RESUMEN
Development of effective human immunodeficiency virus 1 (HIV-1) vaccines requires synergy between innate and adaptive immune cells. Here we show that induction of the transcription factor CREB1 and its target genes by the recombinant canarypox vector ALVAC + Alum augments immunogenicity in non-human primates (NHPs) and predicts reduced HIV-1 acquisition in the RV144 trial. These target genes include those encoding cytokines/chemokines associated with heightened protection from simian immunodeficiency virus challenge in NHPs. Expression of CREB1 target genes probably results from direct cGAMP (STING agonist)-modulated p-CREB1 activity that drives the recruitment of CD4+ T cells and B cells to the site of antigen presentation. Importantly, unlike NHPs immunized with ALVAC + Alum, those immunized with ALVAC + MF59, the regimen in the HVTN702 trial that showed no protection from HIV infection, exhibited significantly reduced CREB1 target gene expression. Our integrated systems biology approach has validated CREB1 as a critical driver of vaccine efficacy and highlights that adjuvants that trigger CREB1 signaling may be critical for efficacious HIV-1 vaccines.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunogenicidad Vacunal/inmunología , Vacunas Virales/inmunología , Vacunas contra el SIDA/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Expresión Génica/inmunología , Vectores Genéticos/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/virología , Humanos , Inmunización/métodos , Primates/inmunología , Primates/virología , Vacunación/métodosRESUMEN
Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To better understand the host response to ZIKV infection, we adapted the 10× Genomics Chromium single-cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human monocyte-derived dendritic cells infected with ZIKV at the single-cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN-dependent and -independent genes (the antiviral module). We modeled the ZIKV-specific antiviral state at the protein level, leveraging experimentally derived protein interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for evaluating the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per-cell basis with experimental protein interaction data. IMPORTANCE: Zika virus (ZIKV) remains a public health threat given its potential for re-emergence and the detrimental fetal outcomes associated with infection during pregnancy. Understanding the dynamics between ZIKV and its host is critical to understanding ZIKV pathogenesis. Through ZIKV-inclusive single-cell RNA sequencing (scRNA-seq), we demonstrate on the single-cell level the dynamic interplay between ZIKV and the host: the transcriptional program that restricts viral infection and ZIKV-mediated inhibition of that response. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool for gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.
Asunto(s)
Células Dendríticas , Análisis de la Célula Individual , Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/fisiología , Infección por el Virus Zika/virología , Infección por el Virus Zika/inmunología , Células Dendríticas/virología , Células Dendríticas/inmunología , ARN Viral/metabolismo , ARN Viral/genética , Interferón Tipo I/metabolismo , Interacciones Huésped-Patógeno , Análisis de Secuencia de ARNRESUMEN
It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees' ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents.
Asunto(s)
Anticuerpos Antivirales/inmunología , Vacunas contra el Dengue/inmunología , Inmunogenicidad Vacunal/inmunología , Células T Auxiliares Foliculares/inmunología , Anticuerpos Neutralizantes/inmunología , Dengue/prevención & control , Femenino , Humanos , Masculino , Vacunas Combinadas/inmunologíaRESUMEN
To what extent can cognitive control, self-regulation, and the underlying midfrontal theta oscillatory activity of preschool children be modified by an ecologically realistic training based on pretend play? To answer this question, 70 children aged 4-6 years (37 boys) were assigned to a training group or a control group using a pairing randomization procedure. Children were administered 20 play sessions over 10 weeks. Benefits were evaluated with a pre-post design. The intervention helped children to engage more in self-regulation within the training activities. However, the intervention did not promote self-regulation outside of the training context, nor did it influence cognitive control and theta activity. These results provide a better understanding of the limitations of an ecologically realistic approach to cognitive control training.
