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Respiratory viral infections remain a leading cause of morbidity and mortality. Using a murine model of human metapneumovirus (HMPV), we identified recruitment of a C1q-expressing inflammatory monocyte population concomitant with viral clearance by adaptive immune cells. Genetic ablation of C1q led to reduced CD8+ T cell function. Production of C1q by a myeloid lineage was necessary to enhance CD8+ T cell function. Activated and dividing CD8+ T cells expressed a C1q receptor, gC1qR. Perturbation of gC1qR signaling led to altered CD8+ T cell IFN-γ production, metabolic capacity, and cell proliferation. Autopsy specimens from fatal respiratory viral infections in children demonstrated diffuse production of C1q by an interstitial population. Humans with severe COVID-19 infection also demonstrated upregulation of gC1qR on activated and rapidly dividing CD8+ T cells. Collectively, these studies implicate C1q production from monocytes as a critical regulator of CD8+ T cell function following respiratory viral infection. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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BACKGROUND & AIMS: Biliary atresia (BA) is poorly understood and leads to liver transplantation (LT), with the requirement for and associated risks of lifelong immunosuppression, in most children. We performed a genome-wide association study (GWAS) to determine the genetic basis of BA. METHODS: We performed a GWAS in 811 European BA cases treated with LT in US, Canadian and UK centers, and 4,654 genetically matched controls. Whole-genome sequencing of 100 cases evaluated synthetic association with rare variants. Functional studies included whole liver transcriptome analysis of 64 BA cases and perturbations in experimental models. RESULTS: A GWAS of common single nucleotide polymorphisms (SNPs), i.e. allele frequencies >1%, identified intronic SNPs rs6446628 in AFAP1 with genome-wide significance (p = 3.93E-8) and rs34599046 in TUSC3 at sub-threshold genome-wide significance (p = 1.34E-7), both supported by credible peaks of neighboring SNPs. Like other previously reported BA-associated genes, AFAP1 and TUSC3 are ciliogenesis and planar polarity effectors (CPLANE). In gene-set-based GWAS, BA was associated with 6,005 SNPs in 102 CPLANE genes (p = 5.84E-15). Compared with non-CPLANE genes, more CPLANE genes harbored rare variants (allele frequency <1%) that were assigned Human Phenotype Ontology terms related to hepatobiliary anomalies by predictive algorithms, 87% vs. 40%, p <0.0001. Rare variants were present in multiple genes distinct from those with BA-associated common variants in most BA cases. AFAP1 and TUSC3 knockdown blocked ciliogenesis in mouse tracheal cells. Inhibition of ciliogenesis caused biliary dysgenesis in zebrafish. AFAP1 and TUSC3 were expressed in fetal liver organoids, as well as fetal and BA livers, but not in normal or disease-control livers. Integrative analysis of BA-associated variants and liver transcripts revealed abnormal vasculogenesis and epithelial tube formation, explaining portal vein anomalies that co-exist with BA. CONCLUSIONS: BA is associated with polygenic susceptibility in CPLANE genes. Rare variants contribute to polygenic risk in vulnerable pathways via unique genes. IMPACT AND IMPLICATIONS: Liver transplantation is needed to cure most children born with biliary atresia, a poorly understood rare disease. Transplant immunosuppression increases the likelihood of life-threatening infections and cancers. To improve care by preventing this disease and its progression to transplantation, we examined its genetic basis. We find that this disease is associated with both common and rare mutations in highly specialized genes which maintain normal communication and movement of cells, and their organization into bile ducts and blood vessels during early development of the human embryo. Because defects in these genes also cause other birth defects, our findings could lead to preventive strategies to lower the incidence of biliary atresia and potentially other birth defects.
