RESUMO
BACKGROUND: The human epidermal growth factor receptors HER1/EGFR and HER2 offer potential targets for treating non-small cell lung cancer (NSCLC). The antitumor efficacy of erlotinib (Tarceva, F. Hoffmann-La Roche, Ltd., Basel, Switzerland), a HER1/EGFR tyrosine-kinase inhibitor, was investigated in relation to HER1/EGFR and HER2 expression in five NSCLC xenograft models. MATERIALS AND METHODS: Tumor-bearing mice were randomized to daily oral erlotinib, 50 mg/kg, or vehicle (controls) for 20-50 days. The antitumor efficacy of erlotinib was measured through tumor volume, serum tumor markers and tumor biomarkers. Tumor HER1/EGFR and HER2 expression were analyzed immunohistochemically. RESULTS: Erlotinib reduced tumor volume in three NSCLC models. It also reduced serum tumor marker levels and the extent of inhibition correlated with tumor growth inhibition. HER1/EGFR and HER2 expression differed between the five tumor models, suggesting that expression level does not predict response to treatment. CONCLUSION: Erlotinib showed differing antitumor activity in five NSCLC models, suggesting that its antitumor effect is independent of HER1/EGFR and HER2 overexpression.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Receptor ErbB-2/metabolismo , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The general feasibility of chimerization of monoclonal antibodies (mAbs) has already been shown for a large number of them. In order to evaluate in vitro parameters relevant to immunosuppressive therapy, we have chimerized and synthesized two anti-CD4 mAbs recognizing two different epitopes on the human T-lymphocyte antigen, CD4. The chimerized mAbs are produced at levels corresponding to those of the original hybridoma cell lines. With respect to activation of human complement, the individual Abs are negative; however, when used in combination, complement activation was performed. When applied in combination, they were found to modulate the CD4 antigen, whereas the individual mAb do not display this property. Individually they mediate an up to 60% inhibition of the mixed lymphocyte reaction (MLR). However, by combination of an anti-CD4 mAb with one directed against the alpha-chain of the human IL2 receptor, nearly 100% inhibition of the MLR was achieved, even with reduced dosage of the mAbs. Our data suggest that the combination of an anti-CD4 mAb and an anti-IL2R alpha chain mAb is more effective with respect to immunosuppression than each mAb by itself, indicating that this mAb cocktail could be a new strategy for immunosuppressive therapy.
Assuntos
Anticorpos/química , Antígenos CD4/imunologia , Receptores de Interleucina-2/imunologia , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Modulação Antigênica , Sequência de Bases , Clonagem Molecular , Ativação do Complemento , Expressão Gênica , Humanos , Hibridomas , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão , TransfecçãoRESUMO
In monocytes, lipopolysaccharide induces synthesis and activity of the 85-kDa cytosolic phospholipase A2. This enzyme releases arachidonic acid and lyso-phospholipids from membranes which are metabolized to eicosanoids and platelet-activating-factor. These lipid mediators increase activity of transcription factors and expression of cytokine genes indicating a function for cytosolic phospholipase A2 in signal transduction and inflammation. We have shown previously that trifluoromethylketone inhibitors of cytosolic phospholipase A2 suppressed interleukin-1beta protein and steady-state mRNA levels in human lipopolysaccharide-stimulated peripheral blood mononuclear leukocytes. In this study, the subcellular mechanisms were analyzed by which trifluoromethylketones interfere with gene expression. We found that they reduced the initial interleukin-1beta mRNA transcription rate through prevention of degradation of inhibitor-kappaB alpha. Consequently, cytosolic activation, nuclear translocation and DNA-binding of nuclear factor-kappaB were decreased. Trifluoromethylketones ameliorate chronic inflammation in vivo. Thus, this therapeutic potency may reside in retention of inactive nuclear factor-kappaB in the cytosol thereby abrogating interleukin-1beta gene transcription.