Asunto(s)
Juego e Implementos de Juego , Autocontrol , Niño , Preescolar , Cognición , Humanos , Masculino , Conducta SocialRESUMEN
In response to microbial stimulation, monocytes can differentiate into macrophages or monocyte-derived dendritic cells (MoDCs) but the molecular requirements guiding these possible fates are poorly understood. In addition, the physiological importance of MoDCs in the host cellular and immune responses to microbes remains elusive. Here, we demonstrate that the nuclear orphan receptor NR4A3 is required for the proper differentiation of MoDCs but not for other types of DCs. Indeed, the generation of DC-SIGN+ MoDCs in response to LPS was severely impaired in Nr4a3-/- mice, which resulted in the inability to mount optimal CD8+ T cell responses to gram-negative bacteria. Transcriptomic analyses revealed that NR4A3 is required to skew monocyte differentiation toward MoDCs, at the expense of macrophages, and allows the acquisition of migratory characteristics required for MoDC function. Altogether, our data identify that the NR4A3 transcription factor is required to guide the fate of monocytes toward MoDCs.
Asunto(s)
Linaje de la Célula/inmunología , Proteínas de Unión al ADN/genética , Células Dendríticas/inmunología , Lipopolisacáridos/farmacología , Monocitos/inmunología , Proteínas del Tejido Nervioso/genética , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Diferenciación Celular , Linaje de la Célula/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/inmunología , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-4/farmacología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Activación de Linfocitos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Noqueados , Monocitos/citología , Monocitos/efectos de los fármacos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/inmunología , Cultivo Primario de Células , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Esteroides/deficiencia , Receptores de Esteroides/inmunología , Receptores de Hormona Tiroidea/deficiencia , Receptores de Hormona Tiroidea/inmunología , Transducción de Señal , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Susceptibility to autoimmune diabetes is a complex genetic trait. Linkage analyses exploiting the NOD mouse, which spontaneously develops autoimmune diabetes, have proved to be a useful tool for the characterization of some of these traits. In a linkage analysis using 3A9 TCR transgenic mice on both B10.BR and NOD.H2k backgrounds, we previously determined that both the Idd2 and Idd13 loci were linked to the proportion of immunoregulatory CD4-CD8- double negative (DN) T cells. In addition to Idd2 and Idd13, five other loci showed weak linkage to the proportion of DN T cells. Of interest, in an interim analysis, a locus on chromosome 12 is linked to DN T cell proportion in both the spleen and the lymph nodes. To determine the impact of this locus on DN T cells, we generated two congenic sublines, which we named Chr12P and Chr12D for proximal and distal, respectively. While 3A9 TCR:insHEL NOD.H2k-Chr12D mice were protected from diabetes, 3A9 TCR:insHEL NOD.H2k-Chr12P showed an increase in diabetes incidence. Yet, the proportion of DN T cells was similar to the parental 3A9 TCR NOD.H2k strain for both of these congenic sublines. A genome-wide two dimensional LOD score analysis reveals genetic epistasis between chromosome 12 and the Idd13 locus. Altogether, this study identified further complex genetic interactions in defining the proportion of DN T cells, along with evidence of genetic epistasis within a locus on chromosome 12 influencing autoimmune susceptibility.
Asunto(s)
Linaje de la Célula , Diabetes Mellitus Tipo 1/genética , Epistasis Genética , Predisposición Genética a la Enfermedad , Linfocitos T/inmunología , Animales , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Femenino , Ligamiento Genético , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones TransgénicosRESUMEN
The development of cognitive control enables children to better resist acting based on distracting information that interferes with the current action. Cognitive control improvement serves different functions that differ in part by the type of interference to resolve. Indeed, resisting to interference at the task-set level or at the response-preparation level is, respectively, associated with cognitive flexibility and inhibition. It is, however, unknown whether the same neural mechanism underlies these two functions across development. Studies in adults have revealed the contribution of midfrontal theta (MFT) oscillations in interference resolution. This study investigated whether MFT is involved in the resolution of different types of interference in two age groups identified as corresponding to different latent structures of executive functions. Preschool (4-6 years) and school children (6-8 years) were tested with a task involving interference at the response level and/or the task-set level while (electroencephalogram) EEG was recorded. Behaviorally, response time and accuracy were affected by task-set. Both age groups were less accurate when the interference occurred at the task-set level and only the younger group showed decreased accuracy when interference was presented at the response-preparation level. Furthermore, MFT power was increased, relative to the baseline, during the resolution of both types of interference and in both age groups. These findings suggest that MFT is involved in immature cognitive control (i.e., preschool and school-ages), by orchestrating its different cognitive processes, irrespective of the interference to resolve and of the level of cognitive control development (i.e., the degree of differentiation of executive functions).
