RESUMEN
BACKGROUND: Inflammation activated by oxidative stress can cause various diseases, such as asthma, rheumatoid arthritis, cancer, diabetes, etc. Plant constituents with sesquiterpene lactones possess antioxidant and anti-inflammatory properties. AIM: To determine the antioxidant and anti-inflammatory potential of isolated phytoconstituent from Cyathocline purpurea Buch-Ham ex D (CP). Don in laboratory animals. Furthermore, to understand the interactions involved in the binding of this compound to cyclooxygenase-2 (COX-2) via computational docking. METHODS: Phytoconstituent was isolated, purified and well characterized (using IR, NMR, and MS) from ethyl acetate fraction of CP methanolic extract. It was then evaluated for its in-vitro antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2) and hydroxyl (OH) radical assays as well as in-vivo anti-inflammatory potential against carrageenan-induced paw edema model in rats. The molecular docking study was performed against the crystal structure of COX-2 to evaluate the binding potential of phytoconstituent towards this enzyme. RESULTS: The isolated compound 6α-hydroxy-4 [14], 10 [15]-guainadien-8α, 12-olide (HGN) showed significant (p<0.001) antioxidant activity with IC50 values of 76µg/mL. Administration of HGN (10 and 20mg/kg) significantly (p<0.001) reduced the increased paw volume after subplantar administration of carrageenan. It also exhibits good binding affinity towards with COX-2 with a docking score of -8.98 and Glide binding energy of -36.488kcal/mol shedding light on the potential mechanism of anti-inflammatory action. CONCLUSIONS: The presence of hydroxyl group in HGN provides a credential to its in-vivo anti-inflammatory and in-vitro antioxidant activities. Furthermore, the good binding affinity of HGN for the active site of COX-2 may open novel vistas in therapeutic option with natural antioxidants like Cyathocline purpurea to treat various inflammatory disorders.
Asunto(s)
Antiinflamatorios/uso terapéutico , Antioxidantes/uso terapéutico , Ciclooxigenasa 2/metabolismo , Edema/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Fitoterapia/métodos , Sesquiterpenos de Guayano/uso terapéutico , Animales , Asteraceae/inmunología , Compuestos de Bifenilo/inmunología , Carragenina/toxicidad , Células Cultivadas , Edema/inducido químicamente , Femenino , Humanos , Peróxido de Hidrógeno/metabolismo , Masculino , Ratones , Estrés Oxidativo/efectos de los fármacos , Picratos/inmunología , Ratas , Ratas WistarRESUMEN
Airway epithelial cells (AECs) express a variety of receptors, which sense danger signals from various aeroallergens/pathogens being inhaled constantly. Proteinase-activated receptor 2 (PAR-2) is one such receptor and is activated by cockroach allergens, which have intrinsic serine proteinase activity. Recently, dual oxidases (DUOX), especially DUOX-2, have been shown to be involved in airway inflammation in response to Toll-like receptor activation. However, the association between PAR-2 and DUOX-2 has not been explored in airways of allergic mice. Therefore, this study investigated the contribution of DUOX-2/reactive oxygen species (ROS) signalling in airway reactivity and inflammation after PAR-2 activation. Mice were sensitized intraperitoneally with intact cockroach allergen extract (CE) in the presence of aluminium hydroxide followed by intranasal challenge with CE. Mice were then assessed for airway reactivity, inflammation, oxidative stress (DUOX-2, ROS, inducible nitric oxide synthase, nitrite, nitrotyrosine and protein carbonyls) and apoptosis (Bax, Bcl-2, caspase-3). Challenge with CE led to up-regulation of DUOX-2 and ROS in AECs with concomitant increases in airway reactivity/inflammation and parameters of oxidative stress, and apoptosis. All of these changes were significantly inhibited by intranasal administration of ENMD-1068, a small molecule antagonist of PAR-2 in allergic mice. Administration of diphenyliodonium to allergic mice also led to improvement of allergic airway responses via inhibition of the DUOX-2/ROS pathway; however, these effects were less pronounced than PAR-2 antagonism. The current study suggests that PAR-2 activation leads to up-regulation of the DUOX-2/ROS pathway in AECs, which is involved in regulation of airway reactivity and inflammation via oxidative stress and apoptosis.
