RESUMO
BACKGROUND: In a subset of patients, anti tumour necrosis factor (TNF) therapeutic antibodies are immunogenic, resulting in the formation of antidrug antibodies (ADAs). Neutralising ADAs compete with TNF for its binding site and reduces the effective serum concentration, causing clinical non-response. It is however unknown to which extent ADAs are neutralising. OBJECTIVES: To study which proportion of antibodies to human(ised) anti-TNF (adalimumab, golimumab, certolizumab) as well as chimeric anti-TNF (infliximab) is neutralising. METHODS: Neutralising capacity of ADAs was assessed using a TNF competition assay in ADA-positive sera of patients treated with adalimumab (n=21), golimumab (n=4), certolizumab (n=9) or infliximab (n=34) sent in to our diagnostic department. RESULTS: In 34 sera with ADAs to adalimumab, golimumab or certolizumab, >97% of the antibodies were neutralising. In 34 sera with ADAs to infliximab >90% of the antibodies were neutralising. Further characterisation of the broader antibody response to infliximab revealed that non-neutralising antibodies to infliximab do not target murine domains, but may bind infliximab-unique domains not involved in TNF binding (located outside the paratope). CONCLUSIONS: Our study shows that ADAs to human(ised) as well as chimeric anti-TNF therapeutic antibodies are largely neutralising. This highly restricted ADA response suggests an immunodominant role for the paratope of anti-TNF therapeutics.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/imunologia , Antirreumáticos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Adalimumab , Anticorpos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos , Certolizumab Pegol , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Infliximab , Polietilenoglicóis , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
The pleiotropic cytokine interleukin 6 (IL-6) plays a role in the pathogenesis of various diseases, such as multiple myeloma, autoimmune and inflammatory diseases and osteoporosis. Therefore, specific inhibitors of IL-6 may have clinical applications. We previously succeeded in developing receptor antagonists of IL-6 that antagonized wild-type IL-6 activity on the human Epstein-Barr virus (EBV)-transformed B cell line CESS and the human hepatoma cell line HepG2. However, these proteins still had agonistic activity on the human myeloma cell line XG-1. We here report the construction of a novel mutant protein of IL-6 in which two different mutations are combined that individually disrupt the association of the IL-6/IL-6 receptor (R) alpha complex with the signaltransducing "beta" chain, gp130, but leave the binding of IL-6 to IL-6R alpha intact. The resulting mutant protein (with substitutions of residues Gln160 to Glu, Thr163 to Pro, and replacement of human residues Lys42-Ala57 with the corresponding residues of mouse IL-6) was inactive on XG-1 cells and weakly antagonized wild-type IL-6 activity on these cells. By introducing two additional substitutions (Phe171Leu, Ser177Arg), the affinity of the mutant protein for IL-6R alpha was increased fivefold, rendering it capable of completely inhibiting wild-type IL-6 activity on XG-1 cells. Moreover, this mutant also antagonized the activity of IL-6, but not that of leukemia inhibitory factor, oncostatin M, or GM-CSF on the human erythroleukemia cell line TF-1, demonstrating its specificity for IL-6. These data demonstrate the feasibility of developing specific IL-6R antagonists. The availability of such antagonists may offer an approach to specifically inhibit IL-6 activity in vivo.