Asunto(s)
Cognición/fisiología , Ritmo Teta/fisiología , Factores de Edad , Niño , Desarrollo Infantil/fisiología , Preescolar , Electroencefalografía/métodos , Función Ejecutiva/fisiología , Femenino , Humanos , Masculino , Tiempo de Reacción/fisiologíaRESUMEN
Natural killer cells constitute potent innate lymphoid cells that play a major role in both tumor immunosurveillance and viral clearance via their effector functions. A four-stage model of NK cell functional maturation has been established according to the expression of CD11b and CD27, separating mature NK (mNK) cells into distinct populations that exhibit specific phenotypic and functional properties. To identify genetic factors involved in the regulation of NK cell functional maturation, we performed a linkage analysis on F2 (B6.Rag1-/- × NOD.Rag1-/- intercross) mice. We identified six loci on chromosomes 2, 4, 7, 10, 11, and 18 that were linked to one or more mNK cell subsets. Subsequently, we performed an in silico analysis exploiting mNK cell subset microarray data, highlighting various genes and microRNAs as potential regulators of the functional maturation of NK cells. Together, the combination of our unbiased genetic linkage study and the in silico analysis positions genes known to affect NK cell biology along the specific stages of NK cell functional maturation. Moreover, this approach allowed us to uncover a novel candidate gene in the regulation of NK cell maturation, namely Trp53 Using mice deficient for Trp53, we confirm that this tumor suppressor regulates NK cell functional maturation. Additional candidate genes revealed in this study may eventually serve as targets for the modulation of NK cell functional maturation to potentiate both tumor immunosurveillance and viral clearance.
Asunto(s)
Regulación de la Expresión Génica , Ligamiento Genético , Células Asesinas Naturales/fisiología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Antígeno CD11b/inmunología , Diferenciación Celular , Procesos de Crecimiento Celular , Células Cultivadas , Simulación por Computador , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos NOD , MicroARNs/genética , MicroARNs/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
NK cells play a crucial role in innate immunity due to their direct cytotoxicity toward tumors, virally infected cells, and stressed cells, and they also contribute to the orchestration of the adaptive response by their ability to produce immunoregulatory cytokines. In secondary lymphoid organs, NK cells compose the third most abundant lymphocyte subset after T cells and B cells. In this study, we perform an unbiased linkage analysis to determine the genetic loci that may limit the size of the NK cell compartment. Specifically, we exploit differences in NK cell proportion and absolute number between the C57BL/6 and the NOD mice. In addition to the previously identified linkage to chromosome 8, we find that a locus on chromosome 17, which encompasses the MHC locus, impacts NK cell number. Moreover, we identify a locus on mouse chromosome 9 that is strongly linked to the proportion and absolute number of NK cells. Using NOD congenic mice, we validate that both the MHC and the chromosome 9 loci influence the proportion and absolute number of NK cells. We have thus identified additional loci specifically linked to the proportion of NK cells and present some of the potential candidate genes comprised within these loci.
Asunto(s)
Inmunidad Adaptativa/genética , Cromosomas Humanos Par 17/inmunología , Cromosomas Humanos Par 8/inmunología , Cromosomas Humanos Par 9/inmunología , Células Asesinas Naturales/inmunología , Animales , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 8/genética , Cromosomas Humanos Par 9/genética , Citometría de Flujo , Ligamiento Genético , Humanos , Ratones , Ratones Endogámicos C57BL/genética , Ratones Endogámicos C57BL/inmunología , Ratones Endogámicos NOD/genética , Ratones Endogámicos NOD/inmunología , Ratones TransgénicosRESUMEN
Pseudoaneurysm due to a swallowed fishbone rarely involves subclavian arteries. A 46-year-old male with nonaberrant right subclavian artery (RSA) presented pseudoaneurysm and brachial plexus septic necrosis. Open surgery with sternotomy and right transverse supraclavicular cervicotomy was done in emergency to achieve revascularization using in situ cryopreserved arterial allograft. Infection severity led to septic allograft rupture that necessitated ligation without new arterial reconstruction. During follow-up, patient remained alive 8 months after surgery. Neurological deficit slowly regressed, and no upper arm ischemic sign appeared.
