RESUMEN
BACKGROUND: Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is caused by UNC13D variants. The clinical manifestations of FHL3 are highly diverse and complex. Some patients exhibit atypical or incomplete phenotypes, making accurate diagnosis difficult. Our study aimed to broaden the understanding of the atypical FHL3 clinical spectrum. METHODS: In our study, we analyzed in detail the clinical features of four Chinese patients with UNC13D variants. Additionally, we conducted a comprehensive review of the existing literature on previously reported atypical manifestations and summarized the findings. RESULTS: Two of our patients presented with muscle involvement, while the other two had hematological involvement; none of them met the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH). However, protein expression and functional analysis ultimately confirmed diagnostic criteria for FHL3 in all patients. From the literature we reviewed, many atypical FHL3 patients had neurological involvement, especially isolated neurological manifestations. At the same time, arthritis and hypogammaglobulinemia were also prone to occur. CONCLUSION: Our study highlights that the expression of the Munc13-4 protein may not fully indicate the pathogenicity of UNC13D variants, whereas CD107a analysis could be more sensitive for disease diagnosis. These findings contribute to a broader understanding of the FHL3 clinical spectrum and may offer new insights into the underlying pathogenesis of UNC13D variants. It is crucial to prioritize the timely and accurate diagnosis of atypical patients, as they may often be overlooked among individuals with rheumatic or hematological diseases.
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Linfohistiocitosis Hemofagocítica , Proteínas de la Membrana , Niño , Femenino , Humanos , Lactante , Masculino , China/epidemiología , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfohistiocitosis Hemofagocítica/genética , Proteínas de la Membrana/genética , Mutación , Fenotipo , AdolescenteRESUMEN
Receptor proteins, such as epidermal growth factor receptor (EGFR), interact with other proteins in the extracellular region of the cell membrane to drive intracellular signalling. Therefore, analysis of extracellular protein-protein interactions (exPPIs) is important for understanding the biological function of receptor proteins. Here, we present an approach using a proximity biotinylation enzyme (AirID) fusion fragment of antigen binding (FabID) to analyse the proximity exPPIs of EGFR. AirID was C-terminally fused to the Fab fragment against EGFR (EGFR-FabID), which could then biotinylate the extracellular region of EGFR in several cell lines. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis indicated that many known EGFR interactors were identified as proximity exPPIs, along with many unknown candidate interactors, using EGFR-FabID. Interestingly, these proximity exPPIs were influenced by treatment with EGF ligand and its specific kinase inhibitor, gefitinib. These results indicate that FabID provides accurate proximity exPPI analysis of target receptor proteins on cell membranes with ligand and drug responses.
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Receptores ErbB , Espectrometría de Masas en Tándem , Fosforilación , Cromatografía Liquida , Ligandos , Receptores ErbB/metabolismo , Factor de Crecimiento Epidérmico/metabolismoRESUMEN
Herein, we report a child with COVID-19 and seemingly no underlying disease, who died suddenly. The autopsy revealed severe anemia and thrombocytopenia, splenomegaly, hypercytokinemia, and a rare ectopic congenital coronary origin. Immunohistochemical analysis demonstrated that the patient had acute lymphoblastic leukemia of the B-cell precursor phenotype (BCP-ALL). The complex cardiac and hematological abnormalities suggested the presence of an underlying disease; therefore, we performed whole-exome sequencing (WES). WES revealed a leucine-zipper-like transcription regulator 1 (LZTR1) variant, indicating Noonan syndrome (NS). Therefore, we concluded that the patient had underlying NS along with coronary artery malformation and that COVID-19 infection may have triggered the sudden cardiac death due to increased cardiac load caused by high fever and dehydration. In addition, multiple organ failure due to hypercytokinemia probably contributed to the patient's death. This case would be of interest to pathologists and pediatricians because of the limited number of NS patients with LZTR1 variants; the complex combination of an LZTR1 variant, BCP-ALL, and COVID-19; and a rare pattern of the anomalous origin of the coronary artery. Thus, we highlight the significance of molecular autopsy and the application of WES with conventional diagnostic methods.