Assuntos
Interleucina-6/farmacologia , Mieloma Múltiplo/imunologia , Receptores de Interleucina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Sequência de Bases , Carcinoma Hepatocelular , Divisão Celular , Linhagem Celular Transformada , Primers do DNA , Herpesvirus Humano 4/genética , Humanos , Interleucina-6/análogos & derivados , Interleucina-6/biossíntese , Interleucina-6/metabolismo , Cinética , Neoplasias Hepáticas , Camundongos , Dados de Sequência Molecular , Mieloma Múltiplo/patologia , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Receptores de Interleucina/fisiologia , Receptores de Interleucina-6 , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
During human immunodeficiency virus infection and allergic diseases, characterized by a dominant T helper (Th) 2 response, overproduction of prostaglandin E2 (PGE2) is observed. In this paper we studied the effect of PGE2 on interleukin (IL)-12 synthesis, because this cytokine has been described to be essential in induction of Th1 responses. IL-12 synthesis was induced in monocytes that were stimulated with Neisseria meningitidis-derived lipopolysaccharide in whole blood cultures. PGE2 almost completely inhibited lipopolysaccharide induced IL-12 production, whereas IL-6 production was only partially inhibited by PGE2. In contrast, the production of IL-10 was approximately twofold enhanced at these conditions. The effects of PGE2 were due to its cAMP-inducing capacity, since they could be mimicked by other cAMP inducers. Recombinant human IL-10 also inhibited IL-12 and IL-6 production. However, the inhibitory effect of PGE2 on IL-12 production was independent of IL-10 since neutralizing anti-IL-10 antibodies were unable to reverse this inhibition. These results suggest that the capacity of an antigen to induce PGE2 synthesis may play a crucial role in the development of either a Th1 or Th2 response.
Assuntos
Dinoprostona/farmacologia , Interleucina-12/antagonistas & inibidores , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Humanos , Técnicas In Vitro , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Interleucina-6/biossíntese , Lipopolissacarídeos/farmacologiaRESUMO
BACKGROUND: Immunogenicity, specifically the onset of antibodies against tumour necrosis factor (TNF) blocking agents, seems to play an important role in non-response to treatment with these drugs. OBJECTIVES: To assess the relation of clinical response of ankylosing spondylitis (AS) to etanercept with etanercept levels, and the presence of antibodies to etanercept. METHODS: Patients with AS were treated with etanercept 25 mg twice weekly, according to the international Assessment in Ankylosing Spondylitis (ASAS) working group consensus statement. Sera were collected at baseline and after 3 and 6 months of treatment. Clinical response was defined as a 50% improvement or as an absolute improvement of 2 points on a (0-10 scale) Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score. Functional etanercept levels were measured by a newly developed ELISA, measuring the binding of etanercept to TNF. Antibodies against etanercept were measured with a two-site assay and antigen binding test. Clinical data were used to correlate disease activity with serum etanercept levels. RESULTS: In all, 53 consecutive patients were included. After 3 months of treatment 40 patients (76%) fulfilled the response criteria. Mean etanercept levels were 2.7 mg/litre and 3.0 mg/litre after 3 and 6 months respectively. Characteristics and etanercept levels of responders and non-responders were similar. No antibodies to etanercept were detected with any of the assays. CONCLUSION: Etanercept levels of responders and non-responders were similar and no antibodies to etanercept were detected with any of the assays. This study indicates that etanercept is much less immunogenic compared with the other TNF-blocking agents.
Assuntos
Antirreumáticos/uso terapêutico , Imunoglobulina G/uso terapêutico , Receptores do Fator de Necrose Tumoral/uso terapêutico , Espondilite Anquilosante/tratamento farmacológico , Espondilite Anquilosante/imunologia , Adulto , Anticorpos/sangue , Reações Antígeno-Anticorpo , Antirreumáticos/sangue , Antirreumáticos/imunologia , Etanercepte , Feminino , Seguimentos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores do Fator de Necrose Tumoral/sangue , Receptores do Fator de Necrose Tumoral/imunologia , Resultado do TratamentoRESUMO
BACKGROUND AND OBJECTIVE: Smoking is associated with increased severity of periodontitis. The underlying mechanisms of this phenomenon are not well understood. The purpose of the present study was to compare the monocyte-derived T cell directing (Th1/Th2) response and pro-inflammatory cytokine production in ex vivo whole blood cell cultures (WBCC) of smoking and non-smoking chronic periodontitis patients. MATERIAL AND METHODS: Venous blood was collected from 29 periodontitis patients (18 non-smokers and 11 smokers) receiving supportive periodontal treatment, and diluted 10-fold for WBCC. The WBCC were stimulated for 18 h with Neisseria meningitidis lipo-oligosaccharide (LOS) or Porphyromonas gingivalis sonic extract (Pg-SE). The production of the T cell directing cytokines interleukin (IL)-12 p40 and IL-10, as well as the pro-inflammatory cytokines IL-1beta, IL-6 and IL-8, was measured in the culture supernatants. RESULTS: After LOS stimulation of WBCC, smokers showed a lower IL-12 p40/IL-10 ratio than non-smokers (P < 0.05). Interleukin-1beta production was significantly lower in smokers compared with non-smokers after stimulation with either LOS or Pg-SE (P < 0.05). Interleukin-6 and IL-8 production was similar in WBCC from both smokers and non-smokers, for both LOS and Pg-SE. CONCLUSION: A more pronounced Th2 response in smoking periodontitis patients may be related to increased severity of the disease.