Assuntos
Hidrocarbonetos Fluorados/farmacologia , Interleucina-1/genética , Cetonas/farmacologia , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Fosfolipases A/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Animais , Citosol/efeitos dos fármacos , Citosol/enzimologia , Sondas de DNA , Citometria de Fluxo , Humanos , Interleucina-1/biossíntese , Interleucina-1/isolamento & purificação , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Fosfolipases A/metabolismo , Fosfolipases A2 , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
To define the isoform of phospholipases A2 active in inflammation we evaluated the effects of low-molecular-weight inhibitors of secretory and cytosolic phospholipases A2. We found that inhibitors of cytosolic phospholipase A2 had therapeutic efficacy in an in vivo model of chronic inflammation (rat adjuvant arthritis), whereas inhibitors of secretory phospholipase A2 had no beneficial effect. In vitro, inhibitors of cytosolic phospholipase A2 diminished surface expression of Mac-1 (CD11b/CD18) beta2-integrin on calcium ionophore-stimulated human blood granulocytes and suppressed synthesis of interleukin-1beta in lipopolysaccharide-stimulated human blood monocytes and U937 cells by reducing mRNA levels. Lipid mediators promote Mac-1 exocytosis and transcription of interleukin-1beta, which further enhances cytosolic phospholipase A2 activity and expression. Thus, superinduction of cytosolic phospholipase A2 may establish a positive feedback loop, converting acute inflammation into chronic inflammation. Consequently, inhibitors of cytosolic phospholipase A2 may prevent inflammation in vivo by interfering with cellular activation and infiltration. We conclude that cytosolic phospholipase A2 but not secretory phospholipase A2 is the predominant enzyme in inflammatory signalling.
Assuntos
Citocinas/biossíntese , Citosol/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inflamação/tratamento farmacológico , Integrinas/biossíntese , Fosfolipases A/antagonistas & inibidores , Animais , Artrite Experimental/tratamento farmacológico , Calcimicina/antagonistas & inibidores , Linhagem Celular , Doença Crônica , Citosol/enzimologia , Depressão Química , Modelos Animais de Doenças , Humanos , Masculino , Peso Molecular , Fosfolipases A2 , Ratos , Ratos Endogâmicos LewRESUMO
Eicosanoids and platelet-activating factor generated upon activation of cytosolic phospholipase A(2) enhance activity of transcription factors and synthesis of proinflammatory cytokines. Here, we show that selective inhibitors and antisense oligonucleotides against this enzyme suppressed expression of the interleukin-1beta gene at the level of transcription and promoter activation in human monocytic cell lines. This inhibitory effect was due to failure of activation of mitogen-activated protein kinases (MAPK) through phosphorylation by upstream mitogen-activated protein kinase kinases (MKK). Consequently, phosphorylation and degradation of inhibitor-kappaBalpha (I-kappaBalpha) and subsequent cytoplasmic mobilization, DNA-binding and the transactivating potential of nuclear factor-kappaB (NF-kB), nuclear factor-interleukin-6 (NF-IL6), activation protein-1 (AP-1) and signal-transducer-and-activator-of-transcription-1 (STAT-1) were impaired. It is concluded, that lipid mediators promote activation of MAPKs, which in turn lead to phosphorylation and liberation of active transcription factors. Since inhibition of cytosolic phospholipase A(2) ameliorates inflammation in vivo, this potency may reside in interference with the MAPK pathway.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/enzimologia , Oligonucleotídeos Antissenso/farmacologia , Fosfolipases A/antagonistas & inibidores , Ácido Araquidônico/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT , Linhagem Celular , Citosol/enzimologia , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Expressão Gênica , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Leucotrieno B4/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/ultraestrutura , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Fator de Ativação de Plaquetas/farmacologia , Fator de Transcrição STAT1 , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
Oligomycin F, a new homologue of oligomycin A (1), was isolated from a Streptomyces species and structure 2 was deduced by NMR methods. Compound 2 is highly active against plant pathogenic fungi and is an extremely potent suppressive agent for various immunological systems.
Assuntos
Imunossupressores/farmacologia , Oligomicinas/química , Oligomicinas/farmacologia , Streptomyces/química , Animais , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fermentação , Humanos , Imunoglobulina G/biossíntese , Imunossupressores/química , Imunossupressores/isolamento & purificação , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Molecular , Oligomicinas/isolamento & purificação , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Células Tumorais CultivadasRESUMO
In HepG2 cells phosphorothioate modified antisense oligonucleotides against a sequence in the Ca2+ binding domain (AS-Ca2+) of type II sPLA2 mRNA restrained IL-6-induced synthesis of sPLA2 protein, sPLA2 mRNA (northern blot), and abolished IL-6 stimulated PGE2 release. An antisense oligonucleotide corresponding to a sequence in the catalytic domain (AS-Cat) of sPLA2 was less effective. The antisense oligonucleotides did not affect albumin synthesis in HepG2 cells, additionally demonstrating their specificity. The corresponding AS-Ca2+ against a homologous part of the rat sPLA2 mRNA depressed rat carrageenin oedema for 60-70%. Identical suppression was achieved by specific low molecular weight inhibitors of sPLA2. Since cyclo- and 5-lipoxygenase inhibitors exerted similar reductions of carrageenin oedema type II sPLA2 dependent eicosanoid formation seems to be a key cascade in this type of inflammation.