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COVID-19 , Síndrome de Noonan , Humanos , Autopsia , Mortalidad del Niño , Síndrome de Liberación de Citoquinas , Fenotipo , Síndrome de Noonan/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive inflammatory disease caused by loss-of-function mutations in both alleles of the ADA2 gene. Most patients with DADA2 exhibit systemic vasculopathy consistent with polyarteritis nodosa, but large phenotypic variability has been reported, and the pathogenesis of DADA2 remains unclear. OBJECTIVES: This study sought to assess the clinical and genetic characteristics of Japanese patients with DADA2 and to gain insight into the pathogenesis of DADA2 by multi-omics analysis. METHODS: Clinical and genetic data were collected from 8 Japanese patients with DADA2 diagnosed between 2016 and 2019. ADA2 variants in this cohort were functionally analyzed by in vitro overexpression analysis. PBMCs from 4 patients with DADA2 were subjected to transcriptome and proteome analyses. Patient samples were collected before and after introduction of anti- TNF-α therapies. Transcriptome data were compared with those of normal controls and patients with other autoinflammatory diseases. RESULTS: Five novel ADA2 variants were identified in these 8 patients and were confirmed pathogenic by in vitro analysis. Anti-TNF-α therapy controlled inflammation in all 8 patients. Transcriptome and proteome analyses showed that upregulation of type II interferon signaling was characteristic of DADA2. Network analysis identified STAT1 as a key regulator and a hub molecule in DADA2 pathogenesis, a finding supported by the hyperactivation of STAT1 in patients' monocytes and B cells after IFN-γ stimulation. CONCLUSIONS: Type II interferon signaling and STAT1 are associated with the pathogenesis of DADA2.
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Adenosina Desaminasa/deficiencia , Agammaglobulinemia/inmunología , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Interferón gamma/inmunología , Leucocitos Mononucleares/inmunología , Factor de Transcripción STAT1/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Adenosina Desaminasa/inmunología , Adolescente , Adulto , Agammaglobulinemia/genética , Agammaglobulinemia/patología , Pueblo Asiatico , Niño , Preescolar , Femenino , Perfilación de la Expresión Génica , Humanos , Lactante , Péptidos y Proteínas de Señalización Intercelular/inmunología , Interferón gamma/genética , Japón , Leucocitos Mononucleares/patología , Masculino , Proteómica , Factor de Transcripción STAT1/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/patologíaRESUMEN
10q26 deletion syndrome is caused by a rare chromosomal abnormality, and patients with this syndrome present with an extensive and heterogeneous phenotypic spectrum. Several genes, such as EMX2 and FGFR2, were identified as the cause genital anomalies and facial dysmorphism in 10q26 deletion syndrome. However, the critical region for 10q26 deletion syndrome is not determined and the precise relationships between the causative genes and the phenotypes are still controversial. WD repeat domain 11 (WDR11), located at 10q25-26, was recently identified as a causative gene in hypogonadotropic hypogonadism, but other clinical phenotypes caused by WDR11 variants have not been identified. In this study, we have identified a WDR11 missense mutation, NM_018117.11: c.2108Gâ¯>â¯A; p.(Arg703Gln); ClinVar accession SCV000852064, in a two-year-old boy with severe growth retardation, ventricular septal defect, and coloboma symptoms. The case suggests that WDR11 is partially responsible for the clinical features of 10q26 deletion syndrome and provides novel insights into the pathophysiology of this syndrome.