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Atresia Biliar , Niño , Animales , Ratones , Humanos , Atresia Biliar/genética , Estudio de Asociación del Genoma Completo , Predisposición Genética a la Enfermedad , Pez Cebra/genética , CanadáRESUMEN
Respiratory viral infections remain a leading cause of morbidity and mortality. Using a murine model of human metapneumovirus (HMPV), we identified recruitment of a C1q-producing inflammatory monocyte population concomitant with viral clearance by adaptive immune cells. Genetic ablation of C1q led to reduced CD8 + T cell function. Production of C1q by a myeloid lineage was sufficient to enhance CD8 + T cell function. Activated and dividing CD8 + T cells expressed a putative C1q receptor, gC1qR. Perturbation of gC1qR signaling led to altered CD8 + T cell IFN-γ production and metabolic capacity. Autopsy specimens from fatal respiratory viral infections in children demonstrated diffuse production of C1q by an interstitial population. Humans with severe COVID-19 infection also demonstrated upregulation of gC1qR on activated and rapidly dividing CD8 + T cells. Collectively, these studies implicate C1q production from monocytes as a critical regulator of CD8 + T cell function following respiratory viral infection.
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BACKGROUND: Ciliary defects cause heterogenous phenotypes related to mutation burden which lead to impaired development. A previously reported homozygous deletion in the Man1a2 gene causes lethal respiratory failure in newborn pups and decreased lung ciliation compared with wild type (WT) pups. The effects of heterozygous mutation, and the potential for rescue are not known. PURPOSE: We hypothesized that survival and lung ciliation, (a) would decrease progressively in Man1a2 +/- heterozygous and Man1a2 -/- null newborn pups compared with WT, and (b) could be enhanced by gestational treatment with N-Acetyl-cysteine (NAC), an antioxidant. METHODS: Man1a2+/- adult mice were fed NAC or placebo from a week before breeding through gestation. Survival of newborn pups was monitored for 24 h. Lungs, liver and tails were harvested for morphology, genotyping, and transcriptional profiling. RESULTS: Survival (p = 0.0001, Kaplan-Meier) and percent lung ciliation (p = 0.0001, ANOVA) measured by frequency of Arl13b+ respiratory epithelial cells decreased progressively, as hypothesized. Compared with placebo, gestational NAC treatment enhanced (a) lung ciliation in pups with each genotype, (b) survival in heterozygous pups (p = 0.017) but not in WT or null pups. Whole transcriptome of lung but not liver demonstrated patterns of up- and down-regulated genes that were identical in living heterozygous and WT pups, and completely opposite to those in dead heterozygous and null pups. Systems biology analysis enabled reconstruction of protein interaction networks that yielded functionally relevant modules and their interactions. In these networks, the mutant Man1a2 enzyme contributes to abnormal synthesis of proteins essential for lung development. The associated unfolded protein, hypoxic and oxidative stress responses can be mitigated with NAC. Comparisons with the developing human fetal lung transcriptome show that NAC likely restores normal vascular and epithelial tube morphogenesis in Man1a2 mutant mice. CONCLUSION: Survival and lung ciliation in the Man1a2 mutant mouse, and its improvement with N-Acetyl cysteine is genotype-dependent. NAC-mediated rescue depends on the central role for oxidative and hypoxic stress in regulating ciliary function and organogenesis during development.
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Large/giant congenital nevi (L/GCMN) are benign neoplasms of the melanocytic neural crest lineage covering extensive areas of skin presenting risk for melanoma. Surgical resection often leads to scarring and trauma. Histone deacetylase inhibitors (iHDACs) as topical therapeutic agents may prove beneficial as an alternative/adjunct to surgery in this disease. Here we describe the effect of in vitro treatment of iHDACs drugs on primary nevocytes isolated from L/GCMN patients. Micropthalmia transcription factor (MITF) expression in L/GCMN patients' lesions was detected by immunohistochemistry, in cultured nevocytes by immunofluorescence, immunoblot and quantitative polymerase chain reaction. Cellular senescence was detected by SA-ß galactosidase activity. Markers for melanocytic differentiation were evaluated by immunoblot analysis and extracted melanin content was estimated spectrophotometrically. Cell death was measured by lactate dehydrogenase (LDH) assay and necrosis confirmed by polymerase (PARP) cleavage and acridine orange staining of the nuclei. MITF was expressed ubiquitously in nevocytes and melanocytes in patients' lesions. In culture, iHDAC treatment suppressed MITF protein and mRNA expression resulting in a senescent-like phenotype with positive ß-galactosidase staining, progressing to necrotic cell death as evidenced by increased LDH activity, appearance of cleaved PARP and necrotic nuclei. This is the first report showing evidence of iHDACs-induced MITF suppression in congenital nevocytes in vitro leading to a morphologic change with positive ß-galactosidase staining, followed by necrotic cell death in nevocytes, indicating that iHDAC drugs could be valuable therapeutic agents for treatment of L/GCMN lesions.