Assuntos
Inflamação/prevenção & controle , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/genética , Acetatos/farmacologia , Albuminas/biossíntese , Animais , Sequência de Bases , Benzopiranos/farmacologia , Carragenina/toxicidade , Linhagem Celular , Primers do DNA/genética , Edema/enzimologia , Edema/prevenção & controle , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Inflamação/enzimologia , Interleucina-6/farmacologia , Fosfolipases A2 , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
5-Hydroxy-3-vinyl-2(5H)-furanone, a potent and selective inhibitor of human synovial phospholipase A2 was isolated from fermentations of a Calyptella species. Its structure as identified by spectroscopic methods is identical to PA 147, an antibiotic previously isolated from a streptomycete. 5-hydroxy-3-vinyl-2(5H)-furanone inhibits the aggregation of human and bovine platelets stimulated by different inducers and exhibits weak antimicrobial activities.
Assuntos
4-Butirolactona/análogos & derivados , Basidiomycota , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/isolamento & purificação , Fosfolipases A/antagonistas & inibidores , Inibidores da Agregação Plaquetária/isolamento & purificação , 4-Butirolactona/química , 4-Butirolactona/isolamento & purificação , 4-Butirolactona/farmacologia , Animais , Basidiomycota/crescimento & desenvolvimento , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Bovinos , Linhagem Celular , Citosol/enzimologia , Inibidores Enzimáticos/farmacologia , Fermentação , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Pâncreas/enzimologia , Fosfolipases A2 , Inibidores da Agregação Plaquetária/farmacologia , Venenos de Serpentes , Membrana Sinovial/enzimologia , Trombina/farmacologia , Células Tumorais CultivadasRESUMO
A new inhibitor of leukotriene biosynthesis, (+)-10 alpha-hydroxy-4-muurolen-3-one (1), was isolated from fermentations of Favolaschia sp. 87129. Its structure was established by spectroscopic methods. The compound exhibited no antifungal or antibacterial activities. The effects of 1 on leukotriene biosynthesis were compared with (+)-T-cadinol, (-)-3-oxo-T-cadinol, and (+)-3 alpha-hydroxy-T-cadinol, three related sesquiterpenes.
Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/toxicidade , Leucócitos/metabolismo , Leucotrienos/biossíntese , Polyporaceae , Sesquiterpenos/química , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Cálcio/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dinoprostona/biossíntese , Dinoprostona/sangue , Fermentação , Células HL-60 , Células HeLa , Humanos , Técnicas In Vitro , Leucemia L1210 , Leucócitos/efeitos dos fármacos , Antagonistas de Leucotrienos , Leucotrieno C4/biossíntese , Leucotrieno C4/sangue , Camundongos , Conformação Molecular , Estrutura Molecular , Ratos , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologiaRESUMO
Simultaneous targeting of epidermal growth factor receptor (EGFR) and Met in cancer therapy is under pre-clinical and clinical evaluation. Here, we report the finding that treatment with EGFR inhibitors of various tumor cells, when stimulated with hepatocyte growth factor (HGF) and EGF, results in transient upregulation of phosphorylated AKT. Furthermore, EGFR inhibition in this setting stimulates a pro-invasive phenotype as assessed in Matrigel-based assays. Simultaneous treatment with AKT and EGFR inhibitors abrogates this invasive growth, hence functionally linking signaling and phenotype. This observation implies that during treatment of tumors a balanced ratio of EGFR and Met inhibition is required. To address this, we designed a bispecific antibody targeting EGFR and Met, which has the advantage of a fixed 2:1 stoichiometry. This bispecific antibody inhibits proliferation in tumor cell cultures and co-cultures with fibroblasts in an additive manner compared with treatment with both single agents. In addition, cell migration assays reveal a higher potency of the bispecific antibody in comparison with the antibodies' combination at low doses. We demonstrate that the bispecific antibody inhibits invasive growth, which is specifically observed with cetuximab. Finally, the bispecific antibody potently inhibits tumor growth in a non-small cell lung cancer xenograft model bearing a strong autocrine HGF-loop. Together, our findings strongly support a combination treatment of EGFR and Met inhibitors and further evaluation of resistance mechanisms to EGFR inhibition in the context of active Met signaling.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Fenótipo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Artrite Experimental/enzimologia , Óxido Nítrico/fisiologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Artrite Experimental/fisiopatologia , Dinoprostona/fisiologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Fator de Ativação de Plaquetas/fisiologia , Ratos , Ratos Endogâmicos LewAssuntos
