RESUMEN
Plasmacytoid dendritic cells are specialized in the production of type I interferon (type I IFN), which promotes antiviral and antitumor responses, as well as autoimmune disorders. Activation of type I IFN secretion depends on the pattern recognition receptors TLR7 and TLR9, which sense microbial RNA and DNA, respectively. Type I IFN production is modulated by several receptors, including the type II C-type lectin domain family 4, member C (CLEC4C). The natural ligand of CLEC4C is unknown. To identify it, here we probed a glycan array with a soluble form of the CLEC4C ectodomain. We found that CLEC4C recognizes complex type sugars with terminal galactose. Importantly, soluble CLEC4C bound peripheral blood leukocytes and tumor cells that express glycans with galactose residues at the non-reducing ends. The positive and negative modulation of galactose residues on cell membranes was paralleled by the regulation of type I IFN secretion by plasmacytoid dendritic cells in co-culture experiments in vitro. These results suggest that the modulation in the expression of non-sialylated oligosaccharides by invading pathogens or transformed cells may affect type I IFN response and immune surveillance.
Asunto(s)
Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligosacáridos/metabolismo , Células Plasmáticas/metabolismo , Receptores Inmunológicos/metabolismo , Células Cultivadas , Células Dendríticas/inmunología , Humanos , Interferón-alfa/genética , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Oligosacáridos/química , Oligosacáridos/genética , Oligosacáridos/inmunología , Células Plasmáticas/inmunología , Estructura Terciaria de Proteína , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunología , Receptor Toll-Like 9/metabolismoRESUMEN
Preterm infants undergo early separation from parents and are exposed to frequent painful clinical procedures, with resultant short- and long-term effects on their neurodevelopment. We aimed to establish whether the mother's voice could provide an effective and safe analgesia for preterm infants and whether endogenous oxytocin (OXT) could be linked to pain modulation. Twenty preterm infants were exposed to three conditions-mother's live voice (speaking or singing) and standard care-in random order during a painful procedure. OXT levels (pg/mL) in saliva and plasma cortisol levels were quantified, and the Premature Infant Pain Profile (PIPP) was blindly coded by trained psychologists. During the mother's live voice, PIPP scores significantly decreased, with a concomitant increase in OXT levels over baseline. The effect on pain perception was marginally significant for singing. No effects on cortisol levels were found. The mother's live voice modulated preterm infants' pain indicators. Endogenous OXT released during vocal contact is a promising protective mechanism during early painful interventions in at-risk populations.
Asunto(s)
Enfermedades del Prematuro/terapia , Oxitocina/análisis , Manejo del Dolor/métodos , Saliva/química , Adulto , Peso Corporal , Femenino , Frecuencia Cardíaca , Humanos , Lactante , Conducta del Lactante , Recien Nacido Prematuro , Madres , Oxitocina/fisiología , Dolor , Dimensión del Dolor , VozRESUMEN
The B19 virus can persist in immunocompromised patients for several months and sometimes even years because of impaired immune response. Viremia in persistent and recurrent infection may range from very low to high titers and may be associated with chronic clinical manifestations, such as chronic anemia. Several recently developed techniques that quantify B19-DNA have improved laboratory diagnosis of the infection and can help guide the choice of treatment in persistent infections (i.e., intravenous immunoglobulin (IVIG) treatment vs immunosuppression reduction). Here we describe the development of a reliable internally controlled quantitative competitive (QC)- polymerase chain reaction (PCR) assay that measures B19-DNA load in serum samples by densitometric analysis of the amplification products for monitoring B19 infection in high-risk patients. A retrospective quantification of B19-DNA in the serum samples from 48 anemic transplanted patients by the QC-PCR assay we developed in our laboratory confirmed the presence of B19-DNA in 11 of 48 samples and showed a viral DNA load between 103 and 108 B19-DNA copies/mL depending on the patients' serostatus (the highest viral load was found in IgM-positive/IgG-negative patients, that is, in patients with active B19 infection at onset). The assay also confirmed B19-DNA negative patients. Our QC-PCR assay may be easily used in monitoring B19 prototype DNA load to follow persistent infections and to better understand the relation between active B19 infection and occurrence of anemia and to assess the efficacy of IVIG therapy or immunosuppression reduction in clearing the virus in high-risk patients.
