RESUMEN
Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a cytotoxic member of the TNF family. Some reports have shown that TRAIL is released from cells in a soluble form. In this work, we have investigated the mechanism of release of TRAIL from monocytes. First, we show that whole gram-positive, gram-negative and mycoplasmal bacteria as well as lipopolysaccharide (LPS), interferon-alpha (IFN-alpha), -beta and -gamma all induced upregulation of TRAIL on the surface of human monocytes. Next, we show that IFN-alpha, -beta and -gamma all induced a dose-dependent release of TRAIL, giving significant amounts of soluble TRAIL after 2 days. Of the bacteria, only the Group B streptococcus COH-1 (GBS) induced release of TRAIL and concomittantly induced IFN-alpha. Monocytes stimulated with GBS or IFN-alpha also showed extensive cell death. When monocyte apoptosis was prevented by interleukin-1, GM-CSF, LPS or the caspase inhibitor zVADfmk, the IFN-alpha-induced release of TRAIL was reduced, whereas agents inducing necrosis caused increased release of TRAIL. LPS also prevented release of TRAIL from GBS-stimulated monocytes. The release of TRAIL from IFN-alpha-stimulated monocytes was reduced by inhibitors of both cysteine and metalloproteases. We conclude that bacteria and IFN induce upregulation of membrane TRAIL and that release of TRAIL is associated with cell death.
Asunto(s)
Interferones/inmunología , Leucina/análogos & derivados , Glicoproteínas de Membrana/inmunología , Monocitos/inmunología , Streptococcus agalactiae/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis , Pruebas Inmunológicas de Citotoxicidad , Citometría de Flujo , Humanos , Leucina/farmacología , Leupeptinas/farmacología , Lipopolisacáridos/inmunología , Activación de Linfocitos/inmunología , Monocitos/microbiología , Inhibidores de Proteasas/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF , Regulación hacia ArribaRESUMEN
BACKGROUND: In studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome-conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis. MATERIALS AND METHODS: Peripheral blood mononuclear cells were stained with fluorochrome-conjugated anti-CD antibodies directly without or with a second-step application of nonconjugated goat anti-mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry. RESULTS: A second-step application of nonconjugated goat anti-mouse IgG antibodies following direct staining with fluorochrome-conjugated anti-CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab')(2) fragments of the goat anti-mouse antibodies showed effects, while Fab fragments did not. CONCLUSIONS: Nonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Antígenos de Superficie/inmunología , Inmunoglobulina G/inmunología , Antígenos Comunes de Leucocito/inmunología , Leucocitos Mononucleares/inmunología , Especificidad de Anticuerpos , Antígenos CD19/inmunología , Sitios de Unión , Complejo CD3/inmunología , Citometría de Flujo/métodos , Fluoresceína-5-Isotiocianato/química , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Reproducibilidad de los ResultadosRESUMEN
Cytokines are important mediators in inflammation, and play a key role in inflammation induced by Gram-negative bacteria. Cytokines are released into serum during different pathological conditions, such as meningococcal disease, and the cytokine levels in serum seem to correlate with fatal outcome of septic shock (1-3). Consequently, detection of cytokines in serum samples from patients with pathological diseases may be of prognostic and clinical value. Cytokines can be detected using bioassays and immunoassays, and this chapter focuses on description of bioassays for detection of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and IL-6 in serum samples.