Quimiotaxia de Leucócito/fisiologia , Leucócitos/enzimologia , Leucócitos/fisiologia , Fosfolipases A/fisiologia , Adulto , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Dinoprostona/biossíntese , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Isoenzimas/metabolismo , Leucócitos/efeitos dos fármacos , Leucotrieno B4/biossíntese , Masculino , Pessoa de Meia-Idade , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fosfolipase C beta , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Prostaglandina-Endoperóxido Sintases/metabolismo , Quinacrina/farmacologia , Fluoreto de Sódio/farmacologia , Compostos de Espiro/farmacologia , Terpenos/farmacologia , Fosfolipases Tipo C/metabolismoRESUMO
Phospholipase A2 (PLA2) has been implicated in the pathogenesis of different diseases. Thus, the pharmacological intervention of PLA2 activity by specific inhibitors is of great therapeutical value in ameliorating pathological conditions. Despite a great number of published data regarding PLA2 inhibitors none has reached clinical application. Since enzyme activity can be greatly influenced by the experimental conditions of the test system used, a potent in vitro enzyme inhibitor does not indicate therapeutic effectiveness per se. In order to enhance the predictable value of an in vitro screening system for PLA2 inhibitors, a battery of test systems each measuring certain parameters should be applied. Considering the complex mechanism(s) of PLA2 it is extremely important to elucidate the exact inhibition mechanism of those compounds, which have passed these first filters. True inhibitors of PLA2 should then be evaluated in suitable ex vivo, in vivo models.
Assuntos
Fosfolipases A/antagonistas & inibidores , Fosfolipases/antagonistas & inibidores , Animais , Cloroquina/farmacologia , Clorpromazina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Pâncreas/enzimologia , Fosfolipases A/fisiologia , Fosfolipases A2 , SuínosRESUMO
Coculture of human peripheral blood mononuclear cells with Fc fragments of human IgG, or the synthetic Fc region-derived peptide, p23, results in the release of oxidative products of arachidonic acid. Prostaglandin E was the major arachidonic acid metabolite found in the culture supernatants. Induction of polyclonal antibody production by Fc fragments and p23 is influenced by the concomitant production of prostaglandin E in culture. Addition of prostaglandin synthetase inhibitors, indomethacin and aspirin, to human peripheral blood mononuclear cell cultures resulted in a significant increase in the amount of polyclonal antibody produced. Moreover, addition of exogenous prostaglandin E to these cultures abrogated the ability of indomethacin to enhance Fc fragment-induced polyclonal antibody production. These results suggest that Fc fragments possess bifunctional immunoregulatory properties.
Assuntos
Ácidos Araquidônicos/sangue , Fragmentos Fab das Imunoglobulinas , Fragmentos Fc das Imunoglobulinas , Imunoglobulina G , Monócitos/imunologia , Prostaglandinas E/sangue , Adulto , Formação de Anticorpos/efeitos dos fármacos , Ácido Araquidônico , Adesão Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Dinoprostona , Feminino , Humanos , Cinética , Masculino , Monócitos/citologia , Oxirredução , Prostaglandinas E/farmacologia , RadioimunoensaioRESUMO
Stimulation of peripheral blood leukocytes with lipopolysaccharide results in the synthesis of inflammatory cytokines including interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha and prostaglandin E2 correlating with an increase in phospholipase A2 activity. Mammalian cells contain several phospholipase A2 isoforms including the 14-kDa secretory isoform and the more recently described high-molecular-mass cytosolic isoform. It is commonly believed that during inflammatory responses secretory phospholipase A2 becomes activated. However, we could not detect secretory phospholipase A2 nor its corresponding mRNA after lipopolysaccharide-induced activation. By contrast, we found increased mRNA levels for cytosolic phospholipase A2 following activation of peripheral blood leukocytes when levels were compared to non-stimulated controls. Our results demonstrate that cytosolic phospholipase A2, rather than the secretory isoform may be the mediator of the lipopolysaccharide-induced inflammatory cascade in human peripheral blood leukocytes.
