RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of Smilax campestris Griseb (Smilacaceae) have been employed in the treatment of several inflammatory diseases as a traditional herbal medicine. However, the cellular and molecular mechanisms involved in the observed effects remain elusive. Macrophages are known to play a central role in inflammatory responses. These cells are activated in response to a diversity of danger signals and produce several mediators of inflammation that eventually regulate the immune response. For all the above mentioned, scientific evidence is required to support the popular use of S. campestris. AIM OF THE STUDY: We aimed to investigate the anti-inflammatory effect of S. campestris aqueous extract (SME) in activated THP-1 human macrophages, on the production of some mediators of inflammation and oxidative stress in order to provide scientific support for its popular use. MATERIALS AND METHODS: The characterization of SME was assessed by HPLC-MS/MS. The production of the pro-inflammatory cytokines and chemokines was evaluated by ELISA. The activity of metalloproteases was evaluated by zymography. The subcellular localization of the NF-κB transcription factor was analysed by Western blot. The superoxide anion and glutathione levels were assessed by flow cytometry. The cytotoxicity induced by SME in THP-1 macrophages was also investigated by the LDH release test. RESULTS: In the present study, we have identified catechin and glycosylated derivatives of quercetin (quercetin-3-O-glucoside, quercetin-3-O-galactoside, rutin and quercetin-3-rhamnoside) as major components of the aqueous SME. We found that SME significantly decreased the production of the pro-inflammatory cytokines tumour necrosis factor (TNF)- α, interleukin (IL)-1ß, IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 and the activity of the metalloproteinase (MMP)-9, in lipopolysaccharide-activated macrophages derived from the monocytic cell line THP-1. Furthermore, SME diminished the expression of NF-κB p65 subunit in the nuclear fraction. In addition, SME decreased the production of superoxide anion in THP-1 macrophages, without altering the levels of reduced glutathione. CONCLUSION: These results suggest that SME exerts its anti-inflammatory effects in human activated macrophages by inhibiting the production of pro-inflammatory cytokines, matrix metalloproteinases and the NF-κB transcription factor pathway along with a reduction of oxidative stress mediators. Moreover, catechin and glycosylated derivatives of were identified by HPLC-MS/MS in SME. Our findings provide scientific support for the traditional use of the S. campestris extracts.
Asunto(s)
Antiinflamatorios/farmacología , Flavonoides/farmacología , Macrófagos/efectos de los fármacos , Extractos Vegetales/farmacología , Smilax/química , Antiinflamatorios/análisis , Antiinflamatorios/aislamiento & purificación , Argentina , Línea Celular , Citocinas/inmunología , Citocinas/metabolismo , Etnofarmacología , Flavonoides/análisis , Flavonoides/aislamiento & purificación , Glutatión/metabolismo , Humanos , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Medicina Tradicional/métodos , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/análisis , Extractos Vegetales/aislamiento & purificación , Superóxidos/metabolismo , Pruebas de Toxicidad , Agua/químicaRESUMEN
Heavy chain antibodies (HCAbs), devoid of the light chains and the CH(1) domain, are present in the serum of camelids. IgG(2) and IgG(3) are HCAbs; whereas IgG(1) has the conventional structure. In order to study the immunological properties of llama HCAbs, from which to date little is known, llamas (Lama glama) HCAbs cDNA were cloned, sequenced and compared with other mammalian Igs. The sequence analysis showed that llama HCAbs cDNA organization is similar to other mammalian Igs and the presence of conserved binding motifs to Protein A, Protein G, FcγRI, FcγRIII and C1q in HCAbs were observed. In a previous work, different IgG isotypes purified by Protein A and Protein G chromatography, were assayed for their ability to fix complement. Both IgG(1) and the total serum were able to fix complement, whereas IgG(2) and IgG(3) fixed complement even in the absence of antigen (anti-complementary activity). Therefore, in this work we performed the complement activating activity of the different IgG isotypes purified under physiological conditions using Sephadex G-150 and their ability to induce hemagglutination. Llamas were immunized with sheep red blood cells (RBC) stroma and the different isotypes were purified from sera. Whole serum and IgG(1) could activate complement; however, HCAbs (IgG(2)+IgG(3)) could not, despite the presence of the C1q binding motif in their primary sequence. Unlike IgG(1), the fraction corresponding to IgG(2)+IgG(3) did not display hemagglutinating activity. Our findings suggest that HCAbs cannot crosslink efficiently with different antigens and that the C1q binding site might be hindered by the proximity of the variable domains.
Asunto(s)
Camélidos del Nuevo Mundo/inmunología , Activación de Complemento/inmunología , Hemaglutinación/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Camélidos del Nuevo Mundo/genética , Clonación Molecular , Activación de Complemento/genética , Secuencia Conservada/genética , Hemaglutinación/genética , Pruebas de Hemaglutinación/veterinaria , Cadenas Pesadas de Inmunoglobulina/inmunología , Alineación de SecuenciaRESUMEN
Hyaluronan (HA) modulates multidrug resistance (MDR) as well as cell migration. Tiam1 is involved in cytoskeleton reorganization during tumor invasion. In this report we show the relationship among HA, Tiam1, migration and MDR in murine lymphoma cell lines. We observed that MDR cells presented higher migratory capacity towards HA in vitro as well as higher constitutive active Tiam1 expression than the sensitive cell line. Besides, HA treatment induced migration towards HA of MDR cell lines through Tiam1 activation by a PI3K-dependent mechanism, showing that disruption of HA signaling would be useful in treatment of MDR hematological malignancies.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ácido Hialurónico/farmacología , Linfoma/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Línea Celular Tumoral , Linfoma/patología , Ratones , Transducción de Señal , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-TRESUMEN
Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chain antibodies, HCAbs). Three IgG isotypes have been identified (IgG(1), IgG(2) and IgG(3)); IgG(2) and IgG(3) are HCAbs whereas the IgG(1) has the conventional structure. Different subtypes of IgG(1) (IgG(1a) and IgG(1b)) and IgG(2) (IgG(2a), IgG(2b) and IgG(2c)) have been classified according to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred as VHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbs has been a limitation for their quantification. In a previous work, we produced specific polyclonal antibodies against IgG(2a), IgG(2b), IgG(2c) and IgG(3) by immunizing rabbits with synthetic and recombinant peptides corresponding to their hinge region. In this work we produced specific antisera against llama IgM and IgG(1). The anti-IgG(1) serum was obtained by immunizing rabbits with a recombinant fusion protein formed by GST fused to the CH(1) domain of the IgG(1). The anti-IgM serum was obtained by immunizing rabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgG and IgG subclasses. Sera from llamas (n=20) analyzed by ELISA gave the following values of immunoglobulins: IgG(1)=6.168+/-1.628 mg/ml; IgG(2)=0.684+/-0.310 mg/ml; IgG(3)=1.232+/-0.410 mg/ml; total IgG=8.933+/-1.815 mg/ml and IgM=1.027+/-0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG(3) subtype is the predominant HCAb. We also analyzed the primary humoral immune response after immunization llamas with different antigens (BSA, BSA-DNP and dextran). Although it has been described that a few VHH clones are very efficient in the interaction with haptens, in this case the response against DNP was characterized by a delayed appearance of HCAbs in comparison with that of IgG(1). No anti-dextran response was observed in any of the isotypes analyzed.
Asunto(s)
Formación de Anticuerpos/fisiología , Antígenos/inmunología , Camélidos del Nuevo Mundo/sangre , Ensayo de Inmunoadsorción Enzimática/veterinaria , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Animales , Camélidos del Nuevo Mundo/inmunología , Dextranos/inmunología , Dinitrobencenos/inmunología , Femenino , Regulación de la Expresión Génica , Inmunoglobulina G/clasificación , Inmunoglobulina M/clasificación , Masculino , Albúmina Sérica Bovina/inmunologíaRESUMEN
We describe an easy method for the production of small recombinant peptides of 8 amino acid residues expressed as a fusion peptide with glutathione S-transferase (GST) and employed in an immunisation schedule to obtain polyclonal antibodies. The chosen peptides corresponded to specific fragments of the hinge regions of llama (Lama glama) IgG2 subisotypes b (2bH) and c (2cH). The DNA sequences encoding each peptide were ligated individually into pGEX-5X-2, which encodes GST. Once purified from a bacterial lysate by glutathione affinity chromatography, GST-2bH and GST-2cH were used to immunize rabbits. In parallel, polyclonal antibodies were generated against specific synthetic fragments of the hinge regions of llama IgG2a (2aH) and IgG3 (3H) coupled to keyhole limpet haemocyanin (KLH). Polyclonal antibodies raised against GST-peptides and KLH-peptides were detected by enzyme-linked immunosorbent assay (ELISA), Western blot (WB) and indirect immunofluorescence (IIF). The results obtained by ELISA demonstrated that monospecific anti-IgG2 and anti-IgG3 antisera were obtained using KLH-2aH, GST-2bH, GST-2cH and KLH-3H as antigens. All antisera showed reactivity with their specific IgG isotype by WB and IIF. This simple and novel recombinant DNA methodology for the generation of two monospecific anti-isotype antisera using small peptides expressed as fusion peptides with GST offers the possibility of large scale peptide production as an alternative to chemical peptide synthesis.
Asunto(s)
Camélidos del Nuevo Mundo/inmunología , Sueros Inmunes/inmunología , Inmunoglobulina G/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Vectores Genéticos/genética , Exones de la Región Bisagra/genética , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Linfocitos/química , Linfocitos/inmunología , Microscopía Fluorescente , Péptidos/genética , Péptidos/inmunología , Conejos , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
The antigenic protein Ro52 was expressed in the E. coli system harboring a 6 x His tag in the form of insoluble inclusion bodies. Direct chemical extraction of the product using 6-8 M urea proved to be effective. Furthermore, the tagged protein was recovered by direct adsorption on Ni2+-loaded commercial adsorbents derivatized with iminodiacetic acid. Screening experiments in small packed columns revealed that selective binding and elution were possible using a denaturing buffer at pH 4.5. The hydrodynamic evaluation of scaled-up fluidized systems showed values for the phi (dynamic zone) parameter in the range 0.95-1.00 for fluidization in buffer and in the range 0.70-0.85 for the biomass-containing feedstock. Removal of macromolecular DNA released by the disrupted biomass was mandatory. Under optimized process conditions good recovery (60-70%) was achieved and a highly purified (95%) product obtained. The purified Ro52 retained its immunoreactivity against sera of patients with systemic lupus erythematosus (SLE) and Sjogren's syndrome-related disorders. The production and application of new recombinant antigens may contribute to increasing the sensitivity and specificity of the detection of anti-Ro antibodies in these autoimmune diseases.