RESUMEN

Flagellin is the major component of the flagellum in gram-positive and -negative bacteria and is also the ligand for the Toll-like receptor 5 (TLR5). The activation of TLR5 promotes the expression of proinflammatory cytokines and chemokines and the subsequent activation of T cells. This study evaluated a recombinant domain from the amino-terminus D1 domain (rND1) of flagellin from Vibrio anguillarum, a fish pathogen, as an immunomodulator in human peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MoDCs). We demonstrated that rND1 induced an upregulation of proinflammatory cytokines in PBMCs, characterized at the transcriptional level by an expression peak of 220-fold for IL-1ß, 20-fold for IL-8, and 65-fold for TNF-α. In addition, at the protein level, 29 cytokines and chemokines were evaluated in the supernatant and were correlated with a chemotactic signature. MoDCs treated with rND1 showed low levels of co-stimulatory and HLA-DR molecules and kept an immature phenotype with a decreased phagocytosis of dextran. We probed that rND1 from a non-human pathogen promotes modulation in human cells, and it may be considered for further studies in adjuvant therapies based on pathogen-associated patterns (PAMPs).

Asunto(s)

Quimiotaxis de Leucocito , Flagelina , Humanos , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Dendríticas , Flagelina/genética , Flagelina/farmacología , Leucocitos Mononucleares/metabolismo , Fenotipo , Proteínas de Unión al GTP rho/metabolismo , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/metabolismo

RESUMEN

The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Purinas/farmacología , Receptor Smoothened/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HT29 , Proteínas Hedgehog/metabolismo , Humanos , Ratones Endogámicos C57BL , Neoplasias/metabolismo , Purinas/química , Purinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/metabolismo

RESUMEN

Chile has one of the worst numbers worldwide in terms of SARS-CoV-2 positive cases and COVID-19-related deaths per million inhabitants; thus, characterization of neutralizing antibody (NAb) responses in the general population is critical to understanding of immunity at the local level. Given our inability to perform massive classical neutralization assays due to the scarce availability of BSL-3 facilities in the country, we developed and fully characterized an HIV-based SARS-CoV-2 pseudotype, which was used in a 96-well plate format to investigate NAb responses in samples from individuals exposed to SARS-CoV-2 or treated with convalescent plasma. We also identified samples with decreased or enhanced neutralization activity against the D614G spike variant compared with the wild type, indicating the relevance of this variant in host immunity. The data presented here represent the first insights into NAb responses in individuals from Chile, serving as a guide for future studies in the country.

Asunto(s)

Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Prueba Serológica para COVID-19 , COVID-19 , Mutación Missense , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Sustitución de Aminoácidos , Animales , COVID-19/sangre , COVID-19/genética , Chile , Chlorocebus aethiops , Femenino , Células HEK293 , Humanos , Masculino , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/sangre , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero

RESUMEN

We synthesized a new family of six 4(3H)quinazolinimines based on the reaction between (E)-N-(2-cyanophenyl)benzimidoyl chloride and substituted anilines reaching the formation of their corresponding C2, N3-substituted quinazoliniminium chlorides. This method provides novel, direct and flexible access to diverse substituted 4(3H)quinazolinimines. New compounds obtained following the proposed synthesis were fully characterized and, including the thirteen 4(3H)quinazolinimines synthesized by this method and previously reported by us, were used to study its cytotoxic effect on neoplastic cell lines. The mechanism involved in cell toxicity was also studied. Results showed that these compounds were highly cytotoxic, in particular on Human Promyelocytic Leukemia cells (HL60) and Chronic Myelogenous Leukemia cells (K562) when compared with conventional antineoplastic drugs such as etoposide and cisplatin. The mechanism associated to cytotoxic effect was mainly apoptosis, which not was decreased by antioxidant addition, thereby suggesting that the compounds exert apoptotic death through a mechanism unrelated with oxidative stress.

Asunto(s)

Antineoplásicos/síntesis química , Quinazolinonas/química , Antineoplásicos/química , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Estrés Oxidativo/efectos de los fármacos , Quinazolinonas/síntesis química , Quinazolinonas/toxicidad , Relación Estructura-Actividad

RESUMEN

Exposure to high levels of glucocorticoids (GCs) during early life induces long-lasting neuroinflammation. GCs induce rapid degranulation of mast cells, which release proinflammatory molecules promoting activation of microglia and astrocytes. The possible involvement of oligodendrocytes, however, remains poorly understood. It was studied whether high GC levels during gestation activates the inflammasome in hippocampal oligodendrocytes of mouse offspring. Oligodendrocytes of control pups showed expression of inflammasome components (NLRP3, ACS, and caspase-1) and their levels were increased by prenatal administration of dexamethasone (DEX), a synthetic GC. These cells also showed high levels of IL-1ß and TNF-α, revealing activation of the inflammasome. Moreover, they showed increased levels of the P2X7 receptor and pannexin1, which are associated to inflammasome activation. However, levels of connexins either were not affected (Cx29) or reduced (Cx32 and Cx47). Nonetheless, the functional states of pannexin1 and connexin hemichannels were elevated and directly associated to functional P2X7 receptors. As observed in DEX-treated brain slices, hemichannel activity first increased in hippocampal mast cells and later in microglia and macroglia. DEX-induced oligodendrocyte hemichannel activity was mimicked by urocortin-II, which is a corticotropin-releasing hormone receptor (CRHR) agonist. Response to DEX and urocortin-II was inhibited by antalarmin (a CRHR blocker) or by mast cells or microglia inhibitors. The increase in hemichannel activity persisted for several weeks after birth and cross-fostering with a control mother did not reverse this condition. It is proposed that activation of the oligodendrocyte inflammasome might be relevant in demyelinating diseases associated with early life exposure to high GC levels. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 625-642, 2017.

Asunto(s)

Glucocorticoides/metabolismo , Hipocampo/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Oligodendroglía/metabolismo , Animales , Animales Recién Nacidos , Conexinas/metabolismo , Femenino , Edad Gestacional , Glucocorticoides/efectos adversos , Inflamación/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Neuroinmunomodulación , Embarazo

RESUMEN

High incidence of Rho Cdc42-GTPase overexpression has been found in Colorectal Cancer (CRC) samples, suggesting its potential role in tumor development. However, no conclusive studies have shown the lack of mutations and/or copy number of Cdc42 gene in this type of samples. To understand mutation/deletion and copy number status of Cdc42 gene, CRC patients were evaluated for both parameters. More than Cdc42 mutants, single-nucleotide variants were found. Analysis of regions flanking the Cdc42 gene showed allelic imbalance; 58.7% were loss of heterozygosity (LOH) positive and 14.8% presented microsatellite instability. The highest LOH percentage was located between microsatellite markers D1S199 and D1S2674, where the Cdc42 gene is located. No association between gender, age, and tumor stage was found. LOH validation through gene dosage analysis showed most CRC patients with allelic imbalance also presented a low gene dosage of Cdc42, although equal amounts of Cdc42 mRNA were detected in all samples. Although changes in Cdc42 expression were not found in any condition, Cdc42 activation was different between high and normal gene dosage samples, but not between samples with normal and low copy number. Low dosage of Cdc42 was also not related to changes in methylation status at the Cdc42 promoter region. Results suggest that low copy of Cdc42 gene is not associated with Cdc42 protein dysfunction in CRC patients.

Asunto(s)

Neoplasias Colorrectales/genética , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Pérdida de Heterocigocidad , Proteína de Unión al GTP cdc42/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Estadificación de Neoplasias , Estudios Retrospectivos , Eliminación de Secuencia , Proteína de Unión al GTP cdc42/deficiencia

RESUMEN

A series of 2,6,9-trisubstituted purine derivatives have been synthesized and investigated for their potential role as antitumor agents. Twelve compounds were obtained by a three step synthetic procedure using microwave irradiation in a pivotal step. All compounds were evaluated in vitro to determine their potential effect on cell toxicity by the MTT method and flow cytometry analysis on four cancer cells lines and Vero cells. Three out of twelve compounds were found to be promising agents compared to a known and effective anticancer drug, etoposide, in three out of four cancer cell lines assayed with considerable selectivity. Preliminary flow cytometry data suggests that compounds mentioned above induce apoptosis on these cells. The main structural requirements for their activity for each cancer cell line were characterized with a preliminary pharmacophore model, which identified aromatic centers, hydrogen acceptor/donor center and a hydrophobic area. These features were consistent with the cytotoxic activity of the assayed compounds.

Asunto(s)

Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Modelos Moleculares , Purinas/química , Purinas/farmacología , Animales , Antineoplásicos/síntesis química , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Estructura Molecular , Purinas/síntesis química , Relación Estructura-Actividad , Células Vero

RESUMEN

Autocrine and paracrine signals coordinate responses of several cell types of the immune system that provide efficient protection against different challenges. Antigen-presenting cells (APCs) coordinate activation of this system via homocellular and heterocellular interactions. Cytokines constitute chemical intercellular signals among immune cells and might promote pro- or anti-inflammatory effects. During the last two decades, two membrane pathways for intercellular communication have been demonstrated in cells of the immune system. They are called hemichannels (HCs) and gap junction channels (GJCs) and provide new insights into the mechanisms of the orchestrated response of immune cells. GJCs and HCs are permeable to ions and small molecules, including signaling molecules. The direct intercellular transfer between contacting cells can be mediated by GJCs, whereas the release to or uptake from the extracellular milieu can be mediated by HCs. GJCs and HCs can be constituted by two protein families: connexins (Cxs) or pannexins (Panxs), which are present in almost all APCs, being Cx43 and Panx1 the most ubiquitous members of each protein family. In this review, we focus on the effects of different cytokines on the intercellular communication mediated by HCs and GJCs in APCs and their impact on purinergic signaling.

Asunto(s)

Células Presentadoras de Antígenos/metabolismo , Conexinas/metabolismo , Citocinas/metabolismo , Animales , Uniones Comunicantes/metabolismo , Humanos

RESUMEN

Natural rubber latex (NRL; Hevea brasiliensis) allergy is an IgE-mediated reaction to latex proteins. When latex glove exposure is the main sensitizing agent, Hev b 5 is one of the major allergens. Dendritic cells (DC), the main antigen presenting cells, modulated with pharmacological agents can restore tolerance in several experimental models, including allergy. In the current study, we aimed to generate DC with tolerogenic properties from NRL-allergic patients and evaluate their ability to modulate allergen-specific T and B cell responses. Here we show that dexamethasone-treated DC (dxDC) differentiated into a subset of DC, characterized by low expression of MHC class II, CD40, CD80, CD86 and CD83 molecules. Compared with LPS-matured DC, dxDC secreted lower IL-12 and higher IL-10 after CD40L activation, and induced lower alloantigenic T cell proliferation. We also show that dxDC pulsed with the dominant Hev b 5 T-cell epitope peptide, Hev b 5(46-65), inhibited both proliferation of Hev b 5-specific T-cell lines and the production of Hev b 5-specific IgE. Additionally, dxDC induced a subpopulation of IL-10-producing regulatory T cells that suppressed proliferation of Hev b 5-primed T cells. In conclusion, dxDC generated from NRL-allergic patients can modulate allergen-specific T-cell responses and IgE production, supporting their potential use in allergen-specific immunotherapy.

Asunto(s)

Alérgenos/inmunología , Células Dendríticas/inmunología , Inmunoglobulina E/inmunología , Hipersensibilidad al Látex/inmunología , Linfocitos T/inmunología , Adulto , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Plantas/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Línea Celular , Proliferación Celular , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Dexametasona/farmacología , Epítopos de Linfocito T/inmunología , Femenino , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Interleucina-10/inmunología , Interleucina-10/metabolismo , Masculino , Persona de Mediana Edad , Péptidos/inmunología , Proteínas de Plantas/inmunología , Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Adulto Joven

RESUMEN

In the central nervous system (CNS), mastocytes and glial cells (microglia, astrocytes and oligodendrocytes) function as sensors of neuroinflammatory conditions, responding to stress triggers or becoming sensitized to subsequent proinflammatory challenges. The corticotropin-releasing hormone and glucocorticoids are critical players in stress-induced mastocyte degranulation and potentiation of glial inflammatory responses, respectively. Mastocytes and glial cells express different toll-like receptor (TLR) family members, and their activation via proinflammatory molecules can increase the expression of connexin hemichannels and pannexin channels in glial cells. These membrane pores are oligohexamers of the corresponding protein subunits located in the cell surface. They allow ATP release and Ca(2+) influx, which are two important elements of inflammation. Consequently, activated microglia and astrocytes release ATP and glutamate, affecting myelinization, neuronal development, and survival. Binding of ligands to TLRs induces a cascade of intracellular events leading to activation of several transcription factors that regulate the expression of many genes involved in inflammation. During pregnancy, the previous responses promoted by viral infections and other proinflammatory conditions are common and might predispose the offspring to develop psychiatric disorders and neurological diseases. Such disorders could eventually be potentiated by stress and might be part of the etiopathogenesis of CNS dysfunctions including autism spectrum disorders and schizophrenia.

Asunto(s)

Sistema Nervioso Central/metabolismo , Mastocitos/citología , Neuroglía/metabolismo , Receptores Toll-Like/metabolismo , Adenosina Trifosfato/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Conexinas/metabolismo , Femenino , Glucocorticoides/metabolismo , Ácido Glutámico/metabolismo , Humanos , Inflamación , Ligandos , Microglía/metabolismo , Embarazo , Complicaciones del Embarazo , Transducción de Señal

RESUMEN

Fas ligation via the ligand FasL activates the caspase-8/caspase-3-dependent extrinsic death pathway. In so-called type II cells, an additional mechanism involving tBid-mediated caspase-9 activation is required to efficiently trigger cell death. Other pathways linking FasL-Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X(7) receptors (P2X(7)Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase-8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X(7)Rs participate in FasL-stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time- and caspase-8-dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL-induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL-induced death. Also, oxidized-ATP or Brilliant Blue G, two P2X(7)R blockers, reduced FasL-induced caspase-9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X(7)R connect functionally via caspase-8 and Panx1 HC-mediated ATP release to promote caspase-9/caspase-3-dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells.

Asunto(s)

Adenosina Trifosfato/fisiología , Apoptosis , Caspasa 8/fisiología , Proteína Ligando Fas/fisiología , Receptores Purinérgicos P2X7/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Apirasa/fisiología , Caspasa 3/fisiología , Caspasa 9/fisiología , Conexinas/fisiología , Humanos , Células Jurkat , Proteínas del Tejido Nervioso/fisiología , Antagonistas del Receptor Purinérgico P2X/farmacología , Colorantes de Rosanilina/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Receptor fas/fisiología

RESUMEN

Multiple applications of nanotechnology, especially those involving highly fluorescent nanoparticles (NPs) or quantum dots (QDs) have stimulated the research to develop simple, rapid and environmentally friendly protocols for synthesizing NPs exhibiting novel properties and increased biocompatibility. In this study, a simple protocol for the chemical synthesis of glutathione (GSH)-capped CdTe QDs (CdTe-GSH) resembling conditions found in biological systems is described. Using only CdCl(2), K(2)TeO(3) and GSH, highly fluorescent QDs were obtained under pH, temperature, buffer and oxygen conditions that allow microorganisms growth. These CdTe-GSH NPs displayed similar size, chemical composition, absorbance and fluorescence spectra and quantum yields as QDs synthesized using more complicated and expensive methods.CdTe QDs were not freely incorporated into eukaryotic cells thus favoring their biocompatibility and potential applications in biomedicine. In addition, NPs entry was facilitated by lipofectamine, resulting in intracellular fluorescence and a slight increase in cell death by necrosis. Toxicity of the as prepared CdTe QDs was lower than that observed with QDs produced by other chemical methods, probably as consequence of decreased levels of Cd(+2) and higher amounts of GSH. We present here the simplest, fast and economical method for CdTe QDs synthesis described to date. Also, this biomimetic protocol favors NPs biocompatibility and helps to establish the basis for the development of new, "greener" methods to synthesize cadmium-containing QDs.

Asunto(s)

Materiales Biocompatibles/síntesis química , Cadmio/química , Glutatión/química , Puntos Cuánticos , Telurio/química , Materiales Biocompatibles/química , Biomimética/métodos , Química Bioinorgánica/métodos , Concentración de Iones de Hidrógeno , Ensayo de Materiales , Nanotecnología/métodos , Espectrometría de Fluorescencia , Espectrometría por Rayos X , Espectrofotometría Infrarroja , Temperatura , Factores de Tiempo

RESUMEN

Ski is a transcriptional regulator that has been considered an oncoprotein given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski-/- mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chromatin/chromosome bridges as the major cause of micronuclei (MN) formation and the subsequent aneuploidy. Although these cells arrested in mitosis after treatment with spindle disrupting drugs and exhibited a delayed metaphase/anaphase transition, spindle assembly checkpoint (SAC) was not sufficient to prevent chromosome missegregation, consistent with a weakened SAC. Our in vivo analysis also showed dynamic metaphase plate rearrangements with switches in polarity in cells arrested in metaphase. Importantly, after ectopic expression of Ski the cells that displayed this metaphase arrest died directly during metaphase or after aberrant cell division, relating SAC activation and mitotic cell death. This increased susceptibility to undergo mitosis-associated cell death reduced the number of MN-containing cells. The presented data support a new role for Ski in the mitotic process and in maintenance of genetic stability, providing insights into the mechanism of tumor suppression mediated by this protein.

Asunto(s)

Transformación Celular Neoplásica/genética , Inestabilidad Cromosómica/genética , Proteínas de Unión al ADN/genética , Fibroblastos/patología , Proteínas Proto-Oncogénicas/genética , Animales , Separación Celular , Células Cultivadas , Embrión de Mamíferos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Immunoblotting , Ratones , Ratones Noqueados , Mitosis/genética , Transcripción Genética

RESUMEN

The use of biological agents such as etanercept, infliximab, adalimumab and anakinra has been recently approved for the treatment of rheumatoid arthritis. All are effective controlling signs and symptoms and inhibiting disease progression. To overcome the problems generated by their high costs and possible participation in reactivating latent infections, other therapeutic tools are being developed. Gene therapy using expression vectors carrying genes coding for specific proteins, may interfere in key points involved in the pathogenesis of the disease. Intra-articular administration of cDNA coding for soluble TNF receptors, IL-1, or IL-1Ra decreases signs of the disease in animal models. Vectors, expressing inhibitors of signal transduction pathways involving to NF-kB and JAK-STAT-3, are effective in modulating joint inflammation in mice. The use of antigen-pulsed antigen presenting cells or dendritic cells (DC) bound to apoptosis-inducing molecules, specifically eliminates autoreactive T cells. Other novel approach attempts the development of T regulatory-inducing tolerogenic DC-based vaccines that inhibit autoreactive T cells, through the secretion of suppressing cytokines or by other mechanisms to be elucidated. Oral tolerance induction to auto-antigens is also a successful experimental strategy under study. Current research aims to control peripheral tolerance in rheumatoid arthritis patients.

Asunto(s)

Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Quimioterapia Combinada , Terapia Genética , Humanos

RESUMEN

Tumor cells treated with IL-10 were shown to have decreased, but peptide-inducible expression of MHC class I, decreased sensitivity to MHC class I-restricted CTL, and increased NK sensitivity. These findings could be explained, at least partially, by a down-regulation of TAP1/TAP2 expression. In this study, IT9302, a nanomeric peptide (AYMTMKIRN), homologous to the C-terminal of the human IL-10 sequence, was demonstrated to mimic these previously described IL-10 effects on MHC class I-related molecules and functions. We observed a dose-dependent down-regulation of MHC class I at the cell surface of melanoma cells after 24-h treatment with IT9302. The IL-10 homologue peptide also caused a dose-dependent inhibition of the IFN-gamma-mediated surface induction of MHC class I in a melanoma cell line. We demonstrated, using Western blot and flow cytometry, that IT9302 inhibits the expression of TAP1 and TAP2 proteins, but not MHC class I H chain or low molecular protein molecules. Finally, peptide-treated melanoma cells were shown to be more sensitive to lysis by NK cells in a dose-dependent way. Taken together, these results demonstrate that a small synthetic peptide derived from IL-10 can mimic the Ag presentation-related effects mediated by this cytokine in human melanomas and increase tumor sensitivity to NK cells, which can be relevant in the designing of future strategies for cancer immune therapy.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/biosíntesis , Neoplasias del Ojo/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/biosíntesis , Interleucina-10/agonistas , Melanoma/metabolismo , Proteínas de Neoplasias/biosíntesis , Oligopéptidos/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Cisteína Endopeptidasas/biosíntesis , Cisteína Endopeptidasas/genética , Citotoxicidad Inmunológica , Relación Dosis-Respuesta a Droga , Neoplasias del Ojo/patología , Genes MHC Clase I , Humanos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/farmacología , Interleucina-10/química , Células Asesinas Activadas por Linfocinas/inmunología , Melanoma/patología , Proteínas de Neoplasias/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes

RESUMEN

Several single-nucleotide polymorphisms (SNPs) have been identified in the TNF-alpha gene promoter. The transition G-->A at position -308 generates the TNF-alpha1 (G/G) and TNF-alpha2 (G/A or A/A) alleles, where the polymorphic TNF-alpha2 allele is associated with a high, in vitro TNF-alpha expression and an increased susceptibility to diverse illnesses. Here we study the association of the -308 TNF-alpha SNP with the susceptibility for developing aggressive periodontitis (AP), AP combined with type 1 diabetes mellitus (DM) and DM. We also explore the TNF-alpha capability expression and the presence of the -308 polymorphism. For this purpose we recruited 27 individuals with AP (AP+ group), 27 individuals with AP combined with DM (AP+/DM+ group), and 27 individuals with DM without signs of periodontitis upon clinical examination (DM+ group). The control group was comprised of 30 subjects. Genotyping for TNF-alpha promoter was performed by PCR-RFLP analysis. For TNF-alpha expression we used a blood culture system.

Asunto(s)

Diabetes Mellitus Tipo 1/genética , Leucocitos/metabolismo , Periodontitis/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Factor de Necrosis Tumoral alfa/genética , Adulto , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Predisposición Genética a la Enfermedad/genética , Humanos , Lipopolisacáridos/farmacología , Masculino , Periodontitis/complicaciones , Periodontitis/metabolismo , Polimorfismo de Nucleótido Simple/fisiología , Regiones Promotoras Genéticas/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

Several studies have demonstrated that diabetes is a risk factor for developing periodontal disease, increasing its prevalence and severity. Furthermore, periodontitis may impair the metabolic control and adequate treatment of diabetic patients. LPS from Gram-negative bacteria penetrates the periodontal tissues and subsequently recruits and activates immune cells. Progression to severe periodontitis with loss of supporting structures is mediated by several factors, including secretion of a broad spectrum of inflammatory and destructive (PGE2). mediators such as cytokines (TNF-alpha, IL-1b and IL-6), chemokines (IL-8) and prostaglandin E2. The aim of this work is to investigate differences in the TNF-a, IL-1b and IL-6 expression and prostaglandin E2 (PGE2) release in blood from diabetic patients with and without aggressive periodontitis (AP) stimulated with lipopolysaccharide (LPS). For this purpose we recruited 29 Type 1 diabetes mellitus (DM) patients, 14 with AP and 15 without AP. Fourteen healthy individuals formed the control group. For cytokine expression and PGE2 secretion, an ex vivo whole blood culture system was used. Cytokines and PGE2 were detected by commercial immunometric assays. A wide range of inter-individual variability in spontaneous and LPS-induced TNF-alpha, IL-1b and IL-6 levels in patient groups and controls was found. The mean of spontaneous and LPS-induced TNF-alpha and IL-1b levels did not differ significantly (p > 0.5) when patients were compared to control individuals. Although not significant, the spontaneous TNF-alpha, IL-1b and IL-6 levels in the group of Type 1 DM with AP were higher than in controls, while in diabetic patients without AP, these values were depressed in comparison with controls. In both groups of patients, the means of LPS-induced IL-6 levels were higher than the controls but the differences observed were not significant (p = 0.07). However, the LPS-induced PGE2 levels varied significantly when all groups were compared (p = 0.007). The means of LPS-induced PGE2 levels for Type 1 diabetic patients with AP (p = 0.0009) and without AP (p = 0.024) were significantly higher than the levels observed for healthy controls. Finally, we conclude that Type 1 diabetic patients with or without AP did not express higher LPS-induced TNF-a, IL-1b and IL-6 levels than controls. However, the PGE2 levels released were significantly higher than those detected in controls.

Asunto(s)

Diabetes Mellitus Tipo 1/sangre , Dinoprostona/metabolismo , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Periodontitis/complicaciones , Adulto , Diabetes Mellitus Tipo 1/complicaciones , Femenino , Humanos , Técnicas In Vitro , Masculino

RESUMEN

It is likely that the murine response to human recombinant TNF alpha (hrTNF alpha) may generate antibodies (Ab) to epitopes present in TNF alpha from other species. Here, we demonstrate that F5 anti-hrTNF alpha monoclonal antibody (mAb) recognizes feline TNF alpha while E8 anti-hrTNF alpha mAb failed to do so. In order to demonstrate that E8 and F5 mAb recognize different epitopes in the hrTNF alpha molecule, a constant concentration of E8 and variable concentrations of F5 were incubated with solid phase bound hrTNF alpha. Binding of E8 and F5 to hrTNF alpha was determined with anti-mu and gamma chain specific Ab. F5 bound equally to hrTNF alpha in the presence or absence of E8 and the same amount of E8 bound to hrTNF alpha, in spite of the presence of F5. When using the E8 and F5 mAb for capturing the TNF alpha from the equine, canine, feline and bovine species, in supernatants of an ex vivo lipopolysaccharide (LPS)-stimulated whole blood cell culture, we only detected the feline TNF alpha by F5 mAb (p = 0.001). By a cytotoxic assay on L929 fibroblasts, we indeed demonstrated the feline TNF alpha production after the LPS stimulus. In an inhibition assay, the human and feline cytokines competed for F5, although the inhibition of native human TNF alpha binding to F5 was significant but only about 20% (p = 0.001). In conclusion, most likely the F5 anti-hrTNF alpha mAb recognizes an epitope in feline TNF alpha. Its immunomodulatory potential in the feline model remains to be studied.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Reacciones Cruzadas/inmunología , Epítopos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Gatos , Bovinos , Línea Celular , Perros , Femenino , Caballos/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Radioinmunoensayo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Especificidad de la Especie , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

In mammals, the pleiotropic biological functions of tumor necrosis factor alpha (TNF-alpha) may include important effects on human reproductive physiology. Thus, chronic anovulation, oligo or amenorrhea, infertility, hyperandrogenism, obesity, insulin resistance and increased TNFalpha serum levels have been observed in women affected by polycystic ovary syndrome (PCOS). Whole blood short - term cell cultures (WBSC) are simple systems where the capacity to produce TNF-alpha by circulating leukocytes, mainly of the macrophage/monocyte lineage, can be accurately quantified. Given the relevance of monocytes/macrophages in the production of TNF-alpha, in this study, in a control-case approach, WBSC from women with PCOS were analyzed in their basal and lipolysaccharide (LPS)- stimulated capacity to produce the cytokine. These measurements did not correlate with the increased serum levels of the cytokine and the normal levels of cortisol, found in PCOS women. Increased serum TNF-alpha levels in PCOS women correlated positively with body mass index and negatively with insulin sensitivity. In spite of the increased serum TNF-alpha levels in PCOS women, basal and LPS stimulated production of the cytokine, by the ex vivo WBSC from these patients, were within normal values.

Asunto(s)

Síndrome del Ovario Poliquístico/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Adolescente , Adulto , Células Sanguíneas/inmunología , Índice de Masa Corporal , Estudios de Casos y Controles , Femenino , Humanos , Técnicas In Vitro , Resistencia a la Insulina , Síndrome del Ovario Poliquístico/sangre , Síndrome del Ovario Poliquístico/patología , Síndrome del Ovario Poliquístico/fisiopatología

RESUMEN

Several single-nucleotide polymorphisms have been identified in the human TNF gene promoter. The polymorphism at position-308 (TNF-308), which involves substituting G for A and designing the TNF2 allele, leads to a higher rate of TNF gene transcription than the wild-type TNF1 allele in in vitro expression studies. It has also been linked to increased susceptibility to a variety of illnesses. Using PCR-RFLP analysis we detected significant differences in the TNF-308 genotypes of Chilean and other populations. We conclude that there is a gradient in the distribution of the TNF2 allele according to ethnicity; we have also hypothesized that populations bearing a higher proportion of the TNF2 allele may have an increased predisposition toward or incidence of several chronic metabolic, degenerative, inflammatory and autoimmune diseases.

Asunto(s)

Humanos , Alelos , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Polimorfismo Genético , Regiones Promotoras Genéticas , Factor de Necrosis Tumoral alfa , Chile , Predisposición Genética a la Enfermedad , Genotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción