RESUMO
With the increasing cost and complexity of drug development, biomarkers will play an increasing role in the early phases. Biomarkers can be classified into target, mechanistic, or outcome with varying degrees of linkage to disease or treatment effect. They can be used to determine proof of concept by characterising the efficacy or safety profiles, or determining differentiation from any competitor drugs. PK/PD modelling of biomarker data for novel and marketed compounds can be used to predict outpatient dose response. Subsequent simulations may replace or reduce the size and cost of larger phase 2b outpatient studies. Two examples of biomarkers and PK/PD modelling used to characterise dose response are presented. Penile plethysmography (RigiScan Plus) in male erectile dysfunction and phenylephrine challenge urethral pressure in benign prostatic hyperplasia are used to reduce time and cost to reach major exploratory development decision points in these indications.
Assuntos
Biomarcadores/química , Avaliação Pré-Clínica de Medicamentos , Pressão Sanguínea , Computadores , Tomada de Decisões , Desenho de Fármacos , Desenho de Equipamento , Disfunção Erétil/diagnóstico , Humanos , Masculino , Fenilefrina/química , Pressão , Uretra/patologia , Sistema Urogenital/efeitos dos fármacosRESUMO
Two-dimensional (2-D) gel analysis was used to examine differences in the levels of 19 plasma proteins: before and after an acute inflammatory reaction (parenteral typhoid vaccination) in normal subjects, between rheumatoid arthritis (RA) patients and normals and in RA patients treated with tenidap (120 mg) and piroxicam (20 mg). Typhoid vaccination increased levels of SAA, haptoglobin alpha1, haptoglobin alpha2, haptoglobin beta and alpha1-anti-chymotrypsin but decreased transthyretin and apolipoprotein E. In RA patients, serum amyloid A (SAA), haptoglobin alpha2, haptoglobin beta, alpha1-antichymotrypsin and C3 proactivator levels were elevated while apolipoprotein A-I, apolipoprotein A-IV, transthyretin, Gc-globulin, alpha2-HS glycoprotein, alpha2-macroglobulin and alpha1-B glycoprotein levels were decreased, compared to normals. Compared to piroxicam, tenidap lowered levels of alpha1-antiprotease and SAA but raised the levels of transthyretin, Gc-globulin, alpha2-HS-glycoprotein and alpha2-macroglobulin in RA patients. C-reactive protein (CRP) could not be quantified on 2-D gels but, when measured by rate nephelometry, levels were reduced after treatment with tenidap compared to piroxicam. The general pattern of the acute phase protein response to an acute inflammatory response to typhoid vaccination is similar to that in the chronic inflammatory condition, RA. The impact of tenidap on both positive and negative acute-phase proteins in RA patients could clearly be distinguished from that of piroxicam.
Assuntos
Proteínas de Fase Aguda/análise , Artrite Reumatoide/imunologia , Proteínas Sanguíneas/análise , Eletroforese em Gel Bidimensional/métodos , Adulto , Fatores Etários , Anti-Inflamatórios não Esteroides/farmacologia , Feminino , Humanos , Indóis/farmacologia , Inflamação , Masculino , Oxindóis , Piroxicam/farmacologia , Fatores Sexuais , Vacinas Tíficas-Paratíficas/imunologiaRESUMO
This study evaluated measures of metalloproteinase gene expression in synovial biopsies as a means of differentiating the activities of two antirheumatic therapies: piroxicam and tenidap. Synovial biopsies and quantitative in situ hybridization for stromelysin (STR), collagenase (COL), tissue inhibitor of metalloproteinase-1 (TIMP), and actin mRNA were performed in a subset of patients with rheumatoid arthritis in a larger doubleblind randomized crossover trial comparing 120 mg/day of tenidap for 6 weeks with 20 mg/day of piroxicam for 6 weeks. There were no consistent differences between tenidap and piroxicam on COL or TIMP mRNA expression, but STR mRNA was significantly lower after tenidap compared with piroxicam (p = 0.037). There were no significant associations between measures of local or systemic clinical activity and STR, COL, or TIMP gene expression. However, changes in STR gene expression were significantly correlated with changes in serum C-reactive protein (p = 0.016). Because tenidap and piroxicam are both potent inhibitors of prostaglandin production, the effect of tenidap on STR gene expression may be due to its additional cytokine modulating activity. Clinical trials with data from synovial biopsies may be useful in evaluating the potential for disease-modifying effects of antirheumatic drugs.