Asunto(s)
Aneurisma Falso/microbiología , Aneurisma Infectado/microbiología , Huesos , Fístula Esofágica/microbiología , Migración de Cuerpo Extraño/etiología , Alimentos Marinos , Infecciones Estreptocócicas/microbiología , Arteria Subclavia/microbiología , Aneurisma Falso/diagnóstico por imagen , Aneurisma Falso/cirugía , Aneurisma Infectado/diagnóstico por imagen , Aneurisma Infectado/cirugía , Antibacterianos/uso terapéutico , Angiografía por Tomografía Computarizada , Fístula Esofágica/diagnóstico por imagen , Fístula Esofágica/cirugía , Migración de Cuerpo Extraño/diagnóstico por imagen , Migración de Cuerpo Extraño/cirugía , Humanos , Masculino , Persona de Mediana Edad , Infecciones Estreptocócicas/diagnóstico por imagen , Infecciones Estreptocócicas/cirugía , Arteria Subclavia/diagnóstico por imagen , Arteria Subclavia/cirugía , Resultado del Tratamiento , Procedimientos Quirúrgicos VascularesRESUMEN
Autoimmune diseases result from a break in immune tolerance. Various mechanisms of peripheral tolerance can protect against autoimmunity, including immunoregulatory CD4(-)CD8(-) double-negative (DN) T cells. Indeed, we have previously shown that diabetes-prone mouse strains exhibit a low proportion of DN T cells relative to that of diabetes-resistant mice, and that a single autologous transfer of DN T cells can impede autoimmune diabetes development, at least in the 3A9 TCR transgenic setting. In this study, we aim to understand the genetic basis for the difference in DN T cell proportion between diabetes-resistant and diabetes-prone mice. We thus perform an unbiased linkage analysis in 3A9 TCR F2 (NOD.H2(k) × B10.BR) mice and reveal that a locus on chromosome 9, which coincides with Idd2, is linked to the proportion of DN T cells in the lymph nodes. We generate two NOD.H2(k).B10-Chr9 congenic mouse strains and validate the role of this genetic interval in defining the proportion of DN T cells. Moreover, we find that the increased proportion of DN T cells in lymphoid organs is associated with a decrease in both diabetes incidence and serum IgG Ab levels. Together, the data suggest that Idd2 is linked to DN T cell proportion and that a physiological increase in DN T cell number may be sufficient to confer resistance to autoimmune diabetes. Altogether, these findings could help identify new candidate genes for the development of therapeutic avenues aimed at modulating DN T cell number for the prevention of autoimmune diseases.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Ganglios Linfáticos/inmunología , Sitios de Carácter Cuantitativo/inmunología , Animales , Diabetes Mellitus Tipo 1/prevención & control , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos TRESUMEN
Plasmacytoid dendritic cells (pDC) compose one of the many distinct dendritic cell subsets. The primary function of pDC is to potently produce type 1 IFNs upon stimulation, which is highly relevant in antiviral responses. Consequently, the ability to manipulate the size of the pDC compartment in vivo may increase the capacity to clear viral infections. In an attempt to identify genetic loci affecting the size of the pDC compartment, defined by both the proportion and absolute number of pDC, we undertook an unbiased genetic approach. Linkage analysis using inbred mouse strains identified a locus on chromosome 7 (Pdcc1) significantly linked to both the proportion and the absolute number of pDC in the spleen. Moreover, loci on either chromosome 11 (Pdcc2) or 9 (Pdcc3) modified the effect of Pdcc1 on chromosome 7 for the proportion and absolute number of pDC, respectively. Further analysis using mice congenic for chromosome 7 confirmed Pdcc1, demonstrating that variation within this genetic interval can regulate the size of the pDC compartment. Finally, mixed bone marrow chimera experiments showed that both the proportion and the absolute number of pDC are regulated by cell-intrinsic hematopoietic factors. Our findings highlight the multigenic regulation of the size of the pDC compartment and will facilitate the identification of genes linked to this trait.