Assuntos
Citocinas/análise , Citosol/enzimologia , Leucócitos/enzimologia , Lipopolissacarídeos/farmacologia , Fosfolipases A/biossíntese , Sequência de Bases , Dinoprostona/análise , Indução Enzimática/efeitos dos fármacos , Humanos , Interleucina-1/análise , Interleucina-6/análise , Isoenzimas/biossíntese , Linfotoxina-alfa/análise , Dados de Sequência Molecular , Fosfolipases A2 , Reação em Cadeia da PolimeraseRESUMO
Lipoprotein from the outer membrane of Escherichia coli constitutes a potent mitogen and polyclonal activator for B lymphocytes of different species. The binding of lipoprotein to murine spleen cells was investigated using water-soluble 125I-labelled citraconylated lipoprotein from E. coli B/r. Our results indicate that the binding of this B-cell mitogen to splenocytes is a saturable, time- and dose-dependent, reversible process; about 9.7 X 10(8) lipoprotein molecules were bound to each cell. The mechanism of the binding of lipoprotein to lymphocytes was investigated by using the synthetic analogue of its N-terminal part, S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-( S)-seryl- (S)-seryl-(S)-asparaginyl-(S)-alanine (tripalmitoyl pentapeptide). This compound had been shown by us previously to be the molecular part of lipoprotein responsible for mitogenicity and exhibited, in all experiments performed, a stimulatory activity towards B lymphocytes comparable, or even superior, to native lipoprotein. Binding proteins for the synthetic N-terminus were enriched by affinity chromatography, using an affinity column prepared by coupling the mitogenic compound to CPG-aminopropyl controlled-pore glass beads by the carbodiimide method. [3H]Leucine-labelled murine spleen cells were solubilized by the nonionic detergent NP40 and applied to the affinity adsorbent. Proteins bound to the column were selectively eluted by a solution of tripalmitoyl pentapeptide, and the fractions were analyzed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and autoradiography. Our results indicate the presence of a major binding protein of Mr 35000 on mouse primary lymphocytes for the biologically active N-terminal structure of lipoprotein, which might play a role as membrane receptor in mitogenic B lymphocyte activation.
Assuntos
Linfócitos B/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Mitógenos/metabolismo , Oligopeptídeos/metabolismo , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Lipopeptídeos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Fotofluorografia , Baço/metabolismoRESUMO
Lipoprotein from the outer membrane of Escherichia coli and a synthetic analogue of its N-terminal lipopeptide part, tripalmitoyl-pentapeptide, constitute potent mitogens and polyclonal activators of murine B-lymphocytes in vitro. When entering the circulation after intravenous administration in experimental animals, they interact with the humoral and cellular elements of the blood, which results in splenomegaly and B-lymphocyte activation in vivo. We investigated lipopeptide-binding proteins in normal mouse serum and on splenocytes. By affinity chromatography using an affinity adsorbent prepared by coupling the lipoprotein analogue to CPG-aminopropyl derivatized glass beads, we could enrich one major binding protein for tripalmitoyl-pentapeptide from mouse serum, which was identified as albumin. Binding proteins on lymphocytes were determined as follows: Spleen cells of C3H/HeJ mice were activated by the B cell mitogen lipoprotein, biosynthetically labelled with [3H]leucine, and solubilized by the nonionic detergent Nonidet P40. From the cell lysate, binding proteins were isolated by affinity chromatography: As analysed by polyacrylamide gel electrophoresis and autoradiography, proteins with molecular masses of 24, 27, 33, 45, 53, 61 and 71 kDa were eluted from the tripalmitoyl-pentapeptide adsorbent. The eluted material was further enriched for glycoproteins by Lens culinaris lectin affinity chromatography, and immunoprecipitation studies were performed with the glycoprotein fractions using alloantisera specific for class I and class II gene products of the H-2k haplotype. We could show that both class I and class II MHC glycoproteins could be enriched on the tripalmitoyl-pentapeptide column. This finding might suggest that, among other proteins, MHC-encoded proteins are involved in lymphocyte activation by a mitogenic lipopeptide.