RESUMO
OBJECTIVE: To determine whether tenidap treatment would allow reduction or replacement of systemic corticosteroid treatment in patients with polymyalgia rheumatica (PMR). METHODS: A 15-week double blind, randomized, multicenter, placebo-controlled study of tenidap sodium (120 mg/day) in patients with symptomatically controlled PMR receiving 10 mg/day prednisone was conducted. After receiving study drug for 3 weeks, prednisone dose was reduced by 2.5 mg/day every 3 weeks. The lowest clinically effective dose of prednisone was recorded as 10, 7.5, 5, 2.5 or 0 mg/day. RESULTS: Thirty-two patients were randomized to tenidap or placebo. As prednisone was reduced more placebo patients experienced an exacerbation of PMR symptoms, elevation of erythrocyte sedimentation rate and increased serum C-reactive protein. Twice as many placebo patients (10 of 16) as tenidap patients (5 of 16) discontinued due to lack of efficacy. The lowest effective dose of prednisone could be determined in 27 of the 32 patients, 11 receiving tenidap and 16 placebo. A significantly (p = 0.027) greater proportion of patients receiving tenidap (5 of 11) than placebo (1 of 16) were able to discontinue prednisone without experiencing a symptomatic flare. CONCLUSION: As prednisone was reduced, symptoms of PMR were controlled better by tenidap than by placebo. Forty-five percent of evaluable patients receiving tenidap were able to discontinue prednisone without a disease flare compared to 6% for placebo.
Assuntos
Indóis/uso terapêutico , Polimialgia Reumática/tratamento farmacológico , Prednisona/administração & dosagem , Idoso , Anti-Inflamatórios não Esteroides/uso terapêutico , Sedimentação Sanguínea , Proteína C-Reativa/análise , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Indóis/efeitos adversos , Masculino , Pessoa de Meia-Idade , Oxindóis , Placebos , Polimialgia Reumática/sangue , Prednisona/uso terapêuticoRESUMO
OBJECTIVE: To compare the effects of tenidap and piroxicam on acute-phase protein and cytokine levels in the blood of rheumatoid arthritis (RA) patients and to explore their associations with clinical disease activity. METHODS: A double-blind, randomized, crossover trial in 49 patients with active RA compared 6 weeks of treatment with tenidap (120 mg/day) versus 6 weeks of treatment with piroxicam (20 mg/day). RESULTS: Median values for C-reactive protein (CRP), Westergren erythrocyte sedimentation rate (ESR), serum amyloid A (SAA) protein, and interleukin-6 (IL-6) were significantly lower after tenidap treatment compared with piroxicam treatment, even in the presence of stable background treatment with prednisone, methotrexate, or prednisone plus methotrexate. The median within-patient treatment differences (after tenidap minus after piroxicam) in the CRP, ESR, SAA, and IL-6 values were -1.7 mg/dl, -10.0 mm/hour, -22.0 micrograms/ml, and -3.7 pg/ml, respectively, and represent -60.4%, -17.7%, -35.5%, and -26.1% of the respective baseline levels. IL-6 levels were positively correlated with CRP and SAA. Plasma IL-1 beta was generally below the level of detection. Tumor necrosis factor alpha levels were similar after tenidap and after piroxicam. Treatment differences for 4 of 7 clinical parameters favored tenidap, but did not reach statistical significance. IL-6, CRP, and ESR were significantly correlated with clinical treatment differences. Tenidap and piroxicam toleration were similar, although tenidap-treated patients exhibited a reversible increase in urinary protein excretion. CONCLUSION: Tenidap was differentiated from piroxicam by lower levels of acute-phase proteins, ESR, and IL-6 after tenidap treatment. These treatment differences were significantly correlated with clinical parameters.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Indóis/uso terapêutico , Piroxicam/uso terapêutico , Sedimentação Sanguínea , Proteína C-Reativa/metabolismo , Estudos Cross-Over , Citocinas/efeitos dos fármacos , Citocinas/fisiologia , Método Duplo-Cego , Feminino , Humanos , Indóis/farmacologia , Interleucina-1/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Oxindóis , Medição da Dor , Proteinúria/metabolismo , Proteína Amiloide A Sérica/metabolismo , Fator de Necrose Tumoral alfa/análiseRESUMO