RESUMEN
Because persistent tumor necrosis factor (TNF)-alpha activation may play a pathogenic role in human immunodeficiency virus infection, TNF component levels were assessed over 78 weeks in plasma and peripheral blood mononuclear cells (PBMC) during highly active antiretroviral therapy (HAART) in 40 HIV-infected patients. HAART induced a significant decline in plasma levels of TNF-alpha and soluble TNF receptors and was associated with a fall in the abnormally increased unstimulated and a rise in the abnormally low Mycobacterium avium complex-purified-protein derivative-stimulated TNF-alpha released from PBMC. However, concentrations of these TNF components were not normalized. Patients with virologic and immunologic treatment failure after 52 weeks had higher levels of several TNF components than other patients early after initiation of therapy, also during periods with adequate virologic response. Although TNF components significantly decreased during HAART, these results support data indicating that full immunologic normalization is not achieved during such therapy. The persistent activation of the TNF system in a subgroup of persons may be involved in treatment failure.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adolescente , Adulto , Fármacos Anti-VIH/administración & dosificación , Recuento de Linfocito CD4 , Quimioterapia Combinada , Femenino , Infecciones por VIH/virología , Humanos , Técnicas In Vitro , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Complejo Mycobacterium avium/inmunología , ARN Viral/sangre , Receptores del Factor de Necrosis Tumoral/sangre , Factores de Tiempo , Insuficiencia del Tratamiento , Tuberculina/inmunologíaRESUMEN
Soluble cytokine receptors (R) may regulate the activities of the cytokines present. We have studied the occurrence of soluble receptors for the cytokines tumor necrosis factor (TNF), interleukin-2 (IL-2) and interleukin-6 (IL-6) in normal pregnant women. Levels of TNFR and IL-2R were higher in retroplacental serum (RPS) than in the corresponding peripheral serum (PS) samples. TNFR, but neither IL-2R nor IL-6R, concentrations in PS from pregnant women were elevated compared to that in nonpregnant women. RPS inhibited TNF-mediated cytotoxicity, with the inhibitory effect being reduced after the removal of 55 kD TNFR from RPS. Our data reveal that soluble cytokine receptors are present in RPS and may contribute to the adjustment of intrauterine cytokine activities during pregnancy.
Asunto(s)
Antígenos CD/sangre , Placenta , Receptores de Interleucina-2/sangre , Receptores de Interleucina/sangre , Receptores del Factor de Necrosis Tumoral/sangre , Muerte Celular , Femenino , Humanos , Embarazo , Receptores de Interleucina-6 , Receptores del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Using quantitative zymography, we measured activity of the type IV collagenases metalloprotease 2 (MMP-2) and MMP-9 in 192 biopsies from colorectal carcinomas, adenomas, and normal bowel. The median level of MMP-9 in samples from Dukes' stage A (n = 18) or C (n = 48) tumors was significantly higher than in stage B carcinomas (n = 65), adenomas (n = 25), and normals (n = 36; P = 0.0001). The median level of active MMP-2 was significantly higher in stage A or C compared with adenomas (P = 0.0001) and normals (P = 0.0001). The median level of inactive MMP-2 was higher in all Dukes' stages compared with normals and adenomas (P = 0.0001). There was a significant increase in inactive MMP-2 from Jass prognostic groups I-IV (P = 0.006) but no correlation with the active enzyme. MMP activity was not related to tumor differentiation, colon versus rectal location, or disease-free, 5-year survival. All groups expressed mRNA for both enzymes, but there were quantitative and locational differences in MMP-2 mRNA expression between normal, benign, and malignant tissues. Thus MMP-2 is controlled at the level of mRNA and protein production and activation in colorectal cancer, and active MMP-2 and MMP-9 enzymes are associated strongly with Dukes' A and C stages of the disease. Variations in MMP levels with the stage or prognostic group of colorectal cancer reflect their differing stromal content.
Asunto(s)
Adenoma/enzimología , Biomarcadores de Tumor/biosíntesis , Carcinoma/enzimología , Colagenasas/biosíntesis , Neoplasias Colorrectales/enzimología , Gelatinasas/biosíntesis , Metaloendopeptidasas/biosíntesis , Secuencia de Bases , Humanos , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pronóstico , ARN Mensajero/biosíntesisRESUMEN
Lipopolysaccharide (LPS), a part of the Gram-negative bacteria cell wall, is a potent inducer of tumor necrosis factor (TNF). TNF is an important mediator in Gram-negative infections such as meningococcal septic shock, but its harmful action can be prevented by the natural occurring soluble (s) TNF receptors (sTNFR) sp55 and sp75. In this study, the effect of LPS on release of sTNFR was investigated. First, we found a selective increase in human whole-blood sp75 TNFR levels following LPS stimulation, accompanied by no increase in sp55. Separating the different blood cell populations, mononuclear cells (PBMC) selectively released sp75 upon LPS stimulation, while LPS induced a minor increase in sp75 release from polymorphonuclear granulocytes. Interestingly, in co-cultures of PBMC and granulocytes, the release of LPS-induced sp75 TNFR was enhanced. Second, adherent monocytes were also found to selectively release sp75 TNFR upon LPS stimulation, where Neisseria meningitidis LPS was found to be 100-1000 times more potent in inducing sp75 release than Escherichia coli LPS. Using flow cytometry, the monocyte membrane distribution of both TNFR were found to be increased after LPS stimulation. Third, human umbilical vein endothelial cells selectively released sp55 TNFR after stimulation with LPS. We conclude that mononuclear and endothelial cells might be the main sources of soluble p75 and p55 TNFR, respectively, observed in Gram-negative sepsis, although these receptors are released in vivo more rapidly than they are in vitro.