Asunto(s)
ADN Viral/sangre , Parvovirus B19 Humano/genética , Reacción en Cadena de la Polimerasa/métodos , Carga Viral/métodos , Adulto , Anticuerpos Antivirales/sangre , Calibración , ADN Viral/genética , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Parvovirus B19 Humano/inmunología , Plásmidos/genética , Plásmidos/normas , Reproducibilidad de los ResultadosRESUMEN
Infections from human polyomaviruses BK and JC (BKV and JCV) occur independently, but concomitant infections and the simultaneous persistence of both viruses have been observed in renal transplant recipients. Several studies have disclosed a correlation between BKV and interstitial nephritis in renal transplant recipients, and an association between JCV and some cases of nephropathy has recently been hypothesized. This article describes the development of a semiquantitative-nested polymerase chain reaction (PCR) assay to simultaneously detect BKV and JCV viral load in urine and serum. The first-round amplification step uses primers that amplify a 385-bp DNA fragment from the "large T antigen" region of both viruses. Samples testing positive in the first step are then run in the second step. In the second-round amplification, different inner primers are used to separately quantify BKV-DNA and/or JCV-DNA. The assay offers several advantages including: (1) rapid submission of clinical samples to screening; (2) verification of the absence of Taq polymerase inhibitors with the use of an internal control; (3) a sensitivity threshold of 10 copies/reaction; and (4) assay running is less labor intensive, cheap, and easy to perform. The assay may be easily used to monitor viral loads versus baseline levels in urine and serum samples from renal transplant recipients to detect those at risk of BKV- or JCV-related nephropathy, and to monitor their response to immunosuppression reduction therapy if it occurs.
Asunto(s)
Virus BK , ADN Viral/sangre , ADN Viral/orina , Virus JC , Infecciones por Polyomavirus/sangre , Infecciones por Polyomavirus/orina , Infecciones Tumorales por Virus/sangre , Infecciones Tumorales por Virus/orina , Virus BK/genética , Femenino , Humanos , Virus JC/genética , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/métodos , Masculino , Nefritis/complicaciones , Nefritis/terapia , Nefritis/virología , Reacción en Cadena de la Polimerasa/métodos , Infecciones por Polyomavirus/etiología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Infecciones Tumorales por Virus/etiología , Carga Viral/métodosRESUMEN
Posttransplant lymphoproliferative disorders (PTLD) are a severe complication arising in solid organ transplant patients. A strong correlation between Epstein-Barr virus (EBV) infection, the grade and type of immunosuppression, and the development of PTLD has been recognized. This article describes the development of a double-step polymerase chain reaction (PCR) assay for the quantification of EBV-deoxyribonucleic acid (DNA) to monitor EBV infection. Screening of samples containing >/=10(3) viral genomes/10(5) peripheral blood mononuclear cells (PBMC) or 100 micro L serum by a semiquantitative PCR assay is followed by quantification of the samples containing a high number of viral genomes in a quantitative-competitive (QC)-PCR assay. Screening by semiquantitative PCR selects samples with a high number of viral genomes for use in the more labor-intensive and expensive QC-PCR assay and thus provides a handy means for quantitative DNA analysis of large numbers of samples. Our double-step PCR assay can be employed in EBV viral load measurement in PBMC and serum samples to monitor transplanted patients at risk to develop PTLD.
Asunto(s)
ADN Viral/sangre , Infecciones por Virus de Epstein-Barr/sangre , Herpesvirus Humano 4/aislamiento & purificación , Leucocitos Mononucleares/virología , Reacción en Cadena de la Polimerasa/métodos , Carga Viral/métodos , Células Cultivadas , ADN Viral/genética , Infecciones por Virus de Epstein-Barr/diagnóstico , Femenino , Herpesvirus Humano 4/genética , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Plasmacytoid dendritic cells (PDC) are a functionally distinct lineage of dendritic cells characterized by the release of large amounts of type I interferon (IFN I). IFN I release is efficiently triggered by viral infection and modulates several aspects of immune reactions including the activation of cytotoxic mechanisms finalized to the elimination of infected cells. In this study, we report that TLR7 and TLR9 ligands can induce the secretion of biologically active TNF-related apoptosis-inducing ligand (TRAIL) by PDC. Accordingly, PDC supernatant is endowed with TRAIL-mediated cytotoxic activity when tested on a TRAIL-sensitive Jurkat cell line. TRAIL production is only partially dependent on the autocrine production of IFN I as documented by the use of a blocking anti-IFNRA antibody and the stimulation with exogenous IFN I. Importantly, both TRAIL secretion and cytotoxic activity of PDC supernatants are completely abolished by BDCA2 ligation. These results provide further insights into the biological role of BDCA-2 and document a negative regulatory pathway of PDC cytotoxic activity that may be relevant in pathological situations such as tumors and autoimmune diseases.