In situ hybridization was used to localize and quantify gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissue. Collagenase, tissue inhibitor of metalloproteinases (TIMP), HLA-DR, and complement (C2 and C3) gene expression was studied in synovial tissue from 23 patients with RA, OA, or other inflammatory arthropathies. Gene expression was highly compartmentalized: Collagenase, TIMP, and C2 messenger RNA (mRNA) were localized primarily to the synovial lining layer; HLA-DR mRNA was prominent in the lining and in some sublining lymphoid aggregates; the C3 probe hybridized only to sublining lymphoid aggregates. Relative mRNA levels were quantified using computer-assisted image analysis. There was significantly more collagenase, C2, C3, and HLA-DR mRNA in RA compared with OA patients. However, TIMP mRNA levels were similar in RA and OA. Expression of collagenase, TIMP, C2, C3, and HLA-DR genes correlated with the degree of synovial inflammation. The effect of intraarticular corticosteroid injection on synovial tissue gene expression was studied using serial percutaneous synovial biopsy samples from the knees of 3 RA patients. Joints were biopsied, injected with triamcinolone, and rebiopsied 1-2 weeks later. Histologic inflammation scores were lower in posttreatment synovia. Collagenase and TIMP mRNA, although abundant in presteroid samples, were nearly undetectable in post-steroid tissues. HLA-DR mRNA levels also were significantly decreased. C2 and C3 hybridization significantly decreased in 2 of 3 patients and 1 of 3 patients, respectively. Hence, clinical response to intraarticular steroid therapy was accompanied by histologic improvement and decreased expression of genes that play a role in articular destruction.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Corticosteroides/farmacologia , Artrite Reumatoide/metabolismo , Proteínas do Sistema Complemento/genética , Glicoproteínas/genética , Antígenos HLA-DR/genética , Colagenase Microbiana/antagonistas & inibidores , Colagenase Microbiana/genética , Osteoartrite/metabolismo , Membrana Sinovial/metabolismo , Corticosteroides/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/genética , Proteínas do Sistema Complemento/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Glicoproteínas/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Injeções Intra-Articulares , Masculino , Colagenase Microbiana/metabolismo , Pessoa de Meia-Idade , Osteoartrite/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Membrana Sinovial/efeitos dos fármacos , Inibidores Teciduais de MetaloproteinasesRESUMO
We studied the effects of gold sodium thiomalate (GST) and a new antirheumatic drug, tenidap sodium ([Z]-5-chloro-2,3-dihydro-3-[hydroxy-2-thienylmethylene]-2-oxo-1H- indole-1-carboxamide, sodium salt), previously known as CP-66,248-2, in a model system of macrophage differentiation using a myelomonocytic cell line. HL-60 cells can be stimulated by vitamin D3 to differentiate along a monocytic pathway. Monocytic HL-60 cells express CD14 (Leu-M3), a macrophage surface marker, and develop the capacity to produce the second complement component (C2) in response to stimulation with cytokines such as gamma-interferon. The effects of GST and tenidap sodium were compared with the effects of dexamethasone and a variety of nonsteroidal antiinflammatory drugs in this model system. We found that GST inhibited the capacity of HL-60 cells to produce C2 but did not inhibit the expression of CD14. Tenidap sodium inhibited C2 production as well as CD14 expression, and it partially reversed the decrease in 3H-thymidine incorporation by HL-60 cells, which accompanies monocytic differentiation. At concentrations that inhibited C2 production by HL-60 cells, tenidap sodium did not inhibit C2 production by monocytes. Neither dexamethasone nor the other nonsteroidal antiinflammatory drugs tested possessed these activities. Thus, both GST and tenidap inhibit markers of monocytic differentiation in HL-60 cells, and this activity may relate to their antirheumatic activities.