Asunto(s)
Antígenos CD/metabolismo , Granulocitos/metabolismo , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Receptores del Factor de Necrosis Tumoral/metabolismo , Células Cultivadas , Endotelio Vascular/metabolismo , Humanos , Receptores Tipo II del Factor de Necrosis TumoralRESUMEN
PROBLEM: To determine the concentration of the two soluble tumor necrosis factor receptors (sTNFR), sp55 and sp75, in healthy pregnant women. METHOD: Serum and urine samples were longitudinally collected from a group of pregnant women (N = 53) five times throughout pregnancy. Maternal and umbilical sera were obtained from some of the deliveries (N = 31). The samples were analysed using ELISA based on two monoclonal antibodies (IV4E and 3H5) against the soluble tumor necrosis factor receptors (sp55 and sp75). RESULTS: Serum concentration of sp55 and sp75 were increased in pregnant women compared to that of nonpregnant controls. Concentration of both sTNFRs increased towards term. Labor was associated with further increase of sp55. Concentrations of sp55 and sp75 in umbilical serum were significantly higher than those of maternal serum. Significant correlations were observed between maternal and umbilical sTNFR concentrations. CONCLUSIONS: The current study suggests that pregnancy is associated with an activation of mechanisms regulating the biological activities of TNF.
Asunto(s)
Trabajo de Parto/sangre , Embarazo/sangre , Embarazo/orina , Receptores del Factor de Necrosis Tumoral/análisis , Femenino , Sangre Fetal/inmunología , Edad Gestacional , Humanos , Trabajo de Parto/inmunología , Trabajo de Parto/orina , Intercambio Materno-Fetal/inmunología , Embarazo/inmunologíaRESUMEN
The relationships between serum levels of soluble tumor necrosis factor receptors (sTNFRs) and other prognostic and immunological parameters in different immunological subgroups of 64 HIV-1 infected patients were studied. In the patient group as a whole, the raised serum levels of sTNFRs were significantly inversely correlated to the numbers of CD4+ and CD8+ lymphocytes and significantly positively correlated with serum levels of neopterin, HIV-1 p24 antigen and the soluble CD8/CD8+ lymphocyte ratio. However, when the patients were classified into three separate immunological subgroups according to the numbers of CD4+ lymphocytes, only serum levels of neopterin were significantly correlated to levels of sTNFRs in all the defined immunological subgroups. These results indicate that HIV-1 infection is associated with a persistent and chronic immune activation in the TNF system manifested by raised serum levels of sTNFRs, which may reflect sustained activation of the immune system particularly in monocytes/macrophages. Further, these results confirm that, when comparing immunological and virological parameters in HIV-1 infection, different results may be obtained in different immunological subgroups of patients.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Factor de Necrosis Tumoral alfa/análisis , Adolescente , Adulto , Biopterinas/análogos & derivados , Biopterinas/sangre , Relación CD4-CD8 , Antígenos CD8/sangre , Estudios de Casos y Controles , Femenino , Infecciones por VIH/virología , Humanos , Masculino , Persona de Mediana Edad , Neopterin , Valor Predictivo de las Pruebas , PronósticoRESUMEN
Accumulating evidence suggests that cytokines are major participants in human reproduction. Cytokines may have beneficial or negative influence on pregnancy outcome, depending on the cytokine level present. Thus, successful reproduction appears to depend on a tight regulation of cytokine activities. The present study raised the question whether normal pregnancy is associated with an activation of native cytokine buffer mechanisms. Soluble interleukin 6 receptors (IL-6Rs) and soluble interleukin 1 receptor antagonists (IL-1RAs) may modify the activity of IL-6 and IL-1, respectively. The production of soluble IL-6R and IL-1RA in pregnancy was studied by assessing the IL-6R and IL-1RA concentrations in serum samples from healthy pregnant women at different gestational ages. At delivery, both maternal and umbilical blood was obtained. Concentrations of IL-6 and IL-1 in the samples were determined to study the influence of cytokines on the activity level of the corresponding buffer mechanism. Serum levels of both IL-6R and IL-1RA were increased in pregnant women, as were levels of IL-6 and IL-1. Cytokine levels did not demonstrate a significant correlation with the concentration of the corresponding activity modifier. IL-1RA and IL-6 increased with gestational age and with labor activity. A significant correlation was observed between the levels of IL-6 and IL-1RA.
Asunto(s)
Interleucina-1/sangre , Interleucina-6/sangre , Embarazo/sangre , Receptores de Interleucina/análisis , Sialoglicoproteínas/sangre , Femenino , Sangre Fetal/química , Edad Gestacional , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Trabajo de Parto/sangre , Estudios Longitudinales , Estudios Prospectivos , Receptores de Interleucina-6RESUMEN
We investigated the in vivo effects of one bolus injection (400 mg/kg) of intravenous immunoglobulin (IVIG) on a number of cytokines, soluble cytokine receptors, and interleukin-1 receptor antagonist (IL-1Ra) in plasma in 12 patients with primary hypogammaglobulinemia. A significant and rapid increase in plasma levels of IL-6, IL-8, and tumor necrosis factor alpha (TNF alpha) was seen within 1 hour after IVIG infusion. This increase was accompanied by a more prolonged elevation in levels of both types of soluble TNF receptors (sTNFRs), which remained elevated throughout the study period (44 hours) although they reached peak levels within 1 hour. After an initial increase in the ratio between TNF alpha and sTNFRs, this ratio decreased to values significantly lower than baseline values 20 and 44 hours postinfusion with approximately 600-fold molar excess of sTNFRs to TNF alpha (trimer). Although only a modest but statistically significant increase in plasma levels of IL-1 beta was seen, IVIG infusion was followed by a marked increase in plasma levels of IL-1Ra with 1,000-fold molar excess of IL-1Ra to IL-1 beta in some patients. The demonstrated effects of IVIG infusion on the cytokine network, particularly the induction of IL-1Ra and sTNFRs release, might be important for the therapeutic effects of IVIG in several immune-mediated disorders.
Asunto(s)
Agammaglobulinemia/terapia , Citocinas/metabolismo , Inmunoglobulinas Intravenosas/administración & dosificación , Receptores de Citocinas/metabolismo , Sialoglicoproteínas/metabolismo , Adulto , Anciano , Femenino , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Masculino , Persona de Mediana Edad , Receptores del Factor de Necrosis Tumoral/metabolismo , Solubilidad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Two EIAs (Medgenix and Quantikine) and a bioassay were used to measure tumor necrosis factor-alpha (TNF alpha) in serum samples from 73 human immunodeficiency virus type 1 (HIV-1)-seropositive patients and in samples from 2 control groups. All clinical groups of HIV-1-infected patients, regardless of concurrent illness, had significantly elevated levels of both types of soluble TNF receptors (sTNFRs) and immunoreactive TNF alpha (Medgenix EIA), with the highest concentrations among the AIDS patients. These TNF parameters were significantly correlated with reduced CD4+ lymphocyte counts. Only a few HIV-1-infected patients had detectable TNF alpha levels measured by the Quantikine EIA. TNF alpha bioactivity was significantly raised only in the AIDS group. Serially measured sTNFRs, expressed as sTNFR slopes, were significantly associated with survival in the patient group. The raised levels of immunoreactive TNF alpha and sTNFRs strongly indicate activation of the TNF alpha system during HIV-1 infection. Levels increase with disease progression and degree of immunodeficiency; thus, serially measured sTNFRs may give useful prognostic information in HIV-1 infection.