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Coloboma/genética , Trastornos del Crecimiento/genética , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Preescolar , Deleción Cromosómica , Cromosomas Humanos Par 10/genética , Coloboma/patología , Predisposición Genética a la Enfermedad , Trastornos del Crecimiento/patología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/patología , Proteínas de Homeodominio/genética , Humanos , Masculino , Fenotipo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Factores de Transcripción/genética , Anomalías Urogenitales/genética , Anomalías Urogenitales/patologíaRESUMEN
Disturbed balance between immune surveillance and tolerance may lead to poor clinical outcomes in some malignancies. In paired analyses of adenocarcinoma and normal mucosa from 142 patients, we found a significant increase of the CD4/CD8 ratio and accumulation of regulatory T cells (Tregs) within the adenocarcinoma. The increased frequency of Tregs correlated with the local infiltration and extension of the tumor. There was concurrent maturation arrest, upregulation of programmed death-1 expression, and functional impairment in CD8+ T cells (CTLs) isolated from the adenocarcinoma. Adenocarcinoma-associated Tregs directly inhibit the function of normal human CTLs in vitro. With histopathological analysis, Foxp3+ Tregs were preferentially located in stroma. Concurrent transcriptome analysis of epithelial cells, stromal cells, and T cell subsets obtained from carcinomatous and normal intestinal samples from patients revealed a distinct gene expression signature in colorectal adenocarcinoma-associated Tregs, with overexpression of CCR1, CCR8, and TNFRSF9, whereas their ligands CCL4 and TNFSF9 were found upregulated in cancerous epithelium. Overexpression of WNT2 and CADM1, associated with carcinogenesis and metastasis, in cancer-associated stromal cells suggests that both cancer cells and stromal cells play important roles in the development and progression of colorectal cancer through the formation of a tumor microenvironment. The identification of CTL anergy by Tregs and the unique gene expression signature of human Tregs and stromal cells in colorectal cancer patients may facilitate the development of new therapeutics against malignancies.
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Adenocarcinoma/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias Colorrectales/inmunología , Linfocitos T Reguladores/inmunología , Escape del Tumor/inmunología , Anciano , Femenino , Humanos , Inmunidad Mucosa/inmunología , Vigilancia Inmunológica/inmunología , Mucosa Intestinal/inmunología , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1RESUMEN
OBJECTIVE: To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) with no NLRP3 mutation. METHODS: A Japanese male child diagnosed as having NOMID was studied. The patient did not have any NLRP3 mutation, even as low-frequency mosaicism. We performed whole-exome sequencing on the patient and his parents. Induced pluripotent stem cells (iPSCs) were established from the patient's fibroblasts. The iPSCs were then differentiated into monocyte lineage to evaluate the cytokine profile. RESULTS: We established multiple iPSC clones from a patient with NOMID and incidentally found that the phenotypes of monocytes from iPSC clones were heterogeneous and could be grouped into disease and normal phenotypes. Because each iPSC clone was derived from a single somatic cell, we hypothesized that the patient had somatic mosaicism of an interleukin-1ß-related gene. Whole-exome sequencing of both representative iPSC clones and the patient's blood revealed a novel heterozygous NLRC4 mutation, p.T177A (c.529A>G), as a specific mutation in diseased iPSC clones. Knockout of the NLRC4 gene using the clustered regularly interspaced short palindromic repeat/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. CONCLUSION: Our findings indicate that the patient has somatic mosaicism of a novel NLRC4 mutation. To our knowledge, this is the first case showing that somatic mutation of NLRC4 causes autoinflammatory symptoms compatible with NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotype dissection for individualized diagnoses.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/genética , Proteínas de Unión al Calcio/genética , Síndromes Periódicos Asociados a Criopirina/genética , Mutación , Humanos , Células Madre Pluripotentes Inducidas , Recién Nacido , Masculino , FenotipoRESUMEN
Zinc transporters play a critical role in spatiotemporal regulation of zinc homeostasis. Although disruption of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology, it is largely unknown which zinc transporters are responsible for the intestinal epithelial homeostasis. Here, we show that Zrt-Irt-like protein (ZIP) transporter ZIP7, which is highly expressed in the intestinal crypt, is essential for intestinal epithelial proliferation. Mice lacking Zip7 in intestinal epithelium triggered endoplasmic reticulum (ER) stress in proliferative progenitor cells, leading to significant cell death of progenitor cells. Zip7 deficiency led to the loss of Olfm4+ intestinal stem cells and the degeneration of post-mitotic Paneth cells, indicating a fundamental requirement for Zip7 in homeostatic intestinal regeneration. Taken together, these findings provide evidence for the importance of ZIP7 in maintenance of intestinal epithelial homeostasis through the regulation of ER function in proliferative progenitor cells and maintenance of intestinal stem cells. Therapeutic targeting of ZIP7 could lead to effective treatment of gastrointestinal disorders.