Assuntos
Periodontite Crônica/imunologia , Citocinas/imunologia , Fumar/imunologia , Adulto , Perda do Osso Alveolar/sangue , Perda do Osso Alveolar/imunologia , Técnicas de Cultura de Células , Linhagem Celular , Periodontite Crônica/sangue , Citocinas/sangue , Feminino , Humanos , Mediadores da Inflamação/imunologia , Interleucina-10/análise , Subunidade p40 da Interleucina-12/análise , Interleucina-1beta/análise , Interleucina-6/análise , Interleucina-8/análise , Lipopolissacarídeos/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Neisseria meningitidis/imunologia , Porphyromonas gingivalis/imunologia , Fumar/sangue , Frações Subcelulares/imunologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
OBJECTIVE: To investigate the in vivo mechanism of non-responding to infliximab treatment of patients with rheumatoid arthritis (RA) and the role of anti-infliximab antibodies by using radiolabeled infliximab. DESIGN: Descriptive and comparative study. METHOD: Two responding and two non-responding RA patients were infused with radiolabeled infliximab. Subsequently imaging investigations and serum analysis were performed at set times. RESULTS: The scintigrams showed that the labelled infliximab was mainly present in the blood until 24 h after infusion. There was a trend of faster blood clearance and higher liver and spleen uptake of 99mTc-infliximab in one non-responding patient. Labelled infliximab was taken up by inflamed joints. The anti-infliximab level was high (1008 and 1641 U/ml) in the non-responders and low or not detectable in the responders. Sucrose gradients of serum revealed antibody complexes in both non-responders. Various sizes of antibody complexes, including very large ones, were observed in one non-responder who developed a serious infusion reaction. CONCLUSION: Infliximab-anti-infliximab immune complexes were found to form in RA non-responders due to the presence of significant quantities of anti-infliximab. This finding may partly explain the failure of the infliximab treatment.