Assuntos
Antígenos H-2/isolamento & purificação , Mitógenos/metabolismo , Oligopeptídeos/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Antígenos H-2/genética , Técnicas In Vitro , Lipopeptídeos , Lipoproteínas/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C3H , Baço/imunologia , Baço/metabolismoRESUMO
Prostaglandins (PG) have been implicated as modulators of both humoral and cellular immune responses. In order to evaluate a possible role for PG in tolerance, the effect of inhibitors of prostaglandin synthesis on tolerance induction and circumvention has been investigated. Injection of deaggregated human gamma-globulin (DHGG) into A/J mice leads to unresponsiveness to a subsequent challenge with immunogenic aggregated human gamma-globulin (AHGG). Administration of indomethacin (IM) or acetylsalicylic acid (ASA) shortly before and after DHGG injection prevents tolerance induction. PGE2 reverses the tolerance overriding effect provided by IM. IM is not able to overcome unresponsiveness when given 10 and 20 days after tolerance induction, at a time point when both T and B lymphocytes are tolerant. As previously shown, lipopolysaccharide (LPS) both inhibits the induction of tolerance to HGG and circumvents tolerant T helper cells late in tolerance when competent B cells are present. In contrast, IM is unable to circumvent T-helper cell tolerance when given at Day 60 after tolerogen, when B cells (but not T cells) are responsive. Furthermore, LPS acts as an adjuvant, B-cell mitogens, inducer of polyclonal Ig secretion, and primes mice when given with tolerogen, while IM has none of these properties. These results indicate a difference between the effects of IM and LPS on tolerance and a possible role of PG in DHGG-mediated tolerance induction.
Assuntos
Aspirina/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Indometacina/farmacologia , Adjuvantes Imunológicos , Animais , Formação de Anticorpos/efeitos dos fármacos , Dinoprostona , Masculino , Camundongos , Camundongos Endogâmicos A , Prostaglandinas E/farmacologia , Linfócitos T/imunologia , Fatores de Tempo , gama-Globulinas/imunologiaRESUMO
Previous data from this laboratory indicated that human Fc gamma fragments induce murine B cells to proliferate and that the induction is macrophage-dependent. To further investigate the role of macrophages in this phenomenon, biologically active Fc gamma fragments from a human IgG1 myeloma protein and the murine macrophage-like cell line P388D1 were utilized. Fc gamma 1 fragments bound specifically and to a single class of receptor on P388D1 cells with a Ka value of 4 X 10(6) M-1 and to approximately 2.4 X 10(5) binding sites/cell. The binding was not effectively inhibited by two immunostimulatory Fc gamma 1 subfragments that were macrophage independent, i.e., pFc' fragments approximating the C gamma 3 domain of IgG1 and synthetic peptides representing residues 335-357 in IgG1. P388D1 cells were able to process Fc gamma 1 fragments but not intact IgG1 into subfragments that were able to induce lymphocyte proliferation in the absence of macrophages. The processing was rapid and resulted in active subfragments of several size classes. These findings not only further document the molecular and cellular events in these systems but underscore the usefulness of the P388D1 cell line in future studies on Fc fragment-induced lymphocyte regulation.
Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Macrófagos/metabolismo , Animais , Especificidade de Anticorpos , Linhagem Celular , Cinética , Ativação Linfocitária , Camundongos , Receptores Fc/antagonistas & inibidores , Receptores Fc/metabolismo , Baço/citologiaRESUMO
The capacity of the C8-substituted guanine ribonucleosides to enhance the in vivo humoral immune response to the protein antigen, human gamma globulin (HGG), in A/J mice was evaluated. It has been shown recently that the C8-substituted guanine ribonucleosides are a new class of potent adjuvant for humoral immune responses to the sheep erythrocyte antigen. The current studies extend these findings to HGG with 8-bromoguanosine (8BrGuo), a representative of this group of nucleosides. The adjuvant activity of 8BrGuo in this system is highly dose and time dependent. Although 8BrGuo enhanced responses when injected either early (Day 0) or late (Day 4 or 5) after immunization, its administration on Day 1 or 2 most often led to no enhancement, suggesting that 8BrGuo may act on two events separated by a resistant stage in an ongoing immune response. The plaque-forming cell (PFC) response to HGG was enhanced optimally at doses as low as 1 mg 8BrGuo/mouse administered either on the day of immunization or 4 days thereafter. In contrast, however, serum anti-HGG antibody concentration assayed by enzyme-linked immunosorbent assay (ELISA) was enhanced only at doses of 10 mg or more, injected on the day of immunization, but doses as low as 1 mg were effective on Day 4. 8BrGuo was also an effective adjuvant when injected after antigen administration in incomplete Freund's adjuvant or when administered by several different routes (intraperitoneal, subcutaneous, oral).