Asunto(s)
Compartimento Celular/inmunología , Cromosomas de los Mamíferos/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Genes Dominantes/inmunología , Animales , Compartimento Celular/genética , Cromosomas de los Mamíferos/genética , Femenino , Genes Dominantes/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Endogámicos NZB , Ratones Noqueados , Quimera por Radiación , Bazo/citología , Bazo/inmunologíaAsunto(s)
Anestesia , Síndrome de Dificultad Respiratoria , Humanos , Pulmón , Proyectos Piloto , SevofluranoRESUMEN
Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To better understand the host response to ZIKV infection, we adapted the 10x Genomics Chromium single cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human moDCs infected with ZIKV at the single cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN dependent and independent genes (antiviral module). We modeled the ZIKV specific antiviral state at the protein level leveraging experimentally derived protein-interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for evaluating the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per cell basis with experimental protein interaction data. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool to gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.
RESUMEN
Protective immunity to dengue virus (DENV) requires antibody response to all four serotypes. Systems vaccinology identifies a multi-OMICs pre-vaccination signature and mechanisms predictive of broad antibody responses after immunization with a tetravalent live attenuated DENV vaccine candidate (Butantan-DV/TV003). Anti-inflammatory pathways, including TGF-ß signaling expressed by CD68low monocytes, and the metabolites phosphatidylcholine (PC) and phosphatidylethanolamine (PE) positively correlate with broadly neutralizing antibody responses against DENV. In contrast, expression of pro-inflammatory pathways and cytokines (IFN and IL-1) in CD68hi monocytes and primary and secondary bile acids negatively correlates with broad DENV-specific antibody responses. Induction of TGF-ß and IFNs is done respectively by PC/PE and bile acids in CD68low and CD68hi monocytes. The inhibition of viral sensing by PC/PE-induced TGF-ß is confirmed in vitro. Our studies show that the balance between metabolites and the pro- or anti-inflammatory state of innate immune cells drives broad and protective B cell response to a live attenuated dengue vaccine.
Asunto(s)
Anticuerpos Antivirales , Vacunas contra el Dengue , Virus del Dengue , Vacunas contra el Dengue/inmunología , Humanos , Virus del Dengue/inmunología , Anticuerpos Antivirales/inmunología , Dengue/inmunología , Dengue/prevención & control , Dengue/virología , Monocitos/inmunología , Monocitos/metabolismo , Formación de Anticuerpos/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/inmunología , Vacunación , Anticuerpos Neutralizantes/inmunologíaRESUMEN
When antigens derived from apoptotic cells are presented by conventional dendritic cells (cDC), T cell tolerance is induced. Surprisingly, the presentation of apoptotic cell antigens by an unconventional DC subset, termed merocytic dendritic cells (mcDC), can reverse T cell anergy. The potency of mcDC at breaking T cell tolerance has been demonstrated in the context of tumors and autoimmunity, suggesting that modulating the number of mcDC in vivo may be of clinical interest. To identify the genetic determinants that define the number of mcDC, we performed a linkage analysis between NOD and C57BL/6 mouse strains, where autoimmune-prone NOD mice show an increased proportion of mcDC relative to the non-autoimmune-prone C57BL/6 mice. We identified a locus on chromosome 2 significantly linked to both the proportion and the absolute number of mcDC in the spleen. Interestingly, the dominant interval on chromosome 2 overlaps with a locus previously associated with diabetes protection, namely Idd13. Using NOD.Idd13 congenic mice, we validate the impact of the Idd13 congenic interval in defining the proportion and number of mcDC in the spleen. These results show that the decreased number of mcDC is conferred by C57BL/6 alleles at the Idd13 locus, which is linked to diabetes resistance.
Asunto(s)
Células Dendríticas/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Animales , Recuento de Células , Mapeo Cromosómico , Células Dendríticas/clasificación , Células Dendríticas/patología , Diabetes Mellitus Tipo 1/patología , Femenino , Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Inmunofenotipificación , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones NoqueadosRESUMEN
Premise: A novel protocol for rapid plant DNA extraction using microneedles is proposed, which supports botanic surveys, taxonomy, and systematics. This protocol can be conducted in the field with limited laboratory skills and equipment. The protocol is validated by sequencing and comparing the results with QIAGEN spin-column DNA extractions using BLAST analyses. Methods and Results: Two sets of DNA extractions were conducted on 13 species spanning various leaf anatomies and phylogenetic lineages: (i) fresh leaves were punched with custom polymeric microneedle patches to recover genomic DNA, or (ii) QIAGEN DNA extractions. Three plastid (matK, rbcL, and trnH-psbA) and one nuclear ribosomal (ITS) DNA regions were amplified and sequenced using Sanger or nanopore technology. The proposed method reduced the extraction time to 1 min and yielded the same DNA sequences as the QIAGEN extractions. Conclusions: Our drastically faster and simpler method is compatible with nanopore sequencing and is suitable for multiple applications, including high-throughput DNA-based species identifications and monitoring.