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Asma/inmunología , Inflamación/inmunología , NADPH Oxidasas/inmunología , Receptor PAR-2/inmunología , Hipersensibilidad Respiratoria/inmunología , Alérgenos/inmunología , Animales , Antiinfecciosos/inmunología , Antiinfecciosos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Asma/metabolismo , Asma/prevención & control , Compuestos de Bifenilo/inmunología , Compuestos de Bifenilo/farmacología , Western Blotting , Cucarachas , Modelos Animales de Enfermedad , Oxidasas Duales , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Inflamación/metabolismo , Inflamación/prevención & control , Masculino , Ratones Endogámicos BALB C , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Compuestos Onio/inmunología , Compuestos Onio/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/inmunología , Piperazinas/inmunología , Piperazinas/farmacología , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Receptor PAR-2/antagonistas & inhibidores , Receptor PAR-2/metabolismo , Hipersensibilidad Respiratoria/metabolismo , Hipersensibilidad Respiratoria/prevención & control , Sistema Respiratorio/inmunología , Sistema Respiratorio/metabolismo , Sistema Respiratorio/patología , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Boscalid is a modern agrochemical belonging to the so-called chemical class of succinate dehydrogenase inhibitor fungicides. With the aim of developing rapid analytical screening methods for this relevant compound, we herein report the synthesis of new boscalid mimics and the study of their suitability for the production of polyclonal antibodies. Aliphatic spacer arms equivalent in length and composition were tethered at two different aromatic rings of the target molecular structure. These haptens, besides being used for immunization, were employed in the development of heterologous competitive enzyme-linked immunosorbent assays (cELISAs) in order to improve assay detectability. Direct and indirect immunoassays were tailored and applied to the determination of samples with incurred boscalid residues. The assays were characterized in terms of sensitivity, specificity, trueness, and precision. Limit of quantification was established at 5 µg kg(-1), coefficients of variation were lower than 20%, and recoveries from spiked samples ranged from 90 to 137%. Finally, ELISA performance was evaluated by Deming regression analysis with tomato and cucumber samples, selecting ultra-performance liquid chromatography-mass spectrometry as the reference method. The results showed that the proposed cELISAs are useful for the routine determination of boscalid fungicides in foods with high-sample throughput and affordable cost.
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Compuestos de Bifenilo/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Contaminación de Alimentos/análisis , Fungicidas Industriales/análisis , Niacinamida/análogos & derivados , Animales , Anticuerpos/inmunología , Anticuerpos Inmovilizados/inmunología , Compuestos de Bifenilo/inmunología , Cucumis sativus/química , Femenino , Haptenos/inmunología , Límite de Detección , Solanum lycopersicum/química , Modelos Moleculares , Niacinamida/análisis , Niacinamida/inmunología , ConejosRESUMEN
Much recent research has demonstrated that honokiol, a phenolic compound originally isolated from Magnolia officinalis, has potent anticancer activities; however, the detailed molecular mechanism of its anti-inflammatory activity has not yet been fully addressed. In this study we demonstrated that honokiol inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha secretion in macrophages, without affecting the activity of the tumor necrosis factor-alpha converting enzyme. At the same time, honokiol not only inhibited nitric oxide expression in LPS-stimulated murine macrophages but also inhibited the LPS-induced phosphorylation of ERK1/2, JNK1/2, and p38. By means of confocal microscope analysis we demonstrated that phosphorylation and membrane translocation of protein kinase C-alpha, as well as NF-kappaB activation, were inhibited by honokiol in LPS-stimulated macrophages. Furthermore, it was found that honokiol neither antagonizes the binding of LPS to cells nor alters the cell surface expression of toll-like receptor 4 and CD14. Our current results have exhaustively described the anti-inflammatory properties of honokiol, which could lead to the possibility of its future pharmaceutical application in the realm of immunomodulation.
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Antiinflamatorios/farmacología , Compuestos de Bifenilo/farmacología , Lignanos/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico/inmunología , Proteína Quinasa C/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/inmunología , Animales , Antiinflamatorios/inmunología , Compuestos de Bifenilo/inmunología , Línea Celular , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Humanos , Lignanos/inmunología , Lipopolisacáridos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/inmunología , FN-kappa B/inmunología , Proteína Quinasa C/inmunología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
CD156 is a member of a family proteins characterized by a disintegrin and a metalloprotease domain (ADAM). These molecules are phylogenically conserved but have individual roles in a variety of cells. Here, Shunsuke Yamamoto and colleagues discuss data suggesting that ADAM family proteins have important roles in the immune system.
Asunto(s)
Antígenos CD , Desintegrinas/inmunología , Metaloendopeptidasas/inmunología , Proteínas ADAM , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Benzoatos/inmunología , Benzoatos/metabolismo , Compuestos de Bifenilo/inmunología , Compuestos de Bifenilo/metabolismo , Adhesión Celular , Movimiento Celular , Desintegrinas/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Datos de Secuencia MolecularRESUMEN
In the present study, we assessed the reactivity with seven anti-angiotensin II monoclonal antibodies of three nonpeptide and one peptide compounds described as selective antagonists of angiotensin II for AT1 (DuP 753, 2-n-butyl-4-chloro-5-(hydroxymethyl)-1-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl] methyl] imidazole; EXP 3174, 2-n-butyl-4-chloro-5-(carboxylic acid)-1-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl] methyl] imidazole) and AT2 receptor sites (CGP42112A, nicotinyl-Tyr-(N alpha-benzyloxycarbonyl-Arg)Lys-His-Pro-Ile-OH; PD123177, 1-[(4-amino-3-methylphenyl) methyl]-5-(diphenyl-acetyl)-4,5,6,7-tetrahydro-1H-imidazol[4,5-c] pyridine 6-carboxylic acid), respectively. These studies were undertaken because the reactivity of the monoclonal antibodies with peptide analogs of angiotensin II and the three-dimensional structure of an angiotensin II-immunoglobulin Fab fragment complex strongly suggested that the conformations identified by the monoclonal antibodies were relevant to those involved in receptor binding as defined by biophysical models supported by structure activity studies. Surprisingly although three of the compounds were described as competitive inhibitors of angiotensin II, binding of the various monoclonal antibodies to either ovalbumin-coupled angiotensin II adsorbed to plastic wells or 125I-labeled angiotensin II in liquid phase was unaffected by any of the nonpeptide antagonists and CGP42112A up to 10(-4) M concentration. The antagonists also failed to bind to rabbit polyclonal anti-angiotensin II antibodies. Direct binding experiments in which solid phase-immobilized angiotensin II and DuP 753 conjugates were incubated with anti-angiotensin II or anti-DuP 753 monoclonal antibodies, did not show any cross-reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , 1-Sarcosina-8-Isoleucina Angiotensina II/inmunología , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Angiotensina II/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Antihipertensivos/farmacología , Compuestos de Bifenilo/inmunología , Imidazoles/inmunología , Imidazoles/farmacología , Técnicas In Vitro , Indicadores y Reactivos , Radioisótopos de Yodo , Losartán , Conformación Molecular , Oligopéptidos/farmacología , Piridinas/farmacología , Ratas , Receptores de Angiotensina/inmunología , Saralasina/inmunología , Saralasina/metabolismo , Tetrazoles/inmunología , Tetrazoles/farmacologíaRESUMEN
Brequinar sodium (BQR) has been shown recently to be a potent immunosuppressive agent. This property has been attributed to the capacity of BQR to inhibit de novo pyrimidine nucleoside biosynthesis and consequently, to blockade the synthesis both of DNA and RNA. The influence of this new immunosuppressant on lymphocyte function has not been fully characterized. To determine the potential efficacy of BQR for the control of antibody-mediated graft rejection, which is of particular significance in the context of xenotransplantation, we have examined the influence of the drug on interleukin-6-dependent IgM production by the human B-cell line, SKW 6.4. At concentrations up to 10 micrograms/ml, BQR did not affect concanavalin A (Con A)-induced human peripheral blood lymphocyte proliferation or IL-6 production by blood mononuclear leucocytes. In contrast, the drug was very effective in inhibiting IL-6-stimulated IgM production by SKW 6.4 cells, with an optimal inhibitory concentration of 0.3 microgram/ml. As expected, addition of exogenous uridine (0.1 mM), the precursor of uridine triphosphate (UTP), reversed the inhibitory effect of BQR on antibody production, while cytidine (0.1 mM) potentiated the inhibitory activity of the drug. It was further demonstrated that the inhibition of IgM production was unrelated to DNA synthesis, indicating that BQR may affect IL-6 signal transduction and IgM production in SKW 6.4 cells independent of any effect on cell proliferation.
Asunto(s)
Linfocitos B/inmunología , Compuestos de Bifenilo/inmunología , Inmunoglobulina M/biosíntesis , Inmunosupresores/inmunología , Interleucina-6/inmunología , Diferenciación Celular , Línea Celular , Concanavalina A/inmunología , Citidina/inmunología , ADN/biosíntesis , Humanos , Pirimidinas/inmunología , Uridina/inmunologíaRESUMEN
Anti-angiotensin II (Ang) antibodies could become important receptor mimicking tools if an antibody with binding properties identical to a particular Ang receptor could be generated. For this purpose, anti-Ang sera from mice were screened for antibodies with structure-affinity relationships similar or identical to a particular Ang receptor. Mice were immunized with BSA-coupled [Sar1]Ang and the sera were screened in ELISA for crossreactivity with the Ang analogues saralasin, L158,809, EXP 3147, DuP 753, DuP 532, PD 123177, PD 123319 and the non-related compounds ACTH, naloxone, and CP 96,345. All sera had at least some cross-reactivity with saralasin and some also with L158,809, a potent non-peptide Ang antagonist, selective for the AT1 site. One serum out of eight recognized most Ang analogues except the AT2 selective PD 123177 and PD 123319. ELISA detection antigens were prepared by two different BSA conjugations: [Sar1]Ang was N-terminally attached and [Sar1,Lys8]Ang was C-terminally attached. Against both detection antigens, the peptide antagonists saralasin and [Sar1,Phe(Br5)8]Ang displaced in a sigmoidal manner the antibodies with an IC50-value of 0.4 mM. L158,809 and EXP 3147 displaced also in a parallel manner, suggesting an apparently homogenous population of binding sites. The selectivity profile of the serum has some resemblance to the AT1 selectivity profile but the observed affinities are too low to suggest AT1 receptor mimicry.
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Angiotensina II/inmunología , Antagonistas de Receptores de Angiotensina , Anticuerpos/inmunología , Hormona Adrenocorticotrópica/inmunología , Secuencia de Aminoácidos , Angiotensina II/análogos & derivados , Animales , Compuestos de Bifenilo/inmunología , Compuestos de Bifenilo/metabolismo , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Imidazoles/inmunología , Imidazoles/metabolismo , Losartán , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Naloxona/inmunología , Piridinas/metabolismo , Saralasina/inmunología , Saralasina/metabolismo , Relación Estructura-Actividad , Tetrazoles/inmunología , Tetrazoles/metabolismoRESUMEN
Two murine monoclonal antibodies were produced to losartan (DuP 753), a nonpeptide angiotensin II receptor antagonist. Using a solid phase competitive enzyme-linked immunosorbent assay (ELISA), each antibody was examined for its ability to bind to a set of losartan analogs that differ structurally in varying degrees. Both antibodies distinguished fine structural changes in the analogs, particularly at the R5 position of the imidazole ring. No cross-reactivity towards either antibody was observed with the natural ligand angiotensin II, the peptide antagonist saralasin, or the AT2 selective nonpeptide antagonist PD123177.
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Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Anticuerpos Monoclonales , Compuestos de Bifenilo/inmunología , Imidazoles/inmunología , Tetrazoles/inmunología , Angiotensina II/antagonistas & inhibidores , Angiotensina II/inmunología , Animales , Especificidad de Anticuerpos , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Losartán , Ratones , Ratones Endogámicos BALB C , Piridinas/inmunología , Saralasina/inmunología , Saralasina/metabolismo , Relación Estructura-ActividadAsunto(s)
Compuestos de Bifenilo/inmunología , Linfocitos/efectos de los fármacos , Bifenilos Polibrominados/inmunología , Animales , Colesterol/sangre , Femenino , Lactancia/efectos de los fármacos , Lipoproteínas/sangre , Intercambio Materno-Fetal , Leche/análisis , Bifenilos Polibrominados/análisis , Embarazo , PorcinosAsunto(s)
Compuestos de Bifenilo/metabolismo , Hidrocarburos Clorados/metabolismo , Sueros Inmunes/farmacología , Bifenilos Policlorados/metabolismo , Animales , Compuestos de Bifenilo/inmunología , Femenino , Hidrocarburos Clorados/inmunología , Ratones , Bifenilos Policlorados/inmunología , Conejos/inmunología , Distribución Tisular/efectos de los fármacosRESUMEN
The intactness of the immune system in cattle exposed to polybrominated biphenyls (PBBs) has been investigated by using several immunoassays. Eighty-seven animals have been studied, 35 control animals (not exposed to PBBs) and 52 animals exposed to PBBs (0.02-30 ppm/g fat equivalent). The immunoassays included a complete blood count, identification of peripheral blood T and B lymphocyte subpopulations, serum immunoglobulin levels (IgG, IgM, and IgA), the in vitro response to lymphocytes to phytolectins (PHA, Con A, PWM), the antibody response to Keyhole limpet hemocyanin (KLH), the cell-mediated response to PPD, and determination of autoantibodies and/or immunosuppressive serum factors. For control and PBB-exposed cattle, there was no statistical difference between the number of circulating erythrocytes or leukocytes, the hematocrit, or hemoglobin content; the percentage or number of T and B lymphocytes; the isotope incorporation index (DNA synthesis) of lymphocytes in response to mitogens; the concentrations of serum immunoglobulins IgG, IgM, or IgA; the mean peak titer to KLH; or in vivo or in vitro immune response to PPD.Additional evaluation of cattle with tissue levels of PBB greater than 3 ppm/g tissue for hematological and immunological parameters revealed no statistical difference from control animals. Other experiments were performed to evaluate serum from cattle exposed to PBBs for autoantibodies to smooth muscle, mitochondrial or nuclear antigens. No evidence for autoantibodies was observed. Further studies were done to examine the cytotoxic and/or immunosuppressive activity of sera from PBB-exposed animals. In these studies, the blastogenic response of lymphocytes from control cattle and humans were evaluated in the presence and absence of serum from animals exposed to PBBs (> 3 ppm/g tissue). No evidence for either a cytotoxic or an immunosuppressive influence of such sera was demonstrable. Our studies indicate that PBB, at the levels studied, does not alter or interfere with lymphocyte surface antigens, the complex nuclear and cytoplasmic events required for mitosis and cell division, or the biological events required for antibody formation and cell-mediated immune reactions. Further, PBB exposure at the levels studied does not predispose cattle to autoantibody production or leucotoxic serum factors.