Asunto(s)
Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Humanos , Células JurkatRESUMEN
Psoriasis is a type I interferon-driven T cell-mediated disease characterized by the recruitment of plasmacytoid dendritic cells (pDC) into the skin. The molecules involved in pDC accumulation in psoriasis lesions are unknown. Chemerin is the only inflammatory chemotactic factor that is directly active on human blood pDC in vitro. The aim of this study was to evaluate the role of the chemerin/ChemR23 axis in the recruitment of pDC in psoriasis skin. Prepsoriatic skin adjacent to active lesions and early lesions were characterized by a strong expression of chemerin in the dermis and by the presence of CD15(+) neutrophils and CD123(+)/BDCA-2(+)/ChemR23(+) pDC. Conversely, skin from chronic plaques showed low chemerin expression, segregation of neutrophils to epidermal microabscesses, and few pDC in the dermis. Chemerin expression was localized mainly in fibroblasts, mast cells, and endothelial cells. Fibroblasts cultured from skin of psoriatic lesions expressed higher levels of chemerin messenger RNA and protein than fibroblasts from uninvolved psoriatic skin or healthy donors and promoted pDC migration in vitro in a chemerin-dependent manner. Therefore, chemerin expression specifically marks the early phases of evolving skin psoriatic lesions and is temporally strictly associated with pDC. These results support a role for the chemerin/ChemR23 axis in the early phases of psoriasis development.
Asunto(s)
Quimiocinas , Células Dendríticas , Psoriasis , Piel , Adulto , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD/metabolismo , Western Blotting , Calcitriol/farmacología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/fisiología , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/citología , Células Dendríticas/metabolismo , Dermatitis Atópica/genética , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Psoriasis/genética , Psoriasis/metabolismo , Psoriasis/patología , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/metabolismo , Piel/patología , Tretinoina/farmacología , HumanosRESUMEN
Activin A, a member of the transforming growth factor-beta superfamily, has a role in tissue repair and inflammation. In our previous studies, we identified by immunohistochemistry DC-SIGN(+) dendritic cells as a source of activin A in vivo. The present study was aimed at investigating activin A production by dendritic cells (DC) in vitro and its function. Here we demonstrate that monocyte-derived DC (Mo-DC) released abundant levels of activin A during the maturation process induced by TLR agonists, bacteria (B. henselae, S. thyphimurium), TNF and CD40L. Activin A was also induced in monocyte-derived Langerhans cells (LC) and in blood myeloid DC by LPS and/or CD40L stimulation, but not in blood plasmacytoid DC following stimulation with influenza virus. Activin A production by DC was selectively down-regulated by anti-inflammatory molecules such as dexamethasone or IL-10. Neutralization of endogenous activin A using its inhibitor follistatin, or the addition of exogenous activin A during LPS maturation did not affect Mo-DC maturation marker expression, cytokine release or allostimulatory function. However, Mo-DC matured with LPS in the presence of exogenous activin A displayed a higher FITC-dextran uptake, similar to that of immature DC. Moreover, activin A promoted monocyte differentiation to DC and reversed the inhibitory effects of IL-6 on DC differentiation of monocytes. These findings demonstrate that different subsets of DC release activin A, a cytokine that promotes DC generation, and affects the ability of mature DC to take up antigens (Ags).
Asunto(s)
Activinas/fisiología , Células Dendríticas/citología , Regulación de la Expresión Génica , Activinas/química , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Folistatina/metabolismo , Humanos , Inflamación , Interleucina-6/metabolismo , Células de Langerhans/citología , Lipopolisacáridos/química , Modelos Biológicos , Monocitos/citología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Langerhans cells (LC) represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFbeta. In vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFbeta. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFbeta family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFbeta. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFbeta, responsible for LC differentiation during inflammatory/autoimmune conditions.
Asunto(s)
Activinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Células de Langerhans/citología , Piel/metabolismo , Antígenos CD/biosíntesis , Antígenos CD1/biosíntesis , Células Cultivadas , Células Dendríticas/citología , Humanos , Técnicas In Vitro , Queratinocitos/citología , Lectinas Tipo C/biosíntesis , Receptores de Lipopolisacáridos/biosíntesis , Lectinas de Unión a Manosa/biosíntesis , Modelos Biológicos , Monocitos/metabolismo , Piel/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Cicatrización de HeridasRESUMEN
Hypertrophic scarring is a skin disorder characterized by persistent inflammation and fibrosis that may occur after wounding or thermal injury. Altered production of cytokines and growth factors, such as TGF-beta, play an important role in this process. Activin A, a member of the TGF-beta family, shares the same intra-cellular Smad signalling pathway with TGF-beta, but binds to its own specific transmembrane receptors and to follistatin, a secreted protein that inhibits activin by sequestration. Recent studies provide evidences of a novel role of activin A in inflammatory and repair processes. The aim of this study was to evaluate the importance of activin A and follistatin expression in the different phases of scar evolution. Immunostaining of sections obtained from active phase hypertrophic scars (AHS) revealed the presence of a high number of alpha-SMA(+) myofibroblasts and DC-SIGN(+) dendritic cells coexpressing activin A. Ex-vivo AHS fibroblasts produced more activin and less follistatin than normal skin or remission phase hypertrophic scar (HS) fibroblasts, both in basal conditions and upon TGF-betas stimulation. We demonstrate that fibroblasts do express activin receptors, and that this expression is not affected by TGF-betas. Treatment of HS fibroblasts with activin A induced Akt phosphorylation, promoted cell proliferation, and enhanced alpha-SMA and type I collagen expression. Follistatin reduced proliferation and suppressed activin-induced collagen expression. These results indicate that the activin/follistatin interplay has a role in HS formation and evolution. The impact of these observations on the understanding of wound healing and on the identification of new therapeutic targets is discussed.
Asunto(s)
Activinas/metabolismo , Cicatriz Hipertrófica/metabolismo , Fibroblastos/metabolismo , Folistatina/metabolismo , Actinas/metabolismo , Receptores de Activinas/metabolismo , Adolescente , Adulto , Anciano , Quemaduras/complicaciones , Quemaduras/metabolismo , Proliferación Celular , Células Cultivadas , Cicatriz Hipertrófica/etiología , Cicatriz Hipertrófica/patología , Células Dendríticas/metabolismo , Femenino , Fibroblastos/fisiología , Humanos , Inmunoensayo , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/metabolismo , Piel/patología , Factor de Crecimiento Transformador beta1/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
OBJECTIVE: To study the relation between human polyomavirus BK (BKV) infection and the risk of developing nephropathy, we monthly investigated the BKV load in urine and serum samples from 15 renal transplant recipients during 6 months in relation with immunosuppressive treatment and renal function. METHODS: BKV-DNA in serum samples was detected by nested PCR. BKV-DNA in urine and positive serum samples was quantified by a PCR protocol developed in our laboratory. RESULTS: Fifty-three percent of the patients had quantifiable BKV-DNA both in urine and serum samples but there was no relation between viruria and viraemia. Seventy-five percent of the patients on FK506 therapy and 71.4% of those on CyA therapy showed activation of BKV infection. No patients developed interstitial nephritis during the study. In ten patients serum creatinine levels were <2 mg/dl for the whole study, even if 80% presented BKV viruria and/ or viraemia. On the other hand, in 4 patients serum creatinine levels reached higher values, but they were BKV viruria and/or viraemia negative during the study. CONCLUSIONS: Our results suggest that viruria and viraemia may reflect independent BKV reactivation in different tissues. The activation of the infection does not seem to be related to the type of immunosuppressive treatment nor to impairment of renal function. To better understand the pathogenetic role of BKV infection in renal transplant recipients further investigations are needed.