RESUMO
In vitro as well as in vivo observations have shown that IL6 plays a key role in the pathogenesis of multiple myeloma. Therefore we started a phase I/II dose escalating study with chimeric monoclonal anti-IL6 antibodies (cMab) in multiple myeloma (MM) patients resistant to second-line chemotherapy. Here we describe the pharmacological data as well as a new method for calculating the endogenous IL6 production. The cMab (CLB IL6/8; Kd: 6.25 x 10(-12) M) was given in two cycles of 14 daily infusions, starting on day 1 and day 28. Daily dose: 5 mg in patients 1-3, 10 mg in patients 4-6, and 20 mg in patients 7-9 (total dose 140, 280, and 560 mg of anti-IL6, respectively). Using the pharmacokinetic data of free IL6 and the binding characteristics of the cMab, the endogenous IL6 production could be calculated from day to day using a one-compartment open model. The median half-life time of this antibody was 17.6 d. No human antichimeric antibodies were induced. Pre-treatment median endogenous IL6 production in the MM patients was 60 micrograms/d (range 13.8-230; normal controls < 7 micrograms/d). During treatment with anti-IL6 cMabs, the endogenous IL6 production immediately decreased in all patients to below 3 micrograms/d and never reached the pre-treatment value during the treatment period, except in two patients who developed an active infection, resulting in an IL6 production of 128 and 1,208 micrograms/d, respectively. We concluded that in MM patients endogenous IL6 production is 2-30 times higher than in healthy individuals. The anti-IL6 cMab strongly suppress this endogenous IL6 production, probably by blocking a positive feed-back loop, but this cMab does not prevent infection-induced IL6 production. The chimeric anti-IL6 Mabs have a long half-life time, a low immunogenicity, and are able to block IL6-dependent processes in vivo.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Interleucina-6/biossíntese , Interleucina-6/imunologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/terapia , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Interleucina-6/farmacocinética , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
An allotypic form of the low affinity IgG Fc receptor Fc gamma RIIa (CD32), termed low responder (LR) because of its weak reactivity with mouse (m) IgG1, interacts efficiently with human (h) IgG2. Fc gamma RIIaLR is the first known human FcR that binds this IgG subclass. In this study, we analyzed the role of Fc gamma RIIa in binding of stable hIgG-subclass dimers, and in induction of T cell mitogenesis using chimeric anti-CD3 mAb. We demonstrate that the functional polymorphism to hIgG2 is expressed on the majority of Fc gamma R-bearing peripheral blood cells: monocytes, neutrophils, and platelets. We were able to assess Fc gamma RII-mediated IgG-binding without interference of other Fc gamma R-classes, by blockade of Fc gamma RI on monocytes, and by using neutrophils of an individual deficient for the Fc gamma RIIIB gene. This study indicates as subclass specificity: hIgG3 >hIgG1,hIgG2 >> hIgG4 for Fc gamma RIIaLR and hIgG3,hIgG1 >> hIgG2 > hIgG4 for Fc gamma RIIaHR. Comparing the serum hIgG levels of individuals homozygous for the two fc gamma RIIa allotypic forms, we observed significantly lower hIgG2 serum levels in individuals expressing the hIgG2-binding LR allotypic form. This observation may implicate that Fc gamma RIIa regulates hIgG subclass production or turnover in man.
Assuntos
Plaquetas/imunologia , Imunoglobulina G/fisiologia , Monócitos/imunologia , Neutrófilos/imunologia , Receptores de IgG/fisiologia , Anticorpos Monoclonais/imunologia , Complexo CD3/fisiologia , Glicosilfosfatidilinositóis/fisiologia , Humanos , Imunoglobulina G/classificação , Ativação Linfocitária , Linfócitos T/imunologiaRESUMO
IL-12 is essential for T helper 1 (Th1) development and inhibits the induction of Th2 responses. Atopic diseases, which are characterized by Th2 responses, are associated with the overproduction of histamine. Here we present evidence that histamine, at physiological concentrations, strongly inhibits human IL-12 p40 and p70 mRNA and protein production by human monocytes. The use of specific histamine receptor antagonists reveals that this inhibition is mediated via the H2 receptor and induction of intracellular cAMP. The inhibition of IL-12 production is independent of IL-10 and IFN-gamma. The observation that histamine strongly reduces the production of the Th1-inducing cytokine IL-12 implies a positive feedback mechanism for the development of Th2 responses in atopic patients.