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Inhibidores de Histona Desacetilasas/uso terapéutico , Nevo Pigmentado/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Factores de Transcripción/efectos de los fármacos , Vorinostat/uso terapéutico , Muerte Celular , Diferenciación Celular , Preescolar , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Lactante , Vorinostat/farmacologíaRESUMEN
BACKGROUND/AIMS: Infectious and genetic factors are invoked, respectively in isolated biliary atresia (BA), or syndromic BA, with major extrahepatic anomalies. However, isolated BA is also associated with minor extrahepatic gut and cardiovascular anomalies and multiple susceptibility genes, suggesting common origins. METHODS: We investigated novel susceptibility genes with genome-wide association, targeted sequencing and tissue staining in BA requiring liver transplantation, independent of BA subtype. Candidate gene effects on morphogenesis, developmental pathways, and ciliogenesis, which regulates left-right patterning were investigated with zebrafish knockdown and mouse knockout models, mouse airway cell cultures, and liver transcriptome analysis. RESULTS: Single nucleotide polymorphisms in Mannosidase-1-α-2 (MAN1A2) were significantly associated with BA and with other polymorphisms known to affect MAN1A2 expression but were not differentially enriched in either BA subtype. In zebrafish embryos, man1a2 knockdown caused poor biliary network formation, ciliary dysgenesis in Kupffer's vesicle, cardiac and liver heterotaxy, and dysregulated egfra and other developmental genes. Suboptimal man1a2 knockdown synergized with suboptimal EGFR signaling or suboptimal knockdown of the EGFR pathway gene, adenosine-ribosylation-factor-6, which had minimal effects individually, to reproduce biliary defects but not heterotaxy. In cultured mouse airway epithelium, Man1a2 knockdown arrested ciliary development and motility. Man1a2 -/- mice, which experience respiratory failure, also demonstrated portal and bile ductular inflammation. Human BA liver and Man1a2 -/- liver exhibited reduced Man1a2 expression and dysregulated ciliary genes, known to cause multisystem human laterality defects. CONCLUSION: BA requiring transplantation associates with sequence variants in MAN1A2. man1a2 regulates laterality, in addition to hepatobiliary morphogenesis, by regulating ciliogenesis in zebrafish and mice, providing a novel developmental basis for multisystem defects in BA.
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Lipoblastomas can occasionally require further molecular confirmation when occurring outside of the usual age groups or demonstrating unusual morphology. We reviewed 28 lipoblastomas with 16 controls. Lipoblastomas were subdivided into myxoid (n = 7), classic (n = 9), or lipoma-like (n = 12) subtypes. PLAG1 immunohistochemistry, PLAG1 fluorescence in situ hybridization (FISH), and targeted RNA sequencing were performed on formalin-fixed paraffin-embedded tissue. Karyotypes were available in a subset of lipoblastomas (n = 9). Gene rearrangements were identified in 17/25 (68%) lipoblastomas, including PLAG1 (15/25, 60%) and HMGA2 (2/25, 8%). Five novel fusion partners (DDX6, KLF10, and KANSL1L with PLAG1 and EP400 and FGD6 with HMGA2) were found. PLAG1 immunohistochemistry was positive (nuclear, moderate/strong) in myxoid and classic subtypes lipoblastomas with preferential expression in mesenchymal cells within myxoid stroma and fibrous septa and negative in all controls. When comparing PLAG1 immunohistochemistry with molecular testing (FISH and/or RNA sequencing and/or karyotype), concordant results were noted in 13/25 (52%) cases, increasing to 15/25 (60%) after slight adjustment of the PLAG1 FISH positive threshold. In myxoid and classic lipoblastomas, PLAG1 immunohistochemistry seems to be a better surrogate marker for PLAG1 rearrangement, as compared with lipoma-like subtypes. In lipoma-like subtypes, targeted RNA sequencing appears to detect PLAG1 fusions better than FISH and immunohistochemistry. The preferential expression of PLAG1 in the mesenchymal and fibroblast-like cells deserves further investigation as the putative cell of origin in lipoblastoma.
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Biomarcadores de Tumor , Inmunohistoquímica , Lipoblastoma/química , Lipoblastoma/genética , Técnicas de Diagnóstico Molecular , Adolescente , Factores de Edad , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Niño , Preescolar , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Femenino , Fusión Génica , Reordenamiento Génico , Predisposición Genética a la Enfermedad , Humanos , Hibridación Fluorescente in Situ , Lactante , Cariotipificación , Lipoblastoma/patología , Lipoblastoma/cirugía , Masculino , Fenotipo , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Análisis de Secuencia de ARN , Adulto JovenRESUMEN
Hepatoblastoma (HBL), the most common childhood liver cancer is cured with surgical resection after chemotherapy or with liver transplantation if local invasion and multifocality preclude resection. However, variable survival rates of 60-80% and debilitating chemotherapy sequelae argue for more informed treatment selection, which is not possible by grading the Wnt-ß-catenin over activity present in most HBL tumors. A hypothesis-generating whole transcriptome analysis shows that HBL tumors removed at transplantation are enriched most for cancer signaling pathways which depend predominantly on epidermal growth factor (EGF) signaling, and to a lesser extent, on aberrant Wnt-ß-catenin signaling. We therefore evaluated whether EGFR, ASAP1, ERBB2 and ERBB4, which signal downstream after ligation of EGF, and which show aberrant expression in several other invasive cancers, would also predict HBL tumor invasiveness. Immunohistochemistry of HBL tumors (n = 60), which are histologically heterogeneous, shows that compared with well-differentiated fetal cells, less differentiated embryonal and undifferentiated small cells (SCU) progressively lose EGFR and ASAP1 expression. This trend is exaggerated in unresectable, locally invasive or metastatic tumors, in which embryonal tumor cells are EGFR-negative, while SCU cells are EGFR-negative and ASAP1-negative. Loss of EGFR-ASAP1 signaling characterizes undifferentiated and invasive HBL. EGFR-expressing HBL tumors present novel therapeutic targeting opportunities.
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Proteínas Adaptadoras Transductoras de Señales/deficiencia , Receptores ErbB/deficiencia , Regulación Neoplásica de la Expresión Génica , Hepatoblastoma/genética , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Niño , Preescolar , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Femenino , Hepatoblastoma/mortalidad , Hepatoblastoma/secundario , Hepatoblastoma/cirugía , Humanos , Lactante , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/cirugía , Trasplante de Hígado , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/cirugía , Masculino , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-4/genética , Receptor ErbB-4/metabolismo , Análisis de Supervivencia , Transcriptoma , Vía de Señalización Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND & AIMS: Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA). METHODS: To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6). RESULTS: Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3' flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10-7), ERK/MAPK and CREB canonical pathways (p<1 x10-34), and functional networks for cellular development and proliferation (p<1 x10-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA. CONCLUSIONS: The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.
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Factores de Ribosilacion-ADP/genética , Atresia Biliar/genética , Factor 6 de Ribosilación del ADP , Animales , Estudios de Casos y Controles , Proliferación Celular/genética , Receptores ErbB/genética , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad/genética , Genotipo , Humanos , Polimorfismo de Nucleótido Simple/genética , Transducción de Señal/genética , Pez Cebra/genéticaRESUMEN
BACKGROUND: The transcription factor, t-bet, promotes inflammatory polarization and intestinal homing of many inflammatory cells. In previous studies, the t-bet and granulysin genes were upregulated in peripheral blood before and after intestine transplantation (ITx) rejection, but not during rejection, possibly because of sequestration in allograft mucosa. Mucosal sequestration of t-bet and granulysin may also explain the presence of inflammatory CD14+ monocyte-derived macrophages (MDM) and immunoglobulin G+ B-cell lineage cells, and loss of mature non-inflammatory CD138+ plasma cells in allograft mucosa during ITx rejection in these previous studies. METHODOLOGY: T-bet-stained and granulysin-stained cells, MDM and CD138+ plasma cells were evaluated with immunohistochemistry in serial biopsies from 17 children, in whom changes in MDM and CD138+ plasma cells were observed previously. RESULTS: T-bet-positive mucosal cells were significantly higher in postperfusion (P = 0.035) and early posttransplant biopsies (P = 0.016) among rejectors, compared with nonrejectors. T-bet-positive cell counts per high-power field (hpf) were (a) positively correlated with MDM counts/hpf in postperfusion (Spearman r = 0.73; P = 0.01) and early posttransplant biopsies (r = 0.54, r = 0.046), and (b) negatively correlated with CD138+B-/pre-plasma cells in early posttransplant biopsies (r = 0.63, P = 0.038). T-bet expression in CD14+ monocytes, CD19+B cells, and several other leukocyte subsets was higher in random blood samples from two rejectors, compared with those from five normal human subjects and three nonrejectors. Scant granulysin-stained mucosal cells precluded additional evaluation of this cytotoxin and its role in ITx rejection. SIGNIFICANCE: The transcription factor, t-bet, primes ITx rejection, and associates with disrupted homeostatic relationships between innate and adaptive immune cells in the allograft mucosa during rejection.
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Rechazo de Injerto/inmunología , Inmunidad Mucosa , Mucosa Intestinal/trasplante , Intestinos/trasplante , Trasplante de Órganos/efectos adversos , Proteínas de Dominio T Box/metabolismo , Inmunidad Adaptativa , Antígenos CD19/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Biomarcadores/metabolismo , Biopsia , Niño , Preescolar , Femenino , Fijadores , Formaldehído , Rechazo de Injerto/patología , Homeostasis , Humanos , Inmunidad Innata , Lactante , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Intestinos/inmunología , Intestinos/patología , Receptores de Lipopolisacáridos/metabolismo , Masculino , Adhesión en Parafina , Sindecano-1/metabolismo , Factores de Tiempo , Fijación del Tejido/métodos , Resultado del TratamientoRESUMEN
Nevocytes (NC) and mastocytes (MC) have different progenitors but share stem cell factor as regulator/activator of NC and for differentiation/proliferation of MC. Both cell types express stem cell factor receptor CD117. We hypothesize that large/giant congenital melanocytic nevi (L/GCMN) may associate with MC hyperplasia. Forty-nine L/GCMN were examined, 12 samples from uninvolved skin of L/GCMN patients and 6 control skin samples studied with Giemsa and immunohistochemistry for CD117 and MC-tryptase. Picrosirius red (PR) was used to assess fibrosis. Digital images were used to count MC/mm(2) using ImageJ software. Western blot (WB) for MC-tryptase in 12 GCMN and 12 non-nevus samples was performed. Analysis of variance (Tukey) and Pearson statistical tests were applied. Increased MCs were observed in nevus tissue (75.1 ± 35.3 MCs/mm(2)) and in uninvolved skin (53.74 ± 27.7 MC/ mm(2)). P â=â 0.109 from patients with L/GCMN, compared with controls from individuals without L/GCMN (28.74 ± 8.4 MC/mm(2)); P â=â 0.001 supported by results of WB analysis for tryptase. A positive trend toward correlation of MC numbers with fibrosis, assessed by PR staining fell short of statistical significance (r â=â 0.245; P â=â 0.086); no difference in fibrosis was found between nevus and non-nevus skin from patients with L/GCMN (P â=â 0.136). We found a higher density of MC, both in normal-appearing skin and nevus areas of L/GCMN patients, compared with control skin samples from individuals without nevi. Given the abnormal wound healing and allergic reactions described in L/GCMN patients, these findings suggest a potential role for MC in the biology of L/GCMN, making them a potential target for therapeutic intervention.
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Mastocitos/patología , Nevo Pigmentado/patología , Neoplasias Cutáneas/patología , Adolescente , Western Blotting , Proliferación Celular , Niño , Preescolar , Femenino , Humanos , Inmunohistoquímica , Lactante , Masculino , Mastocitos/inmunología , Nevo Pigmentado/inmunología , Neoplasias Cutáneas/inmunologíaRESUMEN
Tufting enteropathy (TE) is an uncommon disease causing intractable diarrheas starting in early childhood and resulting in failure to thrive, dependence on total parenteral nutrition, and eventually requiring transplantation for treatment. The diagnosis has been based on histology showing the presence of epithelial "tufts" in the small bowel and colonic mucosa and variable villus alterations with mild to no inflammatory changes and preserved brush border. The gene for TE has been identified to be the EpCAM gene on chromosome 2p21. With Institutional Review Board approval, all cases of intractable diarrhea in children in whom TE was suspected or diagnosed were retrieved from the pathology files (17 patients). Other cases of infantile, neonatal, and childhood diarrhea were also retrieved to serve as controls for the staining studies (total 37 patients). EpCAM/MOC31 antibody staining was performed on all cases. The study cohort comprised 17 patients (13 boys, 4 girls) with a diagnosis of TE ranging in age at diagnosis from 3 months to 9 years, all presenting with protracted diarrhea and/or failure to thrive, usually since birth. Staining with MOC31 was carried out in all but 2 patients (both consults) and was completely negative in the epithelium irrespective of the site of biopsy or resection. In contrast, MOC31 was positive in all other cases tested, giving a sensitivity and specificity of 100% for loss of staining. MOC31 is a diagnostic stain for TE and should be included in the panel in any case of prolonged diarrhea in children to exclude this possibility.
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Antígenos de Neoplasias/análisis , Moléculas de Adhesión Celular/análisis , Colon/química , Diarrea Infantil/metabolismo , Inmunohistoquímica , Mucosa Intestinal/química , Intestino Delgado/química , Síndromes de Malabsorción/metabolismo , Factores de Edad , Biomarcadores/análisis , Biopsia , Niño , Preescolar , Colon/patología , Diarrea Infantil/patología , Regulación hacia Abajo , Molécula de Adhesión Celular Epitelial , Insuficiencia de Crecimiento/metabolismo , Insuficiencia de Crecimiento/patología , Femenino , Humanos , Lactante , Mucosa Intestinal/patología , Intestino Delgado/patología , Síndromes de Malabsorción/patología , Masculino , Valor Predictivo de las Pruebas , Pronóstico , Estudios RetrospectivosRESUMEN
BACKGROUND: Intestinal allograft mucosa undergoes repopulation with host immunocytes. However, critical changes within key immunocyte subsets are not known. METHODS: To explain acute cellular rejection after intestine transplantation (ITx) on the basis of altered mucosal immunocytes, rejecting and rejection-free ITx allografts (n=17) were compared with genome-wide expression arrays. Cells identified by cell/lineage-specific genes were evaluated by immunohistochemistry. The corresponding phenotype and donor-specific alloreactivity were characterized in peripheral blood. Time-dependent changes in candidate cell(s) were evaluated in biopsies from an independent cohort of 12 children with ITx. RESULTS: Among 107 differentially expressed genes, three B-cell lineage-specific genes, CCR10, STAP1, and IGLL1, were down-regulated during ITx rejection and were selected for and achieved technical quantitative reverse transcription polymerase chain reaction replication. Down-regulation of the immunoglobulin (Ig)A+ plasma cell-specific CCR10 gene correlated with decreased mature mucosal CD138+ plasma cell numbers in corresponding biopsy specimens (r=0.761, P=0.006) and inversely correlated with enhanced alloreactivity of CD154+ T-cytotoxic memory cells (r=-0.56, P=0.031), which predict acute cellular rejection with high sensitivity. An independent cohort of serial biopsy specimens from 12 ITx recipients (1) confirmed relative CD138+ plasma cell depletion during rejection (P=0.042) and (2) showed increased IgG+-to-IgA+ cell ratios within 4 hr of reperfusion in rejection-prone allografts (P=0.037) and during ITx rejection (P=0.025), compared with rejection-free allografts. No differences existed late after ITx. Increased peripheral IgG+ CD27+ CD19+ memory B cells (P=0.004) were seen during ITx rejection in archived peripheral blood lymphocyte from test and replication cohorts. CONCLUSIONS: Protracted depletion of the mucosal CD138+ plasma cell barrier and early mucosal infiltration with memory IgG+ cells characterize the rejection-prone intestine allograft. Mucosal IgA+ plasma cell barrier reconstitution may augur resolution of ITx rejection.
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Rechazo de Injerto/patología , Mucosa Intestinal/patología , Intestinos/trasplante , Células Plasmáticas/patología , Enfermedad Aguda , Proteínas Adaptadoras Transductoras de Señales/genética , Antígenos CD19/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Biopsia , Linaje de la Célula/inmunología , Niño , Preescolar , Femenino , Estudio de Asociación del Genoma Completo , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Humanos , Cadenas lambda de