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Compuestos de Bifenilo/inmunología , Bovinos/inmunología , Bifenilos Polibrominados/inmunología , Animales , Anticuerpos , Membrana Celular/inmunología , Células Cultivadas , ADN/biosíntesis , Exposición a Riesgos Ambientales , Pruebas Hematológicas , Inmunoglobulina A , Inmunoglobulina G , Inmunoglobulina M , Linfocitos/inmunología , Mitógenos/farmacologíaAsunto(s)
Dermatitis por Contacto/etiología , Resinas Epoxi/efectos adversos , Aminas/efectos adversos , Aminas/inmunología , Compuestos de Bifenilo/efectos adversos , Compuestos de Bifenilo/inmunología , Reacciones Cruzadas , Resinas Epoxi/inmunología , Anteojos , Femenino , Humanos , Persona de Mediana Edad , Pruebas del ParcheRESUMEN
The effect of elevation of an intracellular cyclic AMP level on in vitro anti-hapten antibody response was studied, by using mesenteric lymph node cells of rabbits which were primed with dinitrophenylated Ascaris antigen (DNP-Asc) or DNP-ragweed antigen (DNP-Rag). The anti-hapten antibody response was induced by stimulation of the primed B cells by either DNP-heterologous carrier conjugate or anti-immunoglobulin (anti-Ig) for 24 hr (first stage), followed by 6-day culture of the activated cells in the presence of nonspecific enhancing factor (second stage). The stimulation with anti-Ig induced IgG anti-hapten antibody response and enhanced the formation of total IgG. Addition of dibutyryl cyclic AMP or aminophylline with anti-Ig or DNP-heterologous carrier during the first stage enhanced IgG anti-hapten antibody response. The optimal concentration of these reagents for the enhancement was 5 x 10(-4) M to 10(-3) M. The presence of 5 x 10(-6) M prostaglandin E1 during the first stage also enhanced the antibody response. Similarly, the presence of dibutyryl cyclic AMP or aminophylline during the stimulation of DNP-Rag-primed cells with homologous antigen (first stage) enhanced the antibody response. If the same concentration of dibutyryl cyclic AMP or aminophylline was added together with enhancing soluble factor during the second stage after the stimulation of the primed cells with anti-Ig, both the antibody response and the formation of IgG were suppressed. The antibody response of DNP-Rag-primed cells stimulated with homologous antigen was also suppressed if dibutyryl cyclic AMP or aminophylline was added during the subsequent culture (second stage). Evidence was obtained that suppression of antibody response by cyclic AMP during the second stage is probably due to inhibition of the proliferation of B cells. Neither of these drugs suppressed the formation of enhancing soluble factor from the carrier-specific cells stimulated with the homologous carrier. The results obtained in the present experiments suggested that stimulation of hapten-primed B cells with anti-gamma chain in the presence of an optimal concentration of dibutyryl cyclic AMP resulted in the formation of a significant amount of IgG anti-DNP antibody without participation of T cells.
Asunto(s)
Anticuerpos Antiidiotipos , Formación de Anticuerpos/efectos de los fármacos , AMP Cíclico/farmacología , Haptenos , Animales , Ascaris/inmunología , Compuestos de Bifenilo/inmunología , Bucladesina/farmacología , Sistema Libre de Células , ADN/biosíntesis , Hemocianinas/inmunología , Fragmentos Fab de Inmunoglobulinas , Técnicas In Vitro , Polen , Conejos , Factores de TiempoRESUMEN
Using dinitrochlorbenzene contact-sensitized guinea pigs, several DNP conjugates have been assayed in the direct macrophage migration inhibition test (MMIT). Although no significant differences could be observed between the carriers used, conjugates prepared from serum proteins and epidermal extracts tended to give the strongest inhibition of macrophage migration. Conjugates prepared from cells cultured in vitro in the presence of hapten did not cause more inhibition than control conjugates prepared in the absence of cell metabolism. The direct MMIT showed statistical differences between sensitized and nonsensitized groups of guinea pigs. However, none of the conjugates permitted conclusions to be drawn with regard to individual animals.