Assuntos
Histamina/farmacologia , Interleucina-12/metabolismo , Monócitos/imunologia , Dinoprostona/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores H2 da Histamina/farmacologia , Humanos , Imidazóis/farmacologia , Interleucinas/metabolismo , Monócitos/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ranitidina/farmacologia , Receptores Histamínicos H2/efeitos dos fármacos , Receptores Histamínicos H2/metabolismo , Tioureia/análogos & derivados , Tioureia/farmacologia , Triprolidina/farmacologiaRESUMO
Interleukin 6 (IL-6) serves as a growth factor for mouse plasmacytomas. As a model for IL-6-mediated growth of plasmacytomas, we study IL-6-dependent B-cell hybridomas, which can be generated through fusion of B lymphocytes with a plasmacytoma cell line, e.g., SP2/0. In the present report, we have investigated the peculiar behavior of B-cell hybridomas with respect to IL-6 dependence. We demonstrate that although newly generated hybridomas are IL-6 dependent, many hybridomas lose this dependency at frequencies as high as 50%, shortly after fusion. We speculated that the loss of IL-6-dependent growth is due to the well-known chromosomal instability of B-cell hybridomas. Consequently, loss of IL-6 dependence is the result of loss of a specific chromosome(s). This model implies the existence of an "IL-6 dependency" gene, the loss of which makes hybridomas capable of proliferating in the absence of IL-6. Because SP2/0 is IL-6 independent, the IL-6-dependent phenotype of B-cell hybridomas, and hence the IL-6 dependency gene, must be derived from the B lymphocyte. We have tested this model by generating human/mouse B-cell hybridomas through fusion of human B lymphocytes with SP2/0. We then analyzed the human chromosome content of 10 IL-6-dependent and 14 IL-6-independent subclones. From that analysis we concluded that the presence of human chromosome 21 correlated with IL-6 dependence. This correlation was confirmed by microcell fusion experiments in which a single copy of chromosome 21 was introduced into IL-6-independent hybridomas, resulting in reconstitution of the IL-6-dependent phenotype. We therefore conclude that chromosome 21 carries an IL-6 dependency gene.
Assuntos
Cromossomos Humanos Par 21/fisiologia , Hibridomas , Interleucina-6/genética , Animais , Linfócitos B , Divisão Celular/genética , Cromossomos Humanos Par 21/genética , Feminino , Humanos , Hibridomas/citologia , Interleucina-6/fisiologia , Cariotipagem , Camundongos , FenótipoRESUMO
Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.
Assuntos
Proteína C-Reativa/biossíntese , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Fígado/metabolismo , Proteína Amiloide A Sérica/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Anticorpos Monoclonais , Células Cultivadas , Dexametasona/farmacologia , Humanos , Interleucina-1/antagonistas & inibidores , Interleucina-6/antagonistas & inibidores , Interleucina-6/imunologia , Cinética , Fígado/efeitos dos fármacos , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Interleukin 6 (IL-6) exerts well-established effects on cells of the immune system as well as on various other cell types. It has been implicated in the control of connective tissue cells in such conditions as rheumatoid arthritis and osteoporosis. We have investigated the effects of recombinant human interleukin-6 (rhIL-6) on human osteoblastlike cells derived from explants of trabecular bone. ROS 17/2.8 cells were used as an additional osteoblastlike cell model system. We were unable to identify any effects of rhIL-6 (5-5000 pg/ml) on the proliferation, alkaline phosphatase activity. osteocalcin production, or release of cytokines or prostaglandins by either osteoblastlike cell model system. Since we have shown previously that these cells release IL-6 in culture, we used a sheep anti-human IL-6 antibody to investigate the possibility that (1) action of added exogenous IL-6 could be masking endogenous production, and (2) endogenous IL-6 may regulate the effects of osteotropic agents on the osteoblastlike cells. Presence of the antibody exerted no detectable effects on 1,25-(OH)2D3-stimulated alkaline phosphatase or on proliferation or TNF production enhanced by IL-1. Thus IL-6 does not appear to be involved in the regulation of osteoblast activity.