RESUMEN
Milli-channel baffle labyrinths are widely used in drip irrigation systems. They induce a pressure drop enabling drip irrigation. However, with a section thickness that is measured in mm2, they are sensitive to clogging, which reduces the performance and service life of a drip irrigation system. The impact of chlorination (1.5 ppm of free chlorine during 1 h application) and pressure flushing (0.18 MPa) on the biofouling of non-pressure-compensating drippers, fed by real reclaimed wastewater, was studied at lab scale using optical coherence tomography. The effect of these treatments on microbial composition (bacteria and eukaryotes) was also investigated by High-throughput DNA sequencing. Biofouling was mainly observed in the inlet, outlet and return areas of the milli-labyrinth channel from drippers. Chlorination reduced biofilm development, particularly in the mainstream of the milli-labyrinth channel, and it was more efficient when combined with pressure flushing. Moreover, chlorination was more efficient in maintaining water distribution uniformity (CU < 95% compared to less than 85% for unchlorinated lines). It reduced more efficiently the bacterial concentration (≈1 log) and the diversity of the bacterial community in the dripper biofilms compared to the pressure flushing method. Chlorination significantly modified the microbial communities, promoting chlorine-resistant bacteria such as Comamonadaceae or Azospira. Inversely, several bacterial groups were identified as sensitive to chlorination such as Chloroflexi and Planctomycetes. Nevertheless, one month after stopping the treatments bacterial diversity recovered and the chlorine-sensitive bacteria such as Chloroflexi phylum and the Saprospiraceae, Spirochaetaceae, Christensenellaceae and Hydrogenophilaceae families re-emerged in conjunction with the growth of biofouling, highlighting the resilience of the bacteria originating from drippers. Based on PCoA analyses, the structure of the bacterial communities still clustered separately from non-chlorinated drippers, showing that the effect of chlorination was still detectable one month after stopping the treatment.
Asunto(s)
Incrustaciones Biológicas , Microbiota , Purificación del Agua , Biopelículas , Halogenación , Humanos , Aguas ResidualesRESUMEN
BACKGROUND: Molecular biology has emerged as a potential companion to histology for the diagnosis of rejection after heart transplantation. Reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) is a technique of targeted gene expression analysis suitable for formalin-fixed paraffin-embedded (FFPE) biopsies. Our aim was to assess RT-MLPA for the diagnosis of allograft rejection in heart transplantation. METHODS: We performed a cross-sectional, case-control, multicenter study. After the selection of a 14-transcript panel (endothelial burden, Natural killer cells, interferon-γ pathway, effector T-cells, and antigen presentation), RT-MLPA was applied to 183 FFPE endomyocardial biopsies (EMB), randomized into a training (nâ¯=â¯113) and a validation (nâ¯=â¯70) series. A two-step class prediction analysis was developed (Linear prediction score-LPS1: rejection vs non-rejection; LPS2: antibody-mediated rejection [AMR] vs acute cellular rejection [ACR]). A study of the agreement between pathology and RT-MLPA was performed. RESULTS: Overall, 48 ACR, 82 AMR, 5 mixed rejection, and 48 non-rejection EMBs were analyzed. Three molecular clusters were delineated by unsupervised hierarchical analysis (molecular non-rejection, ACR, and AMR). AMR was characterized by the high expression of CCL4, GNLY, FCGR3, CXCL11 and ACR by the high expression of CCL18 and ADAMdec. RT-MLPA and histopathology agreed in the final diagnosis in 82.2%, 67.7%, and 76.8% of the EMB in the test, validation, and overall cohort, respectively. Disagreement cases were more common in the case of histologic low-grade rejection and early post-transplant EMB. CONCLUSIONS: RT-MLPA is a suitable technique for targeted gene expression analysis on FFPE EMB with a good overall agreement with the histologic diagnosis of heart allograft rejection.