Assuntos
Tiomalato Sódico de Ouro/farmacologia , Indóis/farmacologia , Macrófagos/citologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colecalciferol/farmacologia , Complemento C2/biossíntese , Inibidores de Ciclo-Oxigenase , Dexametasona/farmacologia , Leucina/metabolismo , Monócitos/metabolismo , Naproxeno/farmacologia , Oxindóis , Timidina/metabolismoRESUMO
A 28-year-old white man presented with neurologic symptoms and skin changes. Subsequent evaluation led to the diagnosis of transverse myelitis of the cervical spine (C8) and linear scleroderma. The progression of neurologic abnormalities prompted treatment with corticosteroids. Neurologic symptoms diminished and the progression of linear skin lesions halted. A review of the literature uncovered considerable evidence for underlying abnormalities of the spine and spinal cord in many patents with linear scleroderma and a paucity of immunologic abnormalities characteristic of progressive systemic sclerosis. Patients presenting with new onset linear scleroderma should be evaluated for underlying neurologic causes.
Assuntos
Mielite Transversa/complicações , Esclerodermia Localizada/etiologia , Adulto , Humanos , Masculino , Hipotonia Muscular/etiologia , Mielite , Mielite Transversa/tratamento farmacológico , Prednisona/uso terapêutico , Esclerodermia Localizada/tratamento farmacológico , Esclerodermia Localizada/patologiaRESUMO
IL-4 was originally described on the basis of its ability to co-stimulate the proliferation of resting B cells treated with anti-IgM. Recently, this cytokine has been shown to have other effects on mast cells, T cells, B cells, and macrophages. We studied the ability of IL-4 to regulate the production of C2 by human monocytes and monocytic cell lines and compared this with stimulation of HLA-DR expression, another recently described activity of IL-4. Responses to IL-4 were compared to IFN-gamma, a cytokine with both activities. IL-4 up-regulated C2 production by human monocytes and this effect was not inhibited by neutralizing anti-IFN-gamma antibody. IL-4 also stimulated C2 production by HL-60 cells that had been pre-treated with vitamin D3 to induce monocytic differentiation. IL-4 did not stimulate C2 production by U937 cells. IFN-gamma, in contrast to IL-4, stimulates C2 production by all three cell types. Although IL-4 increased C2 production by HL-60 cells we could not detect C2 mRNA by Northern blotting. However, co-stimulation of these cells with IL-4 and low concentrations of IFN-gamma resulted in an additive effect on C2 production and a greater increase in C2 mRNA than was seen with IFN-gamma alone. As reported by others, IL-4-stimulated HLA-DR expression by monocytes. In contrast to our findings regarding C2 production, stimulation of HLA-DR expression was inhibited by neutralizing anti-IFN-gamma mAb and IL-4 did not stimulate HLA-DR expression by U937 or HL-60 cells. IFN-gamma stimulated HLA-DR expression by all three cell types. These results identify IL-4 as an additional cytokine able to directly stimulate C2 production by human monocytes and by a monocytic cell line whereas IL-4 stimulation of HLA-DR expression by monocytes appears to be IFN-gamma dependent.
Assuntos
Complemento C2/biossíntese , Antígenos HLA-DR/metabolismo , Interferon gama/farmacologia , Interleucinas/farmacologia , Macrófagos/metabolismo , Monócitos/metabolismo , Anticorpos/fisiologia , Northern Blotting , Linhagem Celular , Humanos , Interferon gama/imunologia , Interleucina-4 , Testes de Neutralização , RNA Mensageiro/isolamento & purificação , Proteínas RecombinantesRESUMO
HL-60 cells, a human promyelocytic cell line, can be induced to differentiate along either monocytic or granulocytic pathways. The production of the second complement component, C2, is a marker of monocytic differentiation and can be up-regulated by cytokine stimulation. We studied the effects of IFN-gamma and vitamin D3, two factors previously shown to induce monocytic differentiation of HL-60 cells, on C2 production and C2 mRNA content. We found that HL-60 cells produce little if any C2 but can be induced to synthesize C2 by IFN-gamma. Vitamin D3 pretreatment followed by IFN-gamma stimulation resulted in earlier and greater production of C2. HL-60 cells did not contain detectable amounts of C2 mRNA unless they were stimulated with IFN-gamma. Pretreatment with vitamin D3 followed by IFN-gamma stimulation resulted in a 147% increase in C2 mRNA content compared with IFN-gamma stimulation alone. These results indicate that the up-regulation of C2 production by IFN-gamma and vitamin D3 is pretranslational although additional posttranslational effects were not excluded. C2 production by these cells is a useful marker of monocytic differentiation.
