RESUMEN
The evaluation of anti-dsDNA antibodies represents one of the essential diagnostic and prognostic marker features in patients affected by Systemic Lupus Erythematosus (SLE). In this study, we have compared immunoblotting (IB) with Crithidia luciliae indirect immunofluorescence test (CLIFT) and chemiluminescent immunoassay (CLIA) in 91 patients referred to our hospital for anti-dsDNA antibodies detection. The concordance and correlation measured by Cohen's kappa and Spearman's coefficient respectively was significant between CLIFT and CLIA (0.70; 0,7404, P < .0001) and among CLIA and IB (0.79; 0,5377, P < 0,0001) and lower between CLIFT and IB (0.55; 0,4373, P <0,0001). Among the 46 IB-positive samples, 14 were positive for either CLIA or CLIFT. It is noteworthy that 11 out of these 14 samples had the final diagnosis of SLE. Thirteen out of fourteen samples were also positive for anti-nucleosome antibodies as measured concomitantly in immunoblotting. While our observations are based on a limited number of samples and will have to be confirmed in a bigger cohort, they underline the contribution of immunoblotting as an additional assay in defining the anti-dsDNA antibody profile in association with other well-established methods such as CLIA and CLIFT.
Asunto(s)
Anticuerpos Antinucleares/sangre , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Immunoblotting/métodos , Mediciones Luminiscentes/métodos , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Programmed death ligand 1 (PD-L1) (also called B7-H1) is a membrane immune-modulatory protein whose overexpression on the surface of tumor cells as well as APCs impairs T-cell-mediated killing. Viruses that establish chronic infections have developed a number of strategies to escape from immune recognition including the up-regulation of PD-L1. This study shows for the first time that the human oncovirus EBV infects human primary monocytes using HLA-DR and induced a strong up-regulation of PD-L1 expression on their surface. Searching for the underlying mechanism/s leading to this immune suppressive effect, we found that EBV activated TLR signaling, increased intracellular ROS, and phosphorylated STAT3. Targeting these molecules partially reverted PD-L1 up-regulation that correlated with an altered cytokine production and a reduction of monocyte cell survival, strongly impairing the antiviral immune response.
Asunto(s)
Antígeno B7-H1/biosíntesis , Monocitos/virología , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/fisiología , Receptores Toll-Like/fisiología , Muerte Celular , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/virología , Depuradores de Radicales Libres/farmacología , Antígenos HLA-DR/inmunología , Humanos , Monocitos/metabolismo , Factor 88 de Diferenciación Mieloide/antagonistas & inhibidores , Factor 88 de Diferenciación Mieloide/fisiología , Fosforilación , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba , Replicación ViralRESUMEN
Quercetin, a bioflavonoid contained in several vegetables daily consumed, has been studied for long time for its antiinflammatory and anticancer properties. Quercetin interacts with multiple cancer-related pathways such as PI3K/AKT, Wnt/ß-catenin and STAT3. These pathways are hyperactivated in primary effusion lymphoma (PEL), an aggressive B cell lymphoma whose pathogenesis is strictly linked to the oncogenic virus Kaposis' Sarcoma-associated Herpesvirus (KSHV). In this study, we found that quercetin inhibited PI3K/AKT/mTOR and STAT3 pathways in PEL cells, and as a consequence, it down-regulated the expression of the prosurvival cellular proteins such as c-FLIP, cyclin D1 and cMyc. It also reduced the release of IL-6 and IL-10 cytokines, leading to PEL cell death. Moreover, quercetin induced a prosurvival autophagy in these cells and increased the cytotoxic effect of bortezomib, a proteasomal inhibitor, against them. Interestingly, quercetin decreased also the expression of latent and lytic KSHV proteins involved in PEL tumorigenesis and up-regulated the surface expression of HLA-DR and calreticulin, rendering the dying cells more likely detectable by the immune system. The results obtained in this study indicate that quercetin, which does not exert any cytotoxicity against normal B cells, may represent a good candidate for the treatment of this aggressive B cell lymphoma, especially in combination with autophagy inhibitors or with bortezomib.
