RESUMEN
Background: Myofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF). Methods: Using WI-38 cells, Fibroblast (FIB) to MYF differentiation was triggered using TGF-ß1 treatment and FIB to LIF differentiation using Metformin treatment. We also analyzed the MYF to LIF and LIF to MYF differentiation by pre-treating the WI-38 cells with TGF-ß1 or Metformin respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IPF patients-derived lung fibroblasts (LF-IPF) cultured in vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells. Results: WI-38 cells and LF-IPF display similar phenotypical and gene expression responses to TGF-ß1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 cells and LF-IPF treated with TGF-ß1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-ß1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the MYF to LIF and LIF to MYF reversible switch using WI-38 cells. Conclusions: WI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research.
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Diferenciación Celular , Fibroblastos , Fibrosis Pulmonar Idiopática , Miofibroblastos , Factor de Crecimiento Transformador beta1 , Humanos , Miofibroblastos/metabolismo , Fibroblastos/metabolismo , Línea Celular , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Pulmón/patología , Pulmón/citología , Transcriptoma , Metformina/farmacología , Plasticidad de la Célula/efectos de los fármacos , FenotipoRESUMEN
Bronchopulmonary dysplasia (BPD) is a neonatal lung disease developing in premature babies characterized by arrested alveologenesis and associated with decreased Fibroblast growth factor 10 (FGF10) expression. One-week hyperoxia (HYX) exposure of newborn mice leads to a permanent arrest in alveologenesis. To test the role of Fgf10 signaling to promote de novo alveologenesis following hyperoxia, we used transgenic mice allowing inducible expression of Fgf10 and recombinant FGF10 (rFGF10) protein delivered intraperitoneally. We carried out morphometry analysis, and IF on day 45. Alveolospheres assays were performed co-culturing AT2s from normoxia (NOX) with FACS-isolated Sca1Pos resident mesenchymal cells (rMC) from animals exposed to NOX, HYX-PBS, or HYX-FGF10. scRNAseq between rMC-Sca1Pos isolated from NOX and HYX-PBS was also carried out. Transgenic overexpression of Fgf10 and rFGF10 administration rescued the alveologenesis defects following HYX. Alveolosphere assays indicate that the activity of rMC-Sca1Pos is negatively impacted by HYX and partially rescued by rFGF10 treatment. Analysis by IF demonstrates a significant impact of rFGF10 on the activity of resident mesenchymal cells. scRNAseq results identified clusters expressing Fgf10, Fgf7, Pdgfra, and Axin2, which could represent the rMC niche cells for the AT2 stem cells. In conclusion, we demonstrate that rFGF10 administration is able to induce de novo alveologenesis in a BPD mouse model and identified subpopulations of rMC-Sca1Pos niche cells potentially representing its cellular target.
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Displasia Broncopulmonar , Hiperoxia , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/metabolismo , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Humanos , Hiperoxia/metabolismo , Recién Nacido , Pulmón/metabolismo , Ratones , Ratones TransgénicosRESUMEN
Oligodendrocytes exist in a heterogenous state and are implicated in multiple neuropsychiatric diseases including dementia. Cortical oligodendrocytes are a glial population uniquely positioned to play a key role in neurodegeneration by synchronizing circuit connectivity but molecular pathways specific to this role are lacking. We utilized oligodendrocyte-specific translating ribosome affinity purification and RNA-seq (TRAP-seq) to transcriptionally profile adult mature oligodendrocytes from different regions of the central nervous system. Weighted gene co-expression network analysis reveals distinct region-specific gene networks. Two of these mature myelinating oligodendrocyte gene networks uniquely define cortical oligodendrocytes and differentially regulate cortical myelination (M8) and synaptic signaling (M4). These two cortical oligodendrocyte gene networks are enriched for genes associated with dementia including MAPT and include multiple gene targets of the regulatory microRNA, miR-142-3p. Using a combination of TRAP-qPCR, miR-142-3p overexpression in vitro, and miR-142-null mice, we show that miR-142-3p negatively regulates cortical myelination. In rTg4510 tau-overexpressing mice, cortical myelination is compromised, and tau-mediated neurodegeneration is associated with gene co-expression networks that recapitulate both the M8 and M4 cortical oligodendrocyte gene networks identified from normal cortex. We further demonstrate overlapping gene networks in mature oligodendrocytes present in normal cortex, rTg4510 and miR-142-null mice, and existing datasets from human tauopathies to provide evidence for a critical role of miR-142-3p-regulated cortical myelination and oligodendrocyte-mediated synaptic signaling in neurodegeneration.
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MicroARNs/genética , Tauopatías/genética , Proteínas tau/genética , Animales , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Corteza Cerebelosa/metabolismo , Corteza Cerebelosa/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Humanos , Ratones , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Oligodendroglía/metabolismo , RNA-Seq , Tauopatías/metabolismo , Tauopatías/patologíaRESUMEN
Noncoding RNAs have been widely recognized as essential mediators of gene regulation. However, in contrast to protein-coding genes, much less is known about the influence of noncoding RNAs on human diseases. Here we examined the association of genetic variants located in primary microRNA sequences and long noncoding RNAs (lncRNAs) with Alzheimer disease (AD) by leveraging data from the largest genome-wide association meta-analysis of late-onset AD. Variants annotated to 5 miRNAs and 10 lncRNAs (in seven distinct loci) exceeded the Bonferroni-corrected significance threshold (p < 1.02 × 10-6 ). Among these, a leading variant (rs2526377:A>G) at the 17q22 locus annotated to two noncoding RNAs (MIR142 and BZRAP1-AS) was significantly associated with a reduced risk of AD and fulfilled predefined criteria for being a functional variant. Our functional genomic analyses revealed that rs2526377 affects the promoter activity and decreases the expression of miR-142. Moreover, differential expression analysis by RNA-Seq in human iPSC-derived neural progenitor cells and the hippocampus of miR-142 knockout mice demonstrated multiple target genes of miR-142 in the brain that are likely to be involved in the inflammatory and neurodegenerative manifestations of AD. These include TGFBR1 and PICALM, of which their derepression in the brain due to reduced expression levels of miR-142-3p may reduce the risk of AD.
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Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad , Variación Genética , MicroARNs/genética , Regiones Promotoras Genéticas , Alelos , Enfermedad de Alzheimer/metabolismo , Animales , Línea Celular , Mapeo Cromosómico , Biología Computacional/métodos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Estudios de Asociación Genética , Estudio de Asociación del Genoma Completo , Hipocampo/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Polimorfismo de Nucleótido Simple , Interferencia de ARN , ARN no TraducidoRESUMEN
Fibroblast growth factors (Fgfs) mediate organ repair. Lung epithelial cell overexpression of Fgf10 postbleomycin injury is both protective and therapeutic, characterized by increased survival and attenuated fibrosis. Exogenous administration of FGF7 (palifermin) also showed prophylactic survival benefits in mice. The role of endogenous Fgfr2b ligands on bleomycin-induced lung fibrosis is still elusive. This study reports the expression of endogenous Fgfr2b ligands, receptors, and signaling targets in wild-type mice following bleomycin lung injury. In addition, the impact of attenuating endogenous Fgfr2b-ligands following bleomycin-induced fibrosis was tested by using a doxycycline (dox)-based inducible, soluble, dominant-negative form of the Fgfr2b receptor. Double-transgenic (DTG) Rosa26(rtTA/+);tet(O)solFgfr2b mice were validated for the expression and activity of soluble Fgfr2b (failure to regenerate maxillary incisors, attenuated recombinant FGF7 signal in the lung). As previously reported, no defects in lung morphometry were detected in DTG (+dox) mice exposed from postnatal days (PN) 1 through PN105. Female single-transgenic (STG) and DTG mice were subjected to various levels of bleomycin injury (1.0, 2.0, and 3.0 U/kg). Fgfr2b ligands were attenuated either throughout injury (days 0-11; days 0-28) or during later stages (days 6-28 and 14-28). No significant changes in survival, weight, lung function, confluent areas of fibrosis, or hydroxyproline deposition were detected in DTG mice. These results indicate that endogenous Fgfr2b ligands do not significantly protect against bleomycin injury, nor do they expedite the resolution of bleomycin-induced lung injury in mice.
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Antibióticos Antineoplásicos/efectos adversos , Bleomicina/efectos adversos , Factor 7 de Crecimiento de Fibroblastos/farmacología , Fibrosis Pulmonar , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Femenino , Ratones , Ratones Transgénicos , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/agonistas , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal/genéticaRESUMEN
Fibroblast growth factor 10 (Fgf10) is a key regulator of diverse organogenetic programs during mouse development, particularly branching morphogenesis. Fgf10-null mice suffer from lung and limb agenesis as well as cecal and colonic atresia and are thus not viable. To date, the Mlcv1v-nLacZ-24 transgenic mouse strain (referred to as Fgf10(LacZ)), which carries a LacZ insertion 114 kb upstream of exon 1 of Fgf10 gene, has been the only strain to allow transient lineage tracing of Fgf10-positive cells. Here, we describe a novel Fgf10(Cre-ERT2) knock-in line (Fgf10(iCre)) in which a Cre-ERT2-IRES-YFP cassette has been introduced in frame with the ATG of exon 1 of Fgf10 gene. Our studies show that Cre-ERT2 insertion disrupts Fgf10 function. However, administration of tamoxifen to Fgf10(iCre); Tomato(flox) double transgenic embryos or adult mice results in specific labeling of Fgf10-positive cells, which can be lineage-traced temporally and spatially. Moreover, we show that the Fgf10(iCre) line can be used for conditional gene inactivation in an inducible fashion during early developmental stages. We also provide evidence that transcription factors located in the first intron of Fgf10 gene are critical for maintaining Fgf10 expression over time. Thus, the Fgf10(iCre) line should serve as a powerful tool to explore the functions of Fgf10 in a controlled and stage-specific manner.
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Desarrollo Embrionario , Factor 10 de Crecimiento de Fibroblastos/genética , Células Madre Mesenquimatosas/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Exones , Silenciador del Gen , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Tamoxifeno/administración & dosificaciónRESUMEN
BACKGROUND: Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature neonates. The pathogenesis of NEC is characterized by an intestinal epithelial injury caused by perinatal insults, leading to the activation of the mucosal innate immune system and exacerbation of the epithelial barrier damage. Cytokines play an important role in mucosal immunity. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that has been shown to play a role in epithelial integrity and modulation of the mucosal immune system. We hypothesized that IL-10 may protect against the development of experimental NEC by blunting the inflammatory response in the intestine. METHODS: Wild-type and IL-10 -/- mice underwent a NEC-inducing regimen of formula feeding in combination with hypoxia and hypothermia (FF+HH). Integrity of the gut barrier was assessed through measurement of epithelial apoptosis, tight junction disruption, and inducible nitric oxide synthase. A total of 5 µg of exogenous IL-10 was administered intraperitoneally to IL-10-/-mouse pups before the initiation of FF+HH to test dependence of gene knockout phenotype on IL-10. RESULTS: IL-10 -/- FF+HH showed more severe morphologic and histologic changes compared with controls as evidenced by increased epithelial apoptosis, decreased junctional adhesion molecule-1 localization, and increased intestinal inducible nitric oxide synthase expression. Administration of exogenous IL-10 alleviated the mucosal injury. CONCLUSIONS: We conclude that IL-10 plays a protective role in the pathogenesis of NEC by attenuating the degree of intestinal inflammation.
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Enterocolitis Necrotizante/tratamiento farmacológico , Enterocolitis Necrotizante/patología , Interleucina-10/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Biopsia con Aguja , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/inmunología , Femenino , Inmunohistoquímica , Inyecciones Intraperitoneales , Interleucina-10/inmunología , Interleucina-10/metabolismo , Mucosa Intestinal/inmunología , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Embarazo , Preñez , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Necrotising enterocolitis (NEC) is one of the most common and fatal intestinal disorders in preterm infants. Breast-fed infants are at lower risk for NEC than formula-fed infants, but the protective components in human milk have not been identified. In contrast to formula, human milk contains high amounts of complex glycans. OBJECTIVE: To test the hypothesis that human milk oligosaccharides (HMO) contribute to the protection from NEC. METHODS: Since human intervention studies are unfeasible due to limited availability of HMO, a neonatal rat NEC model was used. Pups were orally gavaged with formula without and with HMO and exposed to hypoxia episodes. Ileum sections were scored blindly for signs of NEC. Two-dimensional chromatography was used to determine the most effective HMO, and sequential exoglycosidase digestions and linkage analysis was used to determine its structure. RESULTS: Compared to formula alone, pooled HMO significantly improved 96-hour survival from 73.1% to 95.0% and reduced pathology scores from 1.98 ± 1.11 to 0.44 ± 0.30 (p<0.001). Within the pooled HMO, a specific isomer of disialyllacto-N-tetraose (DSLNT) was identified to be protective. Galacto-oligosaccharides, currently added to formula to mimic some of the effects of HMO, had no effect. CONCLUSION: HMO reduce NEC in neonatal rats and the effects are highly structure specific. If these results translate to NEC in humans, DSLNT could be used to prevent or treat NEC in formula-fed infants, and its concentration in the mother's milk could serve as a biomarker to identify breast-fed infants at risk of developing this disorder.
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Enterocolitis Necrotizante/prevención & control , Leche Humana/química , Oligosacáridos/uso terapéutico , Animales , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/mortalidad , Enterocolitis Necrotizante/patología , Cromatografía de Gases y Espectrometría de Masas , Humanos , Oligosacáridos/análisis , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Ineffectiveness of antibiotics in treating neonatal Escherichia coli K1 meningitis and the emergence of antibiotic-resistant strains evidently warrants new prevention strategies. We observed that administration of interleukin (IL)-10 during high-grade bacteremia clears antibiotic-sensitive and -resistant E. coli from blood of infected mice. Micro-CT studies of brains from infected animals displayed gross morphological changes similar to those observed in infected human neonates. In mice, IL-10, but not antibiotic or anti-TNF antibody treatment prevented brain damage caused by E. coli. IL-10 administration elevated CR3 expression in neutrophils and macrophages of infected mice, whereas infected and untreated mice displayed increased expression of FcgammaRI and TLR2. Neutrophils or macrophages pretreated with IL-10 ex vivo exhibited a significantly greater microbicidal activity against E. coli compared with cells isolated from wild-type or IL-10-/- mice. The protective effect of IL-10 was abrogated when CR3 was knocked-down in vivo by siRNA. The increased expression of CR3 in phagocytes was caused by inhibition of prostaglandin E-2 (PGE-2) levels, which were significantly increased in neutrophils and macrophages upon E. coli infection. These findings describe a novel modality of IL-10-mediated E. coli clearance by diverting the entry of bacteria via CR3 and preventing PGE-2 formation in neonatal meningitis.
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Dinoprostona/metabolismo , Escherichia coli/inmunología , Interleucina-10/administración & dosificación , Interleucina-10/farmacología , Antígeno de Macrófago-1/metabolismo , Meningitis por Escherichia coli/inmunología , Fagocitos/microbiología , Animales , Animales Recién Nacidos , Anticuerpos/administración & dosificación , Anticuerpos/farmacología , Bacteriemia/complicaciones , Bacteriemia/inmunología , Bacteriemia/patología , Encéfalo/efectos de los fármacos , Encéfalo/microbiología , Encéfalo/patología , Escherichia coli/efectos de los fármacos , Humanos , Inflamación/inmunología , Inflamación/patología , Interleucina-10/deficiencia , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/microbiología , Meningitis por Escherichia coli/complicaciones , Meningitis por Escherichia coli/microbiología , Meningitis por Escherichia coli/prevención & control , Ratones , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/microbiología , Fagocitos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Análisis de Supervivencia , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Escherichia coli K1 is a leading cause of neonatal meningitis in humans. In this study, we sought to determine the pathophysiologic relevance of inducible nitric oxide (iNOS) in experimental E. coli K1 meningitis. By using a newborn mouse model of meningitis, we demonstrate that E. coli infection triggered the expression of iNOS in the brains of mice. Additionally, iNOS-/- mice were resistant to E. coli K1 infection, displaying normal brain histology, no bacteremia, no disruption of the blood-brain barrier, and reduced inflammatory response. Treatment with an iNOS specific inhibitor, aminoguanidine (AG), of wild-type animals before infection prevented the development of bacteremia and the occurrence of meningitis. The infected animals treated with AG after the development of bacteremia also completely cleared the pathogen from circulation and prevented brain damage. Histopathological and micro-CT analysis of brains revealed significant damage in E. coli K1-infected mice, which was completely abrogated by AG administration. Peritoneal macrophages and polymorphonuclear leukocytes isolated from iNOS-/- mice or pretreated with AG demonstrated enhanced uptake and killing of the bacteria compared with macrophages and polymorphonuclear leukocytes from wild-type mice in which E. coli K1 survive and multiply. Thus, NO produced by iNOS may be beneficial for E. coli to survive inside the macrophages, and prevention of iNOS could be a therapeutic strategy to treat neonatal E. coli meningitis.
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Encéfalo/microbiología , Encéfalo/patología , Escherichia coli/citología , Meningitis/enzimología , Meningitis/microbiología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Fagocitosis , Administración Intranasal , Animales , Animales Recién Nacidos , Encéfalo/diagnóstico por imagen , Encéfalo/enzimología , Supervivencia Celular/efectos de los fármacos , Citocinas/sangre , Modelos Animales de Enfermedad , Escherichia coli/efectos de los fármacos , Femenino , Guanidinas/administración & dosificación , Guanidinas/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Masculino , Meningitis/patología , Meningitis/prevención & control , Ratones , Ratones Endogámicos C57BL , Viabilidad Microbiana/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/microbiología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fagocitosis/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Microtomografía por Rayos XRESUMEN
BACKGROUND: The domestic mite Blomia tropicalis is found in subtropical and tropical environments, and its clinical importance as a sensitizing agent in allergic disease is widely accepted. OBJECTIVE: To investigate the IgE reactivity to allergens present in extracts of the domestic mite B. tropicalis, and compare the IgE responses to these allergens by asthmatics, patients with atopic dermatitis and allergic rhinitis, as well as nonatopic controls. METHODS: Extracts from B. tropicalis were used for skin tests. The B. tropicalis specific IgE in the serum were measured using the FAST Plus Test and immunoblot analysis. RESULTS: A total of 199 volunteers participated in the study. The data show that 18 out of 29 polypeptide bands present in extracts of this mite species were recognized by the allergic and control sera. Of these allergens, four showed a high IgE binding frequency and had relative molecular weights of 104, 80, 68 and 14 kDa. The 14 kDa allergen demonstrated the highest IgE binding frequency. CONCLUSION: Sera from atopic patients reacted to more allergens than sera from patients controls. Extracts from pure bodies of B. tropicalis contain one immunodominant and three important allergens. A common characteristic between all of the sera tested was the high degree of serum IgE reactivity observed to the 14 kDa allergen.
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Asma/sangre , Asma/inmunología , Dermatitis Atópica/sangre , Dermatitis Atópica/inmunología , Inmunoglobulina E/sangre , Ácaros , Rinitis Alérgica Perenne/sangre , Rinitis Alérgica Perenne/inmunología , Extractos de Tejidos/inmunología , Adolescente , Adulto , Animales , Niño , Femenino , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Pruebas CutáneasRESUMEN
The role of cryptopatch aggregates in the development of intestinal intraepithelial lymphocytes (IEL) is a matter of controversy. Therefore, an important question is whether T cells in cryptopatch aggregates are lineally related to IEL. We hypothesized that if gammadelta+ IEL derive from T cells in cryptopatch aggregates, then a clonal relationship would exist between the two populations. To test this hypothesis, we compared the sequence of rearranged TCR gamma variable region 5 genes in gammadelta+ IEL and cryptopatch cells. We purified IEL by FACS and cryptopatch cells were isolated from frozen sections of the intestine by laser-assisted microdissection. PCR showed that TCR gamma variable region 5 was rearranged in gammadelta+ IEL and in CD3+ cryptopatch cells, but not in CD3- cryptopatch cells. DNA sequence analysis showed that the frequency of in-frame junctions in cryptopatch aggregates was at a level consistent with positive selection in both wild-type and athymic nude mice. In addition, the predicted amino acid sequences of V-J junctions present in gammadelta+ IEL and cryptopatch cells were encoded by identical nucleotide sequences. By contrast, the frequency of in-frame joints was significantly reduced in cryptopatch cells isolated from TCR delta-deficient mice, indicating that the enrichment of in-frame joints in cryptopatch cells must normally depend on expression of surface gammadelta TCR. Our results are consistent with the hypothesis that a subset of gammadelta+ IEL are related to T cells in cryptopatch aggregates. The precise role of cryptopatch aggregates in intestinal gammadelta+ T cell homeostasis still needs to be determined.