Assuntos
Interleucina-6/fisiologia , Osteoblastos/fisiologia , Fosfatase Alcalina/metabolismo , Biomarcadores/química , Northern Blotting , Divisão Celular/fisiologia , Células Cultivadas , Dinoprostona/biossíntese , Humanos , Osteocalcina/metabolismo , RNA/isolamento & purificação , Receptores Imunológicos/metabolismo , Receptores de Interleucina-6 , Fator de Necrose Tumoral alfa/biossínteseRESUMO
A monoclonal antibody directed against bovine TSH was obtained by hybridoma technology. This antibody was specific for TSH and did not react with bovine LH and FSH. Affinity chromatography of crude TSH was performed on anti-TSH Sepharose. Bovine TSH was purified in a single step to near homogeneity by this technique, as shown by cation exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified TSH. The biological activity of the hormone was not affected during the purification, as determined by [3H]thymidine incorporation of the TSH-dependent FRTL5 cell line. The results indicate that affinity purification of TSH by means of a monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified TSH.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Tireotropina/imunologia , Animais , Complexo Antígeno-Anticorpo/análise , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Radioisótopos do Iodo , Camundongos , Camundongos Endogâmicos BALB C , Glândula Tireoide , Tireotropina/farmacologiaRESUMO
We have previously shown that L58 in the putative 5th helical region of human interleukin-6 (IL-6) is important for activation of the IL-6 signal transducer gp130 [de Hon et al. (1995) FEBS Lett. 369, 187-191]. To further explore the importance of individual residues in this region for gp130 activation we have now combined Ala substitutions of residues E52, S53, S54, K55, E56, L58 and E60 with other substitutions in IL-6, known to affect gp130 activation (Q160E and T163P). The combination mutant protein with L58A completely lost the capacity to induce the proliferation of XG-1 myeloma cells, and could effectively antagonize wild type IL-6 activity on these cells. Moreover, the data suggest that besides L58, S54 particularly, but also E52, S53, K55 and E56 contribute to gp130 activation.
Assuntos
Antígenos CD/fisiologia , Interleucina-6/química , Interleucina-6/farmacologia , Glicoproteínas de Membrana/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alanina , Antígenos CD/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Clonagem Molecular , Receptor gp130 de Citocina , Escherichia coli , Humanos , Cinética , Leucemia Eritroblástica Aguda , Glicoproteínas de Membrana/efeitos dos fármacos , Modelos Moleculares , Mieloma Múltiplo , Mutagênese Sítio-Dirigida , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
A model of the tertiary structure of human IL-6, derived from the crystal-structure of granulocyte-colony stimulating factor, reveals a 5th helical region in the loop between the first and second alpha-helix. To investigate the importance of this region for biological activity of IL-6, residues Glu-52, Ser-53, Ser-54, Lys-55, Glu-56, Leu-58, and Glu-60 were individually replaced by alanine. IL-6.Leu-58Ala displayed a 5-fold reduced biological activity on the IL-6 responsive human cell lines XG-1 and A375. This reduction in bioactivity was shown to be due to a decreased capacity of the mutant protein to trigger IL-6 receptor-alpha-chain-dependent binding to the IL-6 signal transducer, gp130.
Assuntos
Antígenos CD , Interleucina-6/fisiologia , Leucina/fisiologia , Glicoproteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Divisão Celular , Receptor gp130 de Citocina , Humanos , Hibridomas , Interleucina-6/química , Interleucina-6/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6 , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
The sensitivity and the specificity of the Farr assay for the detection of antibodies to double stranded (ds) DNA depends very much on the reaction conditions. The interaction between ds DNA and anti-ds DNA is inhibited when ionic strength and pH are increased. ds DNA is bound by normal sera at ionic strength lower than 0.11 M NaCl and at physiological ionic strength when the pH is lower than 7.2. Substantial binding of DNA by normal serum takes place in barbitone, borate or Tris-HCl buffers at concentrations of 30 mM or higher, even at a pH higher than 7.2. Such binding is due to Clq and is only partially prevented by heating the serum for 30 min at 56 degrees C, but 10 mM phosphate in the incubation mixture completely prevents it. Standardization of ionic strength, pH, phosphate concentration, incubation volume and DNA-serum ratio enhances the diagnostic usefulness of the Farr assay.