Asunto(s)
Infecciones por VIH/sangre , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Biopterinas/análogos & derivados , Biopterinas/sangre , Linfocitos T CD4-Positivos , Seropositividad para VIH/sangre , Humanos , Recuento de Leucocitos , Neopterin , Receptores del Factor de Necrosis Tumoral/química , SolubilidadRESUMEN
Soluble TNFRs (tumour necrosis factor receptors) inhibit in-vivo and in-vitro bioactivities of TNF, and thus the secretion of soluble TNFRs could be a physiological principle to attenuate the bioactivities of TNF. Two types of TNFR have been identified and both forms can be released from cells. In this study, soluble TNFRs in seminal plasma from three groups of men were analysed: from men with normal semen quality (n = 32), with reduced semen quality (n = 7) and vasectomized men (n = 3). Sensitive and specific enzyme-linked immunosorbent assays were utilized to detect soluble TNFRs in seminal plasma, based on capture antibodies directed against non-TNF-binding sites of the TNFRs and digoxigenin (DIG)-labelled TNF. The mean +/- standard deviation levels of p55 TNFR were 56.4 +/- 20, 64.4 +/- 17 and 45.4 +/- 5 ng/ml in the three groups, respectively. The concentration of p75 TNFR was < 1 ng/ml in all groups. The results suggest an exclusive existence of high amounts of the soluble p55 TNFR in seminal plasma. Seminal plasma from vasectomized men contained p55 TNFR at approximately the same levels as the specimen from the two other groups, indicating that the source of p55 TNFR is not the testis but rather some tissue more distal in the male genital tract, such as the prostate or the seminal vesicles. The soluble p55 TNFR was purified from human seminal plasma, using affinity and gel filtration chromatography. Further characterization of the purified p55 revealed a protein with a molecular weight of approximately 22 kDa, both under reducing and non-reducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Receptores del Factor de Necrosis Tumoral/metabolismo , Semen/metabolismo , Cromatografía de Afinidad , Cromatografía en Gel , Digoxigenina , Ensayo de Inmunoadsorción Enzimática , Humanos , Infertilidad Masculina/metabolismo , Masculino , Peso Molecular , Próstata/metabolismo , Receptores del Factor de Necrosis Tumoral/aislamiento & purificación , Vesículas Seminales/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , VasectomíaRESUMEN
OBJECTIVE: To compare levels of tumor necrosis factor (TNF) and interleukin 1 (IL-1) and 6 (IL-6) with levels of the soluble receptors for TNF in maternal and neonatal urine and amniotic fluid (AF). METHODS: Levels of soluble TNF receptors (p55, p75) in AF and urine from 21 women and their newborns were measured by immunoassay. The amniotic concentrations of IL-1 and IL-6 were assessed by biologic assays, whereas an immunoassay was used to measure TNF levels. The comparison between receptor concentrations in different compartments was performed by one-way analysis of variance, and Student t test was used to compare pairs of groups. Correlation studies were performed when indicated. RESULTS: A high correlation was observed between the concentrations of p55 and p75 in all compartments. The concentration of p55 in AF was significantly higher than that in both maternal and neonatal urine, but the correlation between TNF receptor concentrations in the AF samples and the concomitant levels of cytokines was not statistically significant. CONCLUSION: The high concentration of receptors in the AF compared to those of other biologic compartments suggests that the pregnancy-associated increased receptor shedding takes place in intrauterine tissues. Physiologic levels of cytokines, such as those accompanying normal delivery at term, did not seem to influence the soluble TNF receptor release.