Assuntos
Colecalciferol/administração & dosagem , Complemento C2/biossíntese , Interferon gama/administração & dosagem , Linhagem Celular , Relação Dose-Resposta a Droga , Esquema de Medicação , Humanos , Monócitos/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Gamma-interferon (gamma-IFN) is a T cell-derived lymphokine that has potent macrophage-activating properties. It increases Fc receptor density, increases the formation and release of reactive oxygen intermediates, increases the synthesis and release of complement cascade proteins, especially C2 and factor B, and increases class II (HLA-DR) antigen expression. These effects may play a role in the potentiation of inflammation in rheumatoid arthritis. We examined the possibility that gold sodium thiomalate (GST), an effective treatment for rheumatoid arthritis, would inhibit gamma-IFN-mediated stimulation of monocyte/macrophages. GST in concentrations attainable in vivo was shown to inhibit both spontaneous and gamma-IFN-stimulated C2 production up to 50%. GST inhibition could be only partially overcome with increasing concentrations of gamma-IFN. In addition, GST inhibited gamma-IFN-stimulated HLA-DR expression at the highest concentrations tested (20-50 micrograms/ml). GST alone in low concentrations (0.1-5 micrograms/ml) was found to increase HLA-DR antigen expression as quantitated by several methods, including flow cytometry, cell surface enzyme-linked immunosorbent assay, and Western blotting. This GST-stimulated increase in HLA-DR antigen expression paralleled an increased ability of monocytes to present antigen. The mechanism by which low concentrations of GST stimulate HLA-DR antigen expression is unclear, but was shown by 35S-methionine cell labeling not to involve increased HLA-DR protein synthesis.
Assuntos
Complemento C2/biossíntese , Tiomalato Sódico de Ouro/farmacologia , Antígenos HLA-D/análise , Antígenos HLA-DR/análise , Interferon gama/antagonistas & inibidores , Monócitos/efeitos dos fármacos , Separação Celular , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoensaio , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Monócitos/imunologiaRESUMO
Serum C reactive protein (CRP), IgG, and IgA levels were measured in 22 patients with ankylosing spondylitis (AS) and in 20 patients with rheumatoid arthritis (RA) to study the regulation of these proteins in inflammatory disease states. In both RA and AS the mean CRP, IgG, and IgA levels were raised above normal values. Although IgA and CRP levels showed a significant positive correlation in RA (r = 0.53, p = 0.02), there was no correlation between these values in AS (r = 0.24, p = 0.29). The difference in correlation coefficients between the AS and RA groups was significant at a p = 0.05 level. In RA the raised IgA levels may be another manifestation of the acute phase response, as shown by the good correlation between IgA and CRP in that disease. In AS, however, the IgA levels, although raised, do not correlate with CRP levels, suggesting that the mechanism of increase of IgA in the two diseases is different. Gut mediated immune stimulation has been proposed as a cause of raised IgA levels in AS.
Assuntos
Artrite Reumatoide/imunologia , Proteína C-Reativa/análise , Imunoglobulina A/análise , Imunoglobulina G/análise , Espondilite Anquilosante/imunologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Monocyte complement stimulator (MCS), a product of T lymphocytes, is defined by its ability to stimulate the synthesis and secretion of the second complement component (C2) by monocytes. Most macrophage-activating factor (MAF) activity present in lymphokine-rich culture supernatants has recently been found to be due to interferon-gamma (IFN-gamma). We therefore hypothesized that IFN-gamma may have MCS activity as well. We tested recombinant, E. coli-derived, human IFN-gamma (rIFN-gamma) for its effects on C2 production by adherent peripheral blood monocytes and U937 cells, a human monocytic cell line. Recombinant IFN-gamma in concentrations ranging from 0.1 to 300 U/ml (0.003 to 8.8 ng/ml) stimulates C2 production by both cell populations. Exposure of responding cells for at least 24 hr is required for maximal stimulation. To determine the contribution of IFN-gamma toward total MCS activity in crude lymphokine-rich supernatants, we employed a solid-phase immunoabsorption technique with the use of a monoclonal anti-IFN-gamma antibody. This technique removed all IFN-gamma detectable by a sensitive ELISA, but MCS activity was decreased by only 40 to 50%. Additionally, MCS activity of these supernatants did not correlate with IFN-gamma content as determined by ELISA. By using another method to eliminate IFN-gamma activity, acid dialysis destroyed all rIFN-gamma activity, as measured by stimulation of U937 C2 synthesis, but eliminated only 30 to 67% of MCS activity from crude lymphokine preparations. Thus IFN-gamma stimulates C2 production by monocytes and U937 cells and apparently accounts for some, but not all, MCS activity present in lymphokine-rich supernatants. Other lymphokines are present in such supernatants that also possess this activity.