Assuntos
Anticorpos/análise , DNA/imunologia , Sítios de Ligação de Anticorpos , Soluções Tampão , Humanos , Concentração de Íons de Hidrogênio , Lúpus Eritematoso Sistêmico/imunologia , Concentração Osmolar , Fosfatos/metabolismo , Radioimunoensaio , Cloreto de Sódio/farmacologiaRESUMO
Reduction of IgM antibodies to DNA with mercaptans such as dithioerythrol or 2-mercaptoethanol completely destroys DNA binding in the Farr assay and in the immunofluorescence technique by Crithidia luciliae. In contrast reduction of IgG antibodies to DNA results in a six-fold increase of DNA binding in the Farr assay while no effect on titres in the immunofluorescence technique can be observed. Our results lead to the following conclusions: 1) The Farr assay is selective for high avidity interactions; only a minor part of IgG antibodies to DNA is measured; 2) 7S IgM antibodies to DNA cannot be demonstrated in the Farr assay or the immunofluorescence technique; obviously only multivalent interactions, as obtained with the intact 19S IgM molecule are stable in these assays; 3) reduction of IgG leads to a greater flexibility of this molecule; this facilitates monogamous bivalent binding to DNA; 4) THE PRESENTATION OF DNA in the kinetoplast of Crithidia luciliae favours a monogamous bivalent binding of antibodies to DNA with high avidity; this accounts for occasionally observed discrepancies between anti-DNA activity in the Farr assay and in the immunofluorescence technique.
Assuntos
Anticorpos , DNA/imunologia , Ditioeritritol/farmacologia , Ditiotreitol/análogos & derivados , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Mercaptoetanol/farmacologia , Formação de Anticorpos , Sítios de Ligação de Anticorpos , Eucariotos/imunologia , Imunofluorescência , Humanos , Técnicas Imunológicas , Penicilamina/farmacologiaRESUMO
A series of heavy chain isotype switch variants was derived from a hybridoma cell line secreting monoclonal antibodies specific for horseradish peroxidase. By the combined use of sensitive isotype-specific ELISAs and sequential sublining IgG2b, IgG2a, IgE and Iga anti-peroxidase-producing variants were successively isolated out of IgG1-secreting parental cells. The anti-peroxidase isotype variant antibodies are particularly appropriate for use in studies of the influence of heavy chain isotype in the effector functions of immunoglobulins. The use of variant antibodies with specificity for an enzyme favors their application in immunoassays because an enzyme-conjugated second antibody is not needed. Here we describe two applications of the anti-peroxidase switch variants. First, the variants are compared with respect to their affinity for Staphylococcus protein A. While IgG1 anti-peroxidase showed weak binding, both IgG2 variants strongly bound to protein A, whereas IgE and IgA variants had no affinity for protein A. Next, the switch variants were used to determine the isotype specificity of rat monoclonal antibodies generated to murine IgE.
Assuntos
Anticorpos Monoclonais/imunologia , Peroxidase do Rábano Silvestre/imunologia , Isotipos de Imunoglobulinas/imunologia , Região de Troca de Imunoglobulinas , Peroxidases/imunologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Hibridomas/imunologia , Imunoglobulina E/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Isotipos de Imunoglobulinas/genética , Masculino , Camundongos , Mutação , Ratos , Ratos Endogâmicos , Proteína Estafilocócica A/imunologiaRESUMO
We have investigated whether the Farr assay measures the primary interaction between DNA and anti-DNA. By the use of zonal centrifugation to analyse DNA/anti-DNA complexes and by a detailed study of the quantitative aspects of the Farr assay we demonstrate that: (a) binding of a single IgG molecule to a PM2 DNA molecule (molecular weight 5.9 X 10(6)) renders the latter precipitable in half-saturated ammonium sulphate; (b) two antibody molecules are required for precipitation of T7 DNA (molecular weight 25 X 10(6)); (c) millipore filtration detects binding of a single antibody molecule per molecule of PM2 DNA; (d) theoretically and experimentally the relation between free DNA (F) and the amount of serum (S) can be described by IfF =-cS where c is a constant; (e) it is feasable to quantitate serum anti-DNA activity in terms of units per ml.