Asunto(s)
Líquido Amniótico/química , Recién Nacido/orina , Trabajo de Parto/metabolismo , Receptores de Superficie Celular/análisis , Factor de Necrosis Tumoral alfa/análisis , Femenino , Humanos , Interleucina-1/análisis , Interleucina-6/análisis , Embarazo , Receptores del Factor de Necrosis Tumoral , Solubilidad , Factor de Necrosis Tumoral alfa/orinaRESUMEN
A highly specific and sensitive immunoassay for soluble p55 tumor necrosis factor receptor (TNFR) has been established. The immunoassay was based on a newly developed monoclonal antibody (IV4E) recognizing a non-TNF-binding site of the p55 TNFR. The IV4E antibody immunoprecipitated a 55 kDa TNF binding protein from HL-60 cells. No binding of IV4E to the p75 TNFR could be detected. Bound TNFR to IV4E was detected with digoxigenin (DIG) labeled TNF. This assay could detect down to 300 pg/ml of soluble p55, which represents an 8-10-fold increase in sensitivity compared to earlier developed immunoassays. The assay was specific for soluble p55 TNFR present in serum and cell culture supernatants, since addition of excess unlabeled TNF together with DIG labeled TNF inhibited the signal. TNF concentrations up to 10 ng/ml in the TNFR sample did not affect the assay, indicating that TNFRs can be measured in samples containing TNF. The new immunoassay was used to study the mechanisms underlying the release of soluble p55 TNFR from U937 cells stimulated with TPA. The TPA induced release of soluble p55 TNFR from U937 cells occurred in two phases. First, a rapid increase of soluble p55 was observed after the addition of TPA. Later, the release of p55 occurred at a slower rate, and this release was inhibited by known inhibitors of protein synthesis and intracellular transport. Addition of TPA increased the p55 mRNA expression in U937 cells. The results suggest that TPA induces both release and new synthesis of p55 in U937 cells.
Asunto(s)
Inmunoensayo/métodos , Monocitos/metabolismo , Receptores de Superficie Celular/análisis , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antivirales/farmacología , Northern Blotting , Brefeldino A , Línea Celular , Cicloheximida/farmacología , Ciclopentanos/farmacología , Digoxigenina , Relación Dosis-Respuesta Inmunológica , Citometría de Flujo , Humanos , Hibridomas , Ratones , Ratones Endogámicos BALB C , Monensina/farmacología , Monocitos/efectos de los fármacos , Pruebas de Precipitina , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores del Factor de Necrosis Tumoral , Sensibilidad y Especificidad , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Secretion of soluble cytokine receptors has been suggested as a mechanism of regulating cytokine activity in vivo. In this study, the presence of soluble receptors for tumor necrosis factor-alpha (TNF-alpha) in serum samples from pregnant women and newborn babies was examined by using TNF-alpha receptor-specific immunoassays. Serum from pregnant women contained the 55-kD TNF-alpha receptor p55 [6.4 micrograms/L, interquartile range, i.e. the difference between the 25th and 75th percentile, (IQR) 7.2 micrograms/L], whereas p55 was not detected in control sera from nonpregnant, fertile women. The levels of p55 were also elevated in serum from newborns (3.2 micrograms/L, IQR 0.7 micrograms/L). Furthermore, first-voided urine from newborns contained high levels of p55 (44.6 micrograms/L, IQR 96.6 micrograms/L). The concentration of p55 in the neonatal period was significantly higher than that of children later in childhood (p < 0.05). The concentration of p55 was 7.3 micrograms/L, IQR 13.8 micrograms/L, in urine samples from a group of preschool children (1 mo < age < 5 y) and 6.9 micrograms/L, IQR 2.9 micrograms/L, in urine samples from children in a higher age group (5 y < age < 10 y). The concentration of the 75-kD TNF-alpha receptor in urine did not differ significantly between the different age groups studied. The elevated p55 serum levels of pregnant women and neonates are suggestive of a pregnancy-associated release of soluble receptors. The high concentration of p55 in neonatal urine may reflect a postpartum elimination of soluble receptors shed during pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Niño , Preescolar , Femenino , Humanos , Inmunoensayo/métodos , Lactante , Recién Nacido , Masculino , Embarazo , Receptores del Factor de Necrosis Tumoral , Valores de Referencia , SolubilidadRESUMEN
High concentrations of tumour necrosis factor may threaten the well-being of the foetus. Secretion of soluble cytokine receptors has been suggested as a mechanism for regulating cytokine activity in vivo. In this study pregnancy-related materials were examined for the presence of soluble receptors (p55 and p75) for tumour necrosis factor. In contrast to control materials, serum and urine samples from pregnant women and newborns contained high concentrations of p55, and p55 was highly expressed by villous syncytiotrophoblasts. Thus, the present data suggest that shedding of specific receptors may contribute to the regulation of tumour necrosis factor activity throughout a normal pregnancy.