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Proteínas de Transporte de Catión/genética , Proliferación Celular/genética , Estrés del Retículo Endoplásmico/genética , Zinc/metabolismo , Animales , Apoptosis/genética , Proteínas de Transporte de Catión/biosíntesis , Retículo Endoplásmico/genética , Células Epiteliales/metabolismo , Tracto Gastrointestinal/crecimiento & desarrollo , Tracto Gastrointestinal/metabolismo , Técnicas de Inactivación de Genes , Homeostasis , Mucosa Intestinal/crecimiento & desarrollo , Mucosa Intestinal/metabolismo , Ratones , Organoides/crecimiento & desarrollo , Células de Paneth/metabolismo , Células Madre/metabolismoRESUMEN
Memory CD4(+) T cells promote protective humoral immunity; however, how memory T cells acquire this activity remains unclear. This study demonstrates that CD4(+) T cells develop into antigen-specific memory T cells that can promote the terminal differentiation of memory B cells far more effectively than their naive T-cell counterparts. Memory T cell development requires the transcription factor B-cell lymphoma 6 (Bcl6), which is known to direct T-follicular helper (Tfh) cell differentiation. However, unlike Tfh cells, memory T cell development did not require germinal center B cells. Curiously, memory T cells that develop in the absence of cognate B cells cannot promote memory B-cell recall responses and this defect was accompanied by down-regulation of genes associated with homeostasis and activation and up-regulation of genes inhibitory for T-cell responses. Although memory T cells display phenotypic and genetic signatures distinct from Tfh cells, both had in common the expression of a group of genes associated with metabolic pathways. This gene expression profile was not shared to any great extent with naive T cells and was not influenced by the absence of cognate B cells during memory T cell development. These results suggest that memory T cell development is programmed by stepwise expression of gatekeeper genes through serial interactions with different types of antigen-presenting cells, first licensing the memory lineage pathway and subsequently facilitating the functional development of memory T cells. Finally, we identified Gdpd3 as a candidate genetic marker for memory T cells.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Hidrolasas Diéster Fosfóricas/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos CD4/metabolismo , Comunicación Celular , Diferenciación Celular , Células Cultivadas , Regulación de la Expresión Génica , Inmunocompetencia , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , TranscriptomaRESUMEN
The CCR4-NOT deadenylase complex plays crucial roles in mRNA decay and translational repression induced by poly(A) tail shortening. Although the in vitro activities of each component of this complex have been well characterized, its in vivo role in immune cells remains unclear. Here we show that mice lacking the CNOT3 subunit of this complex, specifically in B cells, have a developmental block at the pro- to pre-B cell transition. CNOT3 regulated generation of germline transcripts in the VH region of the immunoglobulin heavy chain (Igh) locus, compaction of the locus, and subsequent Igh gene rearrangement and destabilized tumor suppressor p53 mRNA. The developmental defect in the absence of CNOT3 could be partially rescued by ablation of p53 or introduction of a pre-rearranged Igh transgene. Thus, our data suggest that the CCR4-NOT complex regulates B cell differentiation by controlling Igh rearrangement and destabilizing p53 mRNA.
Asunto(s)
Linfocitos B/inmunología , Reordenamiento Génico de Cadena Pesada de Linfocito B/inmunología , Estabilidad del ARN/inmunología , ARN Mensajero/inmunología , Factores de Transcripción/inmunología , Proteína p53 Supresora de Tumor/inmunología , Animales , Linfocitos B/citología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Reordenamiento Génico de Cadena Pesada de Linfocito B/genética , Ratones , Ratones Transgénicos , Estabilidad del ARN/genética , ARN Mensajero/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34(+)CD38(+)CD19(+) and CD34(-)CD19(+) cells initiated leukemia, and in MLL-AF9 patients, CD34(-)CD19(+) cells were LICs. In MLL-ENL patients, either CD34(+) or CD34(-) cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34(+)CD38(-)CD19(-)CD33(-) cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34(+)CD38(-)CD19(-)CD33(-) cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4(+) single positive (SP), CD8(+) SP, and CD4(+)CD8(+) double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34(+)CD38(+) and CD34(-) LICs but not in CD34(+)CD38(-)CD19(-)CD33(-) HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia.
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Antígenos CD34/genética , Regulación Leucémica de la Expresión Génica , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animales , Antígeno CD24/genética , Niño , Preescolar , Femenino , Reordenamiento Génico , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Inmunofenotipificación , Lactante , Masculino , Ratones , Ratones Endogámicos NOD , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores de IgG/genética , Tetraspanina 29/genéticaRESUMEN
The immune system is influenced by the vital zinc (Zn) status, and Zn deficiency triggers lymphopenia; however, the mechanisms underlying Zn-mediated lymphocyte maintenance remain elusive. Here we investigated ZIP10, a Zn transporter expressed in the early B-cell developmental process. Genetic ablation of Zip10 in early B-cell stages resulted in significant reductions in B-cell populations, and the inducible deletion of Zip10 in pro-B cells increased the caspase activity in parallel with a decrease in intracellular Zn levels. Similarly, the depletion of intracellular Zn by a chemical chelator resulted in spontaneous caspase activation leading to cell death. Collectively, these findings indicated that ZIP10-mediated Zn homeostasis is essential for early B-cell survival. Moreover, we found that ZIP10 expression was regulated by JAK-STAT pathways, and its expression was correlated with STAT activation in human B-cell lymphoma, indicating that the JAK-STAT-ZIP10-Zn signaling axis influences the B-cell homeostasis. Our results establish a role of ZIP10 in cell survival during early B-cell development, and underscore the importance of Zn homeostasis in immune system maintenance.
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Apoptosis/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas de Transporte de Catión/inmunología , Zinc/metabolismo , Animales , Linfocitos B/citología , Caspasas/metabolismo , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Diferenciación Celular , Supervivencia Celular/inmunología , Citocinas/metabolismo , Homeostasis , Humanos , Quinasas Janus/metabolismo , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Linfopenia/etiología , Linfopenia/inmunología , Linfopenia/metabolismo , Ratones , Ratones Noqueados , Modelos Inmunológicos , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Zinc/deficienciaRESUMEN
Aicardi-Goutières syndrome (AGS) is a rare, genetically determined early-onset progressive encephalopathy. To date, mutations in six genes have been identified as etiologic for AGS. Our Japanese nationwide AGS survey identified six AGS-affected individuals without a molecular diagnosis; we performed whole-exome sequencing on three of these individuals. After removal of the common polymorphisms found in SNP databases, we were able to identify IFIH1 heterozygous missense mutations in all three. In vitro functional analysis revealed that IFIH1 mutations increased type I interferon production, and the transcription of interferon-stimulated genes were elevated. IFIH1 encodes MDA5, and mutant MDA5 lacked ligand-specific responsiveness, similarly to the dominant Ifih1 mutation responsible for the SLE mouse model that results in type I interferon overproduction. This study suggests that the IFIH1 mutations are responsible for the AGS phenotype due to an excessive production of type I interferon.
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Enfermedades Autoinmunes del Sistema Nervioso/genética , ARN Helicasas DEAD-box/genética , Mutación Missense , Malformaciones del Sistema Nervioso/genética , Secuencia de Aminoácidos , Animales , ARN Helicasas DEAD-box/química , Femenino , Humanos , Helicasa Inducida por Interferón IFIH1 , Japón , Masculino , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de AminoácidoRESUMEN
One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell-dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell-dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen.
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Anticuerpos/inmunología , Linfocitos B/inmunología , Células Germinativas/inmunología , Centro Germinal/inmunología , Memoria Inmunológica/inmunología , Transducción de Señal/inmunología , Animales , Anticuerpos/genética , Anticuerpos/metabolismo , Linfocitos B/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Células Germinativas/metabolismo , Centro Germinal/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-6 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transducción de Señal/genéticaRESUMEN
In recent years, advances in the humanized mouse system have led to significantly increased levels of human hematopoietic stem cell (HSC) engraftment. The remaining limitations in human HSC engraftment and function include lymphoid-skewed differentiation and inefficient myeloid development in the recipients. Limited human HSC function may partially be attributed to the inability of the host mouse microenvironment to provide sufficient support to human hematopoiesis. To address this problem, we created membrane-bound human stem cell factor (SCF)/KIT ligand (KL)-expressing NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. hSCF Tg NSG recipients of human HSCs showed higher levels of both human CD45(+) cell engraftment and human CD45(+)CD33(+) myeloid development compared with NSG recipients. Expression of hSCF/hKL accelerated the differentiation of the human granulocyte lineage cells in the recipient bone marrow. Human mast cells were identified in bone marrow, spleen, and gastrointestinal tissues of the hSCF Tg NSG recipients. This novel in vivo humanized mouse model demonstrates the essential role of membrane-bound hSCF in human myeloid development. Moreover, the hSCF Tg NSG humanized recipients may facilitate investigation of in vivo differentiation, migration, function, and pathology of human mast cells.
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Células Madre Hematopoyéticas/citología , Mastocitos/citología , Ratones Transgénicos , Células Mieloides/citología , Regiones Promotoras Genéticas , Factor de Células Madre/metabolismo , Animales , Diferenciación Celular/fisiología , Separación Celular , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Quimera por Trasplante/fisiología , Tolerancia al Trasplante/fisiologíaRESUMEN
Chronic infantile neurological cutaneous and articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease and is caused by a heterozygous germline gain-of-function mutation in the NLRP3 gene. We recently found a high incidence of NLRP3 somatic mosaicism in apparently mutation-negative CINCA/NOMID patients using subcloning and subsequent capillary DNA sequencing. It is important to rapidly diagnose somatic NLRP3 mosaicism to ensure proper treatment. However, this approach requires large investments of time, cost, and labour that prevent routine genetic diagnosis of low-level somatic NLRP3 mosaicism. We developed a routine pipeline to detect even a low-level allele of NLRP3 with statistical significance using massively parallel DNA sequencing. To address the critical concern of discriminating a low-level allele from sequencing errors, we first constructed error rate maps of 14 polymerase chain reaction products covering the entire coding NLRP3 exons on a Roche 454 GS-FLX sequencer from 50 control samples without mosaicism. Based on these results, we formulated a statistical confidence value for each sequence variation in each strand to discriminate sequencing errors from real genetic variation even in a low-level allele, and thereby detected base substitutions at an allele frequency as low as 1% with 99.9% or higher confidence.
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Proteínas Portadoras/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mosaicismo , Alelos , Síndromes Periódicos Asociados a Criopirina/genética , Exones , Variación Genética , Humanos , Modelos Moleculares , Proteína con Dominio Pirina 3 de la Familia NLR , Reacción en Cadena de la PolimerasaRESUMEN
Invariant natural killer T (iNKT) cells possess potent antitumor effects after activation with a specific glycolipid antigen, α-galactosylceramide (αGalCer). A phase I-II clinical study of αGalCer-pulsed dendritic cells (DC) to activate endogenous iNKT cells was previously performed in patients with non-small-cell lung cancer (NSCLC). In this clinical trial, the patients with increased interferon-γ (IFN-γ) production (>two-fold) in PBMC after the DC treatment (good responder group) experienced a prolonged overall survival time in comparison with the poor responder group. We extended the previous study and performed a microarray-based gene expression analysis using peripheral blood CD56(+) cells and CD56(-) CD3(+) T cells from patients enrolled in the above-mentioned clinical study. We sought to identify any biomarkers associated with the immune responses in this immunotherapy trial. Six patient samples corresponding to three subjects in the good responder group and three subjects in the poor responder group were included in the microarray analysis. Genes differentially expressed between pre-treatment and post-treatment samples were selected for analysis. Subsequently, genes that were only expressed in the good responder group or poor responder group were chosen. After these procedures, four selected genes were quantified by reverse transcriptase-polymerase chain reaction in another eight patient samples, and two genes, LTB4DH and DPYSL3, were confirmed to be candidate genes for the predictor of a good immune response. The expression profile of these two genes may be associated with the responsiveness of IFN-γ production after αGalCer-pulsed DC treatment.
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Oxidorreductasas de Alcohol/genética , Perfilación de la Expresión Génica , Proteínas Musculares/genética , Linfocitos T/metabolismo , Adulto , Anciano , Complejo CD3/metabolismo , Antígeno CD56/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/terapia , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Análisis por Conglomerados , Células Dendríticas/inmunología , Células Dendríticas/trasplante , Femenino , Galactosilceramidas/inmunología , Predisposición Genética a la Enfermedad/genética , Humanos , Inmunoterapia Adoptiva , Interferón gamma/metabolismo , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Linfocitos T/inmunologíaRESUMEN
Human acute myeloid leukemia (AML) originates from rare leukemia stem cells (LSCs). Because these chemotherapy-resistant LSCs are thought to underlie disease relapse, effective therapeutic strategies specifically targeting these cells may be beneficial. Here, we report identification of a primary human LSC gene signature and functional characterization of human LSC-specific molecules in vivo in a mouse xenotransplantation model. In 32 of 61 (53%) patients with AML, either CD32 or CD25 or both were highly expressed in LSCs. CD32- or CD25-positive LSCs could initiate AML and were cell cycle-quiescent and chemotherapy-resistant in vivo. Normal human hematopoietic stem cells depleted of CD32- and CD25-positive cells maintained long-term multilineage hematopoietic reconstitution capacity in vivo, indicating the potential safety of treatments targeting these molecules. In addition to CD32 and CD25, quiescent LSCs within the bone marrow niche also expressed the transcription factor WT1 and the kinase HCK. These molecules are also promising targets for LSC-specific therapy.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Leucemia/terapia , Células Madre Neoplásicas/metabolismo , Animales , Trasplante de Médula Ósea/métodos , Ciclo Celular , Linaje de la Célula , Separación Celular , Diseño de Fármacos , Citometría de Flujo , Regulación Leucémica de la Expresión Génica , Humanos , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Ratones , Ratones SCID , Receptores de IgG/biosíntesis , Trasplante HeterólogoRESUMEN
BACKGROUND AND AIM: Endoscopic ultrasonography (EUS) is established as a standard approach for locoregional staging of esophageal cancer. However, only a few published studies have attempted to correlate the station of the abnormal lymph nodes detected by EUS with the definitive histology. We compared EUS and computed tomography (CT) in the initial staging of esophageal squamous cell carcinoma. METHODS: Consecutive patients with esophageal cancer undergoing EUS were evaluated. EUS findings and patient data including histopatology were collected prospectively and analyzed retrospectively. Lymph node locations were divided into three groups; abdominal (A), paraesophageal (B), and thoracic paratracheal (C). RESULTS: A total of 365 consecutive patients underwent EUS and 159 patients underwent esophagectomy without neoadjuvant chemotherapy. Thirty-eight patients were excluded (insufficient EUS, etc.), and 121 patients were enrolled. The overall accuracy of EUS was 64% (sensitivity 68%, specificity 58%, positive predictive value [PPV] 68%), CT was 51% (sensitivity 33%, specificity 75%, PPV 64%), and CT + EUS was 64% (sensitivity 74%, specificity 50%, PPV 66%). The accuracy of EUS was higher than CT in Groups A and C. Sensitivity of CT was lower than that of EUS alone and CT + EUS. CONCLUSIONS: This study has demonstrated that EUS is a more accurate technique than contrast-enhanced CT for detecting abnormal lymph nodes. Sensitivity of CT was lower than that of EUS alone and CT + EUS. But some metastatic lymph nodes in neck and abdominal fields are only detectable by CT. Therefore, both EUS and CT should be undertaken for routine examination prior to treatment of esophageal cancer.
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Carcinoma de Células Escamosas/secundario , Endosonografía , Neoplasias Esofágicas/secundario , Ganglios Linfáticos/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/cirugía , Neoplasias Esofágicas/diagnóstico por imagen , Neoplasias Esofágicas/cirugía , Esofagectomía , Femenino , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
T helper type 2 (T(H)2) bias, which is the propensity of naive CD4(+) T cells to differentiate into interleukin 4 (IL-4)-secreting T(H)2 cells, is a genetic trait that affects susceptibility to infectious, autoimmune and allergic diseases. T(H)2 bias correlates with the amount of IL-4 initially secreted by newly activated helper T cells that feeds back positively through the pathway of the IL-4 receptor and the transcription factors STAT6 and GATA-3 to drive T(H)2 development. Here we identify Mina, a member of the jumonji C (JmjC) protein family, as a genetic determinant of T(H)2 bias. Mina specifically bound to and repressed the Il4 promoter. Mina overexpression in transgenic mice impaired Il4 expression, whereas its knockdown in primary CD4(+) T cells led to Il4 derepression. Our findings collectively provide mechanistic insight into an Il4-regulatory pathway that controls helper T cell differentiation and genetic variation in T(H)2 bias.