Assuntos
Complemento C2/biossíntese , Interferon gama/farmacologia , Linfocinas/farmacologia , Macrófagos/metabolismo , Linhagem Celular , Diálise , Humanos , Técnicas de Imunoadsorção , Cinética , Ativação de Macrófagos , Fatores Ativadores de Macrófagos , Macrófagos/imunologia , Proteínas Recombinantes/farmacologiaRESUMO
The mechanism of action of gold salts in the treatment of rheumatoid arthritis is unknown. Effects of gold on monocyte-macrophage function could be due to inhibition of maturation and differentiation. We found that 3 markers of monocyte differentiation, loss of peroxidase activity, spontaneous synthesis of C2, and spontaneous cytotoxicity for chicken erythrocytes, were all inhibited by gold treatment. This was not a general toxic effect since phorbol myristate acetate could still induce gold-treated monocytes to lyse chicken erythrocytes. Also, phorbol myristate acetate-stimulated superoxide production, a monocyte function not requiring further differentiation, was not inhibited by incubation with gold. Lymphokine-stimulated cytotoxicity for nucleated target cells, another function of monocytes, was inhibited only partially for certain target cells and not at all for others. These data suggest that gold has the capacity to selectively inhibit some monocyte functions which are associated with macrophage differentiation.
Assuntos
Tiomalato Sódico de Ouro/farmacologia , Monócitos/efeitos dos fármacos , Linhagem Celular , Complemento C2/biossíntese , Testes Imunológicos de Citotoxicidade , Humanos , Linfocinas/farmacologia , Ativação de Macrófagos , Monócitos/fisiologia , Neoplasias/imunologia , Peroxidase/metabolismo , Superóxidos/metabolismo , Tiomalatos/farmacologiaRESUMO
The capacity of purified tryptase from human lung mast cells to metabolize human fibrinogen, fibrin, and plasminogen was evaluated. Tryptase (5 micrograms/ml) inactivated the thrombin-induced clotting activity of fibrinogen (100 micrograms/ml) with essentially similar t 1/2 values of 4.6 min in the absence of heparin and 5.8 min in the presence of heparin (20 micrograms/ml) that were not appreciably different than with lysine-Sepharose-purified plasmin (5 micrograms/ml). Fibrinogen treated with tryptase together with heparin lost all detectable clotting activity by 4 hr at 37 degrees C, whereas fibrinogen treated with tryptase alone resulted in destruction of only 80% of fibrinogen clotting equivalents after 16 hr. Tryptase alone was observed to cleave only the alpha-chains of fibrinogen by electrophoresis of tryptase-treated, denatured, and reduced fibrinogen in polyacrylamide gradient gels. Tryptase together with heparin cleaved first the alpha-chain and then the beta-chain, the latter cleavage corresponding to complete loss of fibrinogen clotting activity by 4 hr. No fibrinogen fragments with anticoagulant activity were generated by tryptase. In contrast, plasmin left no residual clotting activity after 4 hr of incubation and generated fibrinogen fragments with anticoagulant activity. Plasmin sequentially cleaved the alpha, beta, and gamma subunits of fibrinogen. Tryptase alone (6 micrograms/ml) or together with heparin (20 micrograms/ml) failed to activate plasminogen (0.6 mg/ml) after a 60-min incubation at 37 degrees C. Addition of urokinase to tryptase-treated or untreated plasminogen resulted in essentially identical plasmin activities (0.32 and 0.34 U/ml, respectively), indicating that tryptase neither activates nor destroys plasminogen. Tryptase (700 ng) also failed to substantially solubilize cross-linked fibrin (2.6 micrograms) or the corresponding amount of fibrinogen bound to plastic microtiter plates with or without heparin. The failure to solubilize fibrinogen and, possibly, fibrin is consistent with the observation that the apparent m.w. by SDS polyacrylamide gel electrophoresis of unreduced fibrinogen is not appreciably altered by prior treatment with tryptase, even though cleavage of alpha-and beta-chains is revealed after reduction. Fibrinogenolysis by tryptase complements other mast cell mediators with anticoagulant properties such as heparin and suggests a significant prevention of coagulation by activated mast cells.
Assuntos
Fibrinogênio/metabolismo , Fibrinólise , Pulmão/citologia , Mastócitos/enzimologia , Peptídeo Hidrolases/metabolismo , Testes de Coagulação Sanguínea , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinolisina/farmacologia , Heparina/farmacologia , Humanos , Cinética , Peptídeo Hidrolases/farmacologia , Ativadores de Plasminogênio/farmacologiaRESUMO
Autoantibody-secreting hybridomas were produced by somatic cell fusion of B lymphocytes from a patient with systemic lupus erythematosus with two different human myeloma lines. Selection of hybrids formed from one of these cell lines was performed by using aminopterine-containing culture medium as this cell line was deficient in hypoxanthine-guanine-phosphoribosyl transferase (HGPRT). The second myeloma line was not HGPRT-deficient but instead was treated with diethylpyrocarbonate, which assured death of unfused myeloma cells. This novel technique has wide applicability. Hybridomas were found to secrete antibodies to native DNA and to extractable nuclear antigen. The binding specificities of one IgM anti-DNA antibody was characterized and found to be specific for double-stranded DNA and had particular binding affinity for poly(dG) . poly(dC).
Assuntos
Anticorpos Antinucleares/análise , Anticorpos Monoclonais/análise , Autoanticorpos/análise , DNA/imunologia , Hibridomas/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Linfócitos B/imunologia , Linhagem Celular , Células Clonais/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Métodos , Mieloma Múltiplo/imunologia , Testes de PrecipitinaRESUMO
Human monocytes synthesize large amounts of the second complement component (C2) after incubation with a T-lymphocyte product called monocyte complement stimulator (MCS). The human monocyte-like cell line, U937, also synthesizes C2 and can be stimulated to increase this synthesis by lymphokine-rich culture supernates. Additionally, phorbol myristate acetate (PMA), an agent which induces maturational changes in other macrophage-like cell lines, also stimulates C2 synthesis by U937 cells. Lymphokine and PMA stimulation of C2 secretion by U937 are both reversibly inhibitable by cycloheximide. At optimal concentrations for stimulation of C2 synthesis, PMA inhibits [3H]thymidine incorporation by U937 indicating that increased C2 is not due to increased numbers of U937 cells.
Assuntos
Complemento C2/biossíntese , Linfocinas/fisiologia , Monócitos/imunologia , Forbóis/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Divisão Celular , Linhagem Celular , Cicloeximida/farmacologia , Dimetil Sulfóxido/farmacologia , Humanos , Monócitos/citologiaRESUMO
The ability of gold sodium thiomalate to inhibit production of the second complement component (C2) by monocytes stimulated by a lymphokine (monocyte complement stimulator is demonstrated. This gold salt inhibits C2 production irreversibly if monocytes are incubated with it before or during lymphokine stimulation. Thiomalic acid is not inhibitory. Monocytes already stimulated by lymphokine are resistant to inhibition of C2 production by gold sodium thiomalate. Gold salts do not reduce monocyte viability, phagocytic ability (latex heads) accessory cell function (as measured by the ability to present antigen to autologous lymphocytes), or capacity to act as stimulating cells in mixed leukocyte culture. Gold sodium thiomalate's inhibition of monocyte responsiveness to lymphokine may be significant in explaining the therapeutic benefit of gold salts in rheumatoid arthritis.