Asunto(s)
Embarazo/inmunología , Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Humanos , Inmunohistoquímica , Recién Nacido , Placenta/inmunología , Placenta/metabolismo , Resultado del Embarazo , Receptores de Superficie Celular/inmunología , Receptores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Secretion of soluble cytokine receptors has been suggested as a mechanism for regulation of cytokine activity in vivo. The present investigation was performed to study whether secretion of soluble TNF (tumor necrosis factor) receptors (TNFRs) might be associated with pregnancy. There are two known molecular species of the TNFR, the 55-kDa TNFR and the 75-kDa TNFR. The 75-kDa, as well as the 55-kDa TNFR, was detected in urine from pregnant women, whereas only the 75-kDa TNFR was detected in urine from the non-pregnant group. The concentration of TNFRs in urine increased towards term and was reduced in association with spontaneous delivery. The soluble forms of both TNFRs were also detected in amniotic fluid. Collectively, the data suggest that secretion of soluble TNFRs during pregnancy might be a defence mechanism for the protection of the fetus against TNF action.
Asunto(s)
Líquido Amniótico/metabolismo , Embarazo/metabolismo , Receptores de Superficie Celular/metabolismo , Femenino , Humanos , Intercambio Materno-Fetal/inmunología , Intercambio Materno-Fetal/fisiología , Peso Molecular , Embarazo/inmunología , Embarazo/orina , Receptores de Superficie Celular/química , Receptores del Factor de Necrosis Tumoral , Solubilidad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Immunoassays were established for the detection of the 55 kDa and 75 kDa tumour necrosis factor receptor (TNFR) fragments present in urine. The immunoassays were based on pairs of monoclonal TNFR antibodies directed against different epitopes of the 55 kDa and 75 kDa TNFRs. The immunoassays were judged to be specific for unoccupied TNFR since the signals were inhibited by adding recombinant human or murine TNF-alpha, and to a lesser extent by rTNF-beta (LT). Other cytokines such as IL-1 beta, IL-2 or rIFN-gamma did not affect the signal. In a preliminary screening it was found that urines from febrile patients contained higher amounts of 55 kDa and 75 kDa TNFR fragments than did urine from non-febrile individuals. The immunoassays could be used to monitor the purification of the two types of TNFR from the same febrile urine. Furthermore, the sensitivity and the speed of the assay could be increased by the use of magnetic beads as a solid support in the assay.
Asunto(s)
Receptores de Superficie Celular/análisis , Factor de Necrosis Tumoral alfa/metabolismo , Fiebre/metabolismo , Humanos , Inmunoensayo , Receptores del Factor de Necrosis TumoralRESUMEN
A sensitive and rapid immunoassay for the detection of tumor necrosis factor (TNF) has been developed. Magnetic monodisperse polymer particles (M-280 Dynabeads) used as solid phase material, were coated with a neutralizing mouse monoclonal antibody to TNF. The coated Dynabeads were shown to have a more rapid binding capacity for recombinant (r) TNF as compared to standard immunowells coated with antibodies to TNF. The amount of TNF bound to the Dynabeads was quantified using either a polyclonal antibody to TNF or a mouse monoclonal antibody to TNF. The antibodies used for detection were either labelled with 125I or peroxidase. The linear assay range for the TNF standard curve was form 62 to 4000 pg/ml, and the assay time was less than 60 min. The sensitivity could be increased 5-8-fold by increasing the sample volume from 0.1 to 2 ml.