Asunto(s)
Antineoplásicos Fitogénicos/metabolismo , Apoptosis , Autofagia , Regulación hacia Abajo , Linfoma de Efusión Primaria/metabolismo , Quercetina/metabolismo , Transducción de Señal , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/efectos adversos , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Bortezomib/agonistas , Bortezomib/farmacología , Línea Celular Tumoral , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Agonismo de Drogas , Humanos , Interleucinas/antagonistas & inhibidores , Interleucinas/metabolismo , Linfoma de Efusión Primaria/tratamiento farmacológico , Linfoma de Efusión Primaria/inmunología , Linfoma de Efusión Primaria/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quercetina/efectos adversos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies.
Asunto(s)
Proteínas Portadoras/metabolismo , Hiperglucemia/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Apoptosis , Glucemia , Línea Celular Tumoral , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Modelos Biológicos , Proteína Fosfatasa 2/metabolismo , Proteolisis , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Type 2 is the type of diabetes with higher prevalence in contemporary time, representing about 90% of the global cases of diabetes. In the course of diabetes, several complications can occur, mostly due to hyperglycemia and increased reactive oxygen species (ROS) production. One of them is represented by an increased susceptibility to microbial infections and by a reduced capacity to clear them. Therefore, knowing the impact of hyperglycemia on immune system functionality is of utmost importance for the management of the disease. In this study, we show that medium containing high glucose reduced the in-vitro differentiation of monocytes into functional DCs and their activation mediated by PAMPs or DAMPs. Most importantly, the same effects were mediated by the hyperglycemic sera derived by type 2 diabetic patients, mimicking a more physiologic condition. DC dysfunction caused by hyperglycemia may be involved in the inefficient control of infections observed in diabetic patients, given the pivotal role of these cells in both the innate and adaptive immune response. Searching for the molecular mechanisms underlying DC dysfunction, we found that canonical Wnt/ß-catenin and p38 MAPK pathways were activated in the DCs differentiated either in the presence of high glucose or of hyper-glycemic sera. Interestingly, the activation of these pathways and the DC immune dysfunction were partially counteracted by the anti-oxidant quercetin, a flavonoid already known to exert several beneficial effects in diabetes.
Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Diabetes Mellitus Tipo 2/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Especies Reactivas de Oxígeno/inmunología , Vía de Señalización Wnt/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Glucemia , Línea Celular , Células Dendríticas/patología , Diabetes Mellitus Tipo 2/patología , Humanos , Suero , beta Catenina/inmunologíaRESUMEN
The association of Epstein-Barr virus (EBV) with plasmacytoid malignancies is now well established but how the virus influences microRNA expression in such cells is not known. We have used multiple myeloma (MM) cell lines to address this issue and find that an oncomiR, miR-21 is induced after in vitro EBV infection. The PU.1 binding site in miR-21 promoter was essential for its activation by the virus. In accordance with its noted oncogenic functions, miR-21 induction in EBV infected MM cells caused downregulation of p21 and an increase in cyclin D3 expression. EBV infected MM cells were highly tumorigenic in SCID mice. Given the importance of miR-21 in plasmacytoid malignancies, our findings that EBV could further exacerbate the disease by inducing miR-21 has interesting implications both in terms of diagnosis and future miR based therapeutical approaches for the virus associated plasmacytoid tumors.
Asunto(s)
Linfocitos B/metabolismo , Diferenciación Celular/genética , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/genética , MicroARNs/genética , Animales , Sitios de Unión/genética , Ciclina D3/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación hacia Abajo/genética , Herpesvirus Humano 4 , Ratones , Ratones SCID , Mieloma Múltiple/genética , Mieloma Múltiple/virología , Regiones Promotoras GenéticasRESUMEN
It has been shown that Epstein-Barr virus (EBV) is able to alter the immune response towards self-antigens and may enhance risk of autoimmune diseases such as systemic lupus erythematosus (SLE) in genetically predisposed individuals. In this study, we evaluated the specific antibody immune response against EBV in patients with anti-nuclear autoantibodies (ANA) in comparison with ANA-negative healthy controls. For this purpose, 92 patients with an high anti-ANA reactivity with or without concomitant extractable nuclear antigen (ENA) or double stranded DNA (dsDNA) positivity were selected and compared with 146 healthy donors. We found that anti-EBV-VCA and EA IgG concentrations were significantly higher in ANA-positive patients in comparison to the controls (VCA P<0.0001 and EA P<0,03) as well as in those ANA-positive patients that showed a concomitant ENA positivity (P=0.0002). Interestingly, elevated anti-EBNA-1 IgG was found in a group of patients who had anti SSA/Ro antibodies. Anti-VCA IgM Abs were more frequently found in those patients with a very high titer of ANA (P=0.06); moreover detection of anti-VCA IgM/IgG in absence of anti-EBNA-1 IgG was more frequent in the patient than in the control group. Both these conditions correlate with a recent EBV infection or reactivation. The data suggest that EBV, particularly during acute infection or in its reactivation phase, could be involved in the ANA and ENA autoantibody formation.
Asunto(s)
Anticuerpos Antinucleares/sangre , Anticuerpos Antivirales/sangre , Herpesvirus Humano 4/inmunología , Antígenos Virales/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangreRESUMEN
Kaposis's sarcoma associated herpesvirus (KSHV) has been reported to infect, among others, monocytes and dendritic cells DCs impairing their function. However, the underlying mechanisms remain not completely elucidated yet. Here we show that DC exposure to active or UV-inactivated KSHV resulted in STAT3 phosphorylation. This effect, partially dependent on KSHV-engagement of DC-SIGN, induced a high release of IL-10, IL-6 and IL-23, cytokines that in turn might maintain STAT3 in a phosphorylated state. STAT3 activation also correlated with a block of autophagy in DCs, as indicated by LC3II reduction and p62 accumulation. The IL-10, IL-6 and IL-23 release and the autophagic block could be overcome by inhibiting STAT3 activation, highlighting the role of STAT3 in mediating such effects. In conclusion, here we show that STAT3 activation can be one of the molecular mechanisms leading to KSHV-mediated DC dysfunction, that might allow viral persistence and the onset of KSHV-associated malignancies.
Asunto(s)
Autofagia/fisiología , Células Dendríticas/fisiología , Células Dendríticas/virología , Herpesvirus Humano 8/fisiología , Interleucinas/biosíntesis , Factor de Transcripción STAT3/metabolismo , Línea Celular , Células Dendríticas/citología , Humanos , Interleucina-10/biosíntesis , Interleucina-23/biosíntesis , Interleucina-6/biosíntesis , Estadística como AsuntoRESUMEN
The B cell lymphomas associated with Epstein-Barr virus (EBV) are not limited to any specific stage of B cell differentiation but covers widely different B cell phenotypes. In vitro infection of the virus negative tumors with a recombinant EBV strain has provided important insights into virus-tumor interaction. Here, we investigated the interaction between EBV and terminally differentiated tumor derived B cells, namely multiple myeloma (MM). The in vitro EBV infected MM expressed restricted viral latency. Acquisition of the virus was accompanied by a partial reprogramming to a mature B cell phenotype. Thus, the plasma cell markers syndecan-1 (CD138), Blimp1 and MUM1 were downregulated, while expression of HLADR, CIITA and TCL1, which are normally not expressed in plasmacytoid cells, was upregulated. The silenced transcription factor gene encoding Pax5 and its target BLNK were activated. Significantly, the free lambda light chains secreted in the medium were reduced in EBV infected MM clones. Collectively, these results suggest that the restricted EBV latency can cause at least partial phenotypic reversion of terminally differentiated B tumor cells. We suggest that the restricted EBV latent gene expression may not only be the consequence but the cause of the mature B cell phenotype, actively participating in the virus persistence.
Asunto(s)
Linfocitos B/virología , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/fisiología , Mieloma Múltiple/patología , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Linfocitos B/patología , Diferenciación Celular , Genes Virales , Herpesvirus Humano 4/genética , Humanos , Cadenas lambda de Inmunoglobulina/biosíntesis , Cadenas lambda de Inmunoglobulina/genética , Factores Reguladores del Interferón/biosíntesis , Factores Reguladores del Interferón/genética , MicroARNs/biosíntesis , MicroARNs/genética , Proteínas de Mieloma/biosíntesis , Proteínas de Mieloma/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Factor de Transcripción PAX5/biosíntesis , Factor de Transcripción PAX5/genética , Fenotipo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Sindecano-1/biosíntesis , Sindecano-1/genética , Células Tumorales Cultivadas/patología , Células Tumorales Cultivadas/virología , Latencia del VirusRESUMEN
Functional impairment of dendritic cells (DC) appears to be one of the mechanisms responsible for tumor escape from the control of the immune system. DC isolated from tumor-bearing animals and cancer patients with solid or with hematological malignancies have phenotypic and functional abnormalities. In addition, supernatants from in vitro cultured tumor cells have been shown to interfere with DC differentiation from CD34+ and monocyte precursors. Primary effusion lymphoma (PEL) is a Human Herpesvirus-8 (HHV-8)-associated tumor, which releases several cytokines such as IL-6, IL-10 and VEGF and its growth seems to be dependent on them in vitro or in vivo. In the present study, we found that these cytokines released by PELs have also an important role in interfering with the in vitro differentiation of immature DC (iDC) from CD14+ monocytes. The iDC obtained in the presence of PEL supernatants showed reduction of FITC-dextran uptake, reduction of MLR allostimulatory activity and altered expression of surface molecules, suggesting that cytokines released by PEL adversely affect DC differentiation.
Asunto(s)
Diferenciación Celular , Citocinas/metabolismo , Células Dendríticas/citología , Linfoma de Efusión Primaria/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , FenotipoRESUMEN
To investigate the role of Epstein-Barr virus (EBV) in the pathogenesis of primary effusion lymphoma (PEL), we infected human herpesvirus 8 (HHV-8+) but EBV- PEL lines BC-3, CRO-AP/6, and CRO-AP/3 cells with the recombinant Akata EBV strain. All EBV-infected clones expressed EBER-1, EBNA-1, and LMP2A. The expression of LMP1 and LMP2B was variable. None, however, expressed EBNA2-6. The surface markers CD30, CD74, and syndecan-1 were down-regulated in EBV convertants. EBV-infected BC-3 and CRO-AP/6 cells were highly tumorigenic in severe combined immunodeficiency (SCID) mice in contrast to their respective EBV- parental cells. However, neither the parental cells nor the virus-converted counterparts expressed TCL1. The results showing that PEL cells on in vitro EBV infection do not sustain latency III despite the absence of immune pressure indicate that the choice of EBV latent gene expression program is cell dependent. The data suggest an important role of EBV in the pathogenesis of PEL.
Asunto(s)
Infecciones por Virus de Epstein-Barr/virología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/patogenicidad , Linfoma/virología , Animales , Línea Celular Tumoral , Infecciones por Virus de Epstein-Barr/patología , Expresión Génica , Infecciones por Herpesviridae/genética , Humanos , Linfoma/patología , Ratones , Ratones SCID , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas/genética , Recombinación Genética , Trasplante HeterólogoRESUMEN
Several reports have suggested an association of human herpesvirus 6 (HHV-6) with multiple sclerosis. Autoreactive T lymphocytes directed against myelin components seem to contribute to the pathogenesis of the disease. It has been suggested that molecular mimicry between viral and self-antigens might be one of the mechanisms that determine the onset of several autoimmune diseases. Following this hypothesis, the purpose of the present study was to evaluate if HHV-6 could play a role in activating T cells capable of cross-reaction with an important myelin component, the myelin basic protein. T cell lines were established from 22 multiple sclerosis patients and from 16 healthy controls, and their capability to react to both virus and myelin basic protein antigens was compared. The analysis of T cell cross-reactivity in patients and controls did not show significant differences in the HHV-6 ability to activate myelin basic protein-reactive T cells. Similarly, the evaluation of the humoral immune response to HHV-6 in patients and controls did not mirror any abnormality in the HHV-6 status in multiple sclerosis patients. Therefore, although the findings of activity in vitro of T cell lines with dual specificity are consistent with the hypothesis of molecular mimicry, the lack of differences in cross-reactivity between patients and controls do not support molecular mimicry as an important mechanism in the physiopathology of this disease.