we describe a similar analysis of the P-selectin lectin domain which demonstrates that a homologous region of this glycoprotein's lectin motif is involved with carbohydrate recognition and cell binding. In addition, we present evidence that is inconsistent with a biological role for either 2'6-linked sialic acid or sulfatide in P-selectin-mediated adhesion. These results suggest that a common region of the E- and P-selectin lectin domains appears to mediate carbohydrate recognition and cell adhesion.
Assuntos
Moléculas de Adesão Celular/fisiologia , Adesão Celular , Glicolipídeos/metabolismo , Glicoproteínas da Membrana de Plaquetas/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Moléculas de Adesão Celular/química , Selectina E , Citometria de Fluxo , Humanos , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Selectina-P , Glicoproteínas da Membrana de Plaquetas/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
E-selectin elicits cell adhesion by binding to the cell surface carbohydrate, sialyl Lewis X (sLe(x)). We evaluated the effects of mutations in the E-selectin lectin domain on the binding of a panel of anti-E-selectin mAbs and on the recognition of immobilized sLe(x) glycolipid. Functional residues were then superimposed onto a three-dimensional model of the E-selectin lectin domain. This analysis demonstrated that the epitopes recognized by blocking mAbs map to a patch near the antiparallel beta sheet derived from the NH2 and COOH termini of the lectin domain and two adjacent loops. Mutations that affect sLe(x) binding map to this same region. These results thus define a small region of the E-selectin lectin domain that is critical for carbohydrate recognition.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular/fisiologia , Antígenos CD15/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sítios de Ligação/fisiologia , Moléculas de Adesão Celular/química , Análise Mutacional de DNA , Selectina E , Imunofluorescência , Humanos , Antígenos CD15/química , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Conformação Proteica , Coelhos , Receptores Imunológicos/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A growing percentage of the population is resistant to two key hormones - insulin and leptin - as a result of increased obesity, often leading to significant health consequences such as type 2 diabetes. Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of signalling by both of these hormones, so that inhibitors of this enzyme may provide promise for correcting endocrine abnormalities in both diabetes and obesity. As with other tyrosine phosphatases, identification of viable drug candidates targeting PTP1B has been elusive because of the nature of its active site. Beginning with novel phosphotyrosine mimetics, we have designed some of the most potent PTP1B inhibitors. However, their highly acidic structures limit intrinsic permeability and pharmacokinetics. Ester prodrugs of these inhibitors improve their drug-like properties with the goal of delivering these nanomolar inhibitors to the cytoplasm of cells within target tissues. In addition to identifying prodrugs that is able to deliver active drugs into cells to inhibit PTP1B and increase insulin signalling, these compounds were further modified to gain a variety of cleavage properties for targeting activity in vivo. One such prodrug candidate improved insulin sensitivity in ob/ob mice, with lowered fasting blood glucose levels seen in the context of lowered fasting insulin levels following 4 days of intraperitoneal dosing. The results presented in this study highlight the potential for design of orally active drug candidates targeting PTP1B, while also delineating the considerable challenges remaining.
Assuntos
Pró-Fármacos/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Administração Oral , Animais , Biomimética , Glicemia/análise , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Desenho de Fármacos , Jejum , Insulina/sangue , Leptina/sangue , Masculino , Camundongos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Ratos , Ratos Zucker , Transdução de Sinais/efeitos dos fármacosRESUMO
We examined expression and cytotoxic triggering capability of the three Fc receptors for IgG (Fc gamma R) on human monocytes, PMNs and myeloid cell lines after in vitro culture with various cytokines. Fc gamma R expression was evaluated using specific anti-Fc gamma R monoclonal antibodies (mAb). The cytotoxic capability of each Fc gamma R was examined after the effector cells were treated with the recombinant cytokines IFN-gamma. TNF alpha, GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, or IL-6. Hybridoma cell lines (HC) bearing antibody directed to Fc gamma RI (HC 32), Fc gamma RII (HC IV.3) or Fc gamma RIII (HC 3G8) were used as targets, as were chicken erythrocytes (CE) sensitized with heteroantibodies composed of anti-Fc gamma R mAbs (32, IV.3, 3G8) linked to anti-CE antibody. Only IFN-gamma treatment significantly increased Fc gamma R expression and then only Fc gamma RI. IFN-gamma dramatically up-regulated Fc gamma RI expression on all cells tested. However, ADCC was enhanced by treatment with a number of cytokines other than IFN-gamma. GM-CSF, TNF, and IFN-gamma treatment enhanced killing of HC 32 and HC IV.3 by in vitro cultured monocytes. G-CSF treatment enabled PMNs to kill HC through Fc gamma RII, whereas PMN killing of HC through Fc gamma RIII could not be induced by any of the cytokines studied. Although only IFN-gamma treatment increased ADCC of CE by monocytes, GM-CSF treatment as well as IFN-gamma treatment augmented ADCC of CE by PMNs. In addition to IFN-gamma treatment, IL-6 treatment enabled U937 cells to lyse CE. Whereas IFN-gamma-treated U937 cells killed CE through both Fc gamma RI and Fc gamma RII, IL-6-treated U937 cells killed CE only through Fc gamma RI. In addition to IFN-gamma treatment, G-CSF treatment enabled HL-60 cells to lyse CE through both Fc gamma RI and Fc gamma RII. These results demonstrate that although IFN-gamma appears unique in regulating Fc gamma R expression on myeloid cells, cytokines other than IFN-gamma affect ADCC by these cells in a receptor-specific manner.
Assuntos
Antígenos de Diferenciação/análise , Fatores Biológicos/farmacologia , Imunoglobulina G , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Receptores Fc/análise , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos CD/análise , Antígenos CD/fisiologia , Antígenos de Diferenciação/fisiologia , Células Cultivadas , Citocinas , Humanos , Leucemia Promielocítica Aguda/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Receptores Fc/fisiologia , Receptores de IgGRESUMO
Through interaction with antibody, IgG Fc receptors provide an interface between specific humoral immunity and Fc gamma R-bearing host cells. Fc gamma R trigger such diverse functions as immune complex clearance, phagocytosis of opsonized pathogens, reactive oxygen intermediate and enzyme secretion, and antibody-dependent cellular cytotoxicity (ADCC). Moreover, Fc gamma R are the exclusive trigger molecules for tumor cell killing by human myeloid cells. Studies of Fc gamma R function have been aided by the use of bispecific antibodies to link cells or pathogens to specific host cell molecules, including Fc gamma R. These reagents have permitted determination of the role of Fc gamma R in ADCC of the protozoan, Toxoplasma gondii, by human effector cells. This approach has also indicated that Fc gamma R do not serve as entry points for viruses such as dengue virus and HIV. Taken together, these results provide insight into the utility of manipulating Fc gamma R function in the therapy of cancer and infectious disease.
Assuntos
Infecções/imunologia , Neoplasias/imunologia , Receptores de IgG/fisiologia , Citotoxicidade Celular Dependente de Anticorpos , Dengue/imunologia , Infecções por HIV/imunologia , Humanos , Toxoplasmose/imunologiaRESUMO
There has been some controversy as to whether or not B cells can kill target cells through their Fc receptors. To address this, we have examined the ability of human B cells from a variety of sources to lyse hybridoma cells with specificity for either the B cell Fc gamma RII or Fc epsilon RII using a reverse killing assay, as well as their ability to lyse opsonized chicken erythrocytes using a classic ADCC assay. Tonsil B cells, chronic lymphocytic leukemia B cells, and Epstein-Barr virus-induced B cells, even after preactivation with a cocktail of cytokines, all failed to lyse any of these targets. We conclude that Fc gamma RII and Fc epsilon RII on human B cells are not cytotoxic trigger molecules.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação/imunologia , Linfócitos B/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Receptores Fc/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular Transformada , Galinhas , Citocinas/imunologia , Humanos , Hibridomas/imunologia , Ativação Linfocitária , Receptores de IgE , Receptores de IgGRESUMO
A novel pyridothiophene inhibitor of PTP1B was discovered by rational screening of phosphotyrosine mimics at high micromolar concentrations. The potency of this lead compound has been improved significantly by medicinal chemistry guided by X-ray crystallography and molecular modeling. Excellent consistency has been observed between structure-activity relationships and structural information from PTP1B-inhibitor complexes.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Tiofenos/farmacologia , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
The three types of IgG FcR (Fc gamma RI, Fc gamma RII, Fc gamma RIII) on human leukocytes play an important role in elimination of antibody-coated infectious agents. To further understand the role of the different Fc gamma R in mediating this killing, we examined the ability of human myeloid and lymphoid cells to kill the protozoan Toxoplasma gondii in the presence of antitoxoplasma IgG or bispecific antibodies. Although human myeloid cells (monocytes, macrophages, neutrophils, and eosinophils) all lysed unsensitized T. gondii, killing by these cells was significantly enhanced by opsonization with antitoxoplasma rabbit IgG. Human lymphocytes, however, did not lyse T. gondii unless the parasites were coated with antibody. The role of antibody and Fc gamma R in mediating ADCC of T. gondii was then examined using bispecific antibodies made by chemically cross-linking Fab fragments of antitoxoplasma antibodies to Fab fragments of antibodies specific for human leukocyte surface Ag, including Fc gamma R. Thus, simultaneous binding of these bispecifics to parasites and effector cells allowed an evaluation of killing when T. gondii were targeted to each Ag independently. Bispecifics which targeted T. gondii to Fc gamma RI, II or III enhanced lysis by monocytes. However, similar results were obtained with bispecifics targeting T. gondii to non-Fc gamma R Ag (CD11b or beta 2-microglobulin) on monocytes. Likewise, polymorphonuclear leukocytes mediated significantly more lysis in the presence of bispecifics linking T. gondii to Fc gamma RII, Fc gamma RIII, or the two non-Fc gamma R Ag CD11b and beta 2-microglobulin. Thus, although human myeloid cells did not require antibody-Fc gamma R triggering to kill T. gondii, antibody appeared to enhance lysis by capturing and directing the parasites to the effector cell surface. Human lymphocytes, in contrast, mediated significant lysis of T. gondii only in the presence of bispecifics targeting T. gondii to Fc gamma RIII, indicating a requirement for specific triggering of Fc gamma RIII for killing by large granular lymphocytes. Consequently, using bispecifics to compare targeting to specific Ag, both non-Fc gamma R and Fc gamma R, allowed determination of the role of antibody-Fc gamma R interactions in T. gondii killing. In addition, these studies demonstrate the potential of bispecifics in determining the role of specific Ag in killing of or infection by pathogens.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Diferenciação/fisiologia , Receptores Fc/fisiologia , Toxoplasma/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Citotoxicidade Imunológica , Humanos , Imunidade Celular , Técnicas In Vitro , Linfócitos/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Receptores de IgGRESUMO
To further understand the mechanism(s) of antibody-dependent cell-mediated cytotoxicity (ADCC) by various effector populations, we have examined the extracellular Ca++ and Mg++ requirements for ADCC performed by lymphocytes, monocytes, polymorphonuclear leukocytes and peritoneal macrophages. We have used the anti-Fc gamma R-bearing hybridoma cell lines (HC) as self directed targets for ADCC to analyse the triggering ability of each of the three defined Fc gamma R; Fc gamma RI, Fc gamma RII, and Fc gamma RIII. Lymphocyte killing of the anti-Fc gamma RIII bearing HC (HC 3G8) was Ca++ dependent, but Mg++ independent. In contrast, monocytes and PMN killed the anti-Fc gamma RI- (HC 32) and the anti-Fc gamma RII- (HC IV.3) bearing HC in a Mg++-dependent, Ca++-independent fashion. In addition, freshly prepared monocytes were able to kill HC 3G8 in a Mg++-dependent, Ca++-independent fashion, indicating that low levels of Fc gamma RIII may be functionally detected on monocytes. Peritoneal macrophages were able to kill all three of the anti-Fc gamma R bearing HC in a Mg++-dependent, Ca++-independent fashion. Thus, the same target is lysed by myeloid cells in the presence of Mg++ without Ca++ and by lymphoid cells in the presence of Ca++ without Mg++. These results suggest that at least two distinct mechanisms of ADCC exist that depend on the type of effector cell mediating antibody-dependent killing and not necessarily on the Fc gamma R type triggered.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos de Diferenciação/imunologia , Ligação Competitiva , Antígenos CD18 , Cálcio/fisiologia , Cátions Bivalentes , Linhagem Celular , Feminino , Humanos , Hibridomas/imunologia , Magnésio/fisiologia , Receptores Fc/imunologia , Receptores de IgG , Receptores de Adesão de Leucócito/imunologiaRESUMO
It is known that antibodies to dengue viruses at subneutralizing concentrations enhance dengue virus infection of Fc gamma R+ cells. This phenomenon called antibody-dependent enhancement (ADE) occurs when virus-antibody complexes bind to the Fc gamma R via the Fc portion of the Ig. It has been hypothesized that ADE may be responsible for the pathogenesis of the severe manifestations of dengue virus infection including dengue hemorrhagic fever/dengue shock syndrome. To further analyze the mechanisms of ADE, we prepared bispecific antibodies by chemically cross-linking antidengue virus antibodies to antibodies specific for Fc gamma RI or Fc gamma RII and the non-Fc R molecules beta2 microglobulin, CD15 or CD33 and examined whether these bispecific antibodies could enhance infection. Bispecific antibodies targeting dengue virus to Fc gamma RI or Fc gamma RII enhanced dengue virus infection, consistent with previous reports using conventional antibodies. Furthermore, bispecific antibodies targeting dengue virus to beta2 microglobulin, CD15 or CD33 also enhanced dengue virus infection. Bispecific antibody mediated ADE was inhibited by pretreating the cells with the appropriate blocking mAb. These results indicate that cell surface molecules other than Fc gamma R can mediate ADE and suggest that the Fc gamma R does not provide a unique signal necessary for enhanced infection. We hypothesize that directing dengue virus to the cell surface by a bispecific antibody facilitates the interaction between dengue virus and its receptor, thereby increasing its infectivity.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos de Superfície/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Isoanticorpos/imunologia , Receptores Virais/fisiologia , Anticorpos Antivirais/química , Complexo Antígeno-Anticorpo/metabolismo , Antígenos CD/fisiologia , Antígenos de Diferenciação/fisiologia , Antígenos de Diferenciação Mielomonocítica/fisiologia , Linhagem Celular , Humanos , Técnicas In Vitro , Isoanticorpos/química , Antígenos CD15 , Receptores Fc/fisiologia , Receptores de IgG , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Microglobulina beta-2/fisiologiaRESUMO
Antibody-dependent enhancement (ADE) of dengue virus infection occurs when neutralizing antibodies at sub-neutralizing concentrations or non-neutralizing antibodies form complexes with the virus. These virus-antibody complexes can then attach to a Fc gamma receptor-bearing cell, via the Fc portion of the immunoglobulin, resulting in an increased number of infected cells. ADE may be responsible in part for the most severe clinical manifestations of dengue virus infection which include haemorrhage and shock. Three classes of human Fc gamma receptors exist, Fc gamma RI, Fc gamma RII and Fc gamma RIII. In this study, we examined the effects of neuraminidase on ADE of dengue virus infection mediated by the low-affinity Fc gamma RII. K562 cells, which express only Fc gamma RII, treated with neuraminidase resulted in augmentation of ADE of dengue virus infection by human anti-dengue antibodies. This augmented ADE of infection could be blocked by anti-Fc gamma RII monoclonal antibody IV.3. Incubation of neuraminidase-treated K562 cells with IgG-coated human red blood cells resulted in an increase in the percentage of rosette formations compared with the untreated K562 cells. A bispecific antibody directed against Fc gamma RII and dengue virus (IV.3 x 2H2) enhanced virus infection. Neuraminidase also augmented ADE mediated by this antibody, but to a much lesser degree (by 50%) compared with that seen using conventional human anti-dengue antibody (by 200 to 300%). Fluorescence-activated cell sorting analysis of neuraminidase-treated K562 cells showed that the number of Fc gamma RII-specific antibodies that bind to Fc gamma RII increases by 15 to 20% after treatment with neuraminidase. These results indicate that neuraminidase augments ADE of dengue virus infection and that the augmented ADE is mediated through Fc gamma RII.
Assuntos
Anticorpos Antivirais/metabolismo , Vírus da Dengue/fisiologia , Neuraminidase/metabolismo , Receptores de IgE/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Humanos , Receptores de IgE/imunologia , Células Tumorais Cultivadas , Replicação ViralRESUMO
Optimal immune responses require both an antigen-specific and a co-stimulatory signal. The shared ligands B7-1 and B7-2 on antigen-presenting cells deliver the co-stimulatory signal through CD28 and CTLA-4 on T cells. Signalling through CD28 augments the T-cell response, whereas CTLA-4 signalling attenuates it. Numerous animal studies and recent clinical trials indicate that manipulating these interactions holds considerable promise for immunotherapy. With the consequences of these signals well established, and details of the downstream signalling events emerging, understanding the molecular nature of these extracellular interactions becomes crucial. Here we report the crystal structure of the human CTLA-4/B7-1 co-stimulatory complex at 3.0 A resolution. In contrast to other interacting cell-surface molecules, the relatively small CTLA-4/B7-1 binding interface exhibits an unusually high degree of shape complementarity. CTLA-4 forms homodimers through a newly defined interface of highly conserved residues. In the crystal lattice, CTLA-4 and B7-1 pack in a strikingly periodic arrangement in which bivalent CTLA-4 homodimers bridge bivalent B7-1 homodimers. This zipper-like oligomerization provides the structural basis for forming unusually stable signalling complexes at the T-cell surface, underscoring the importance of potent inhibitory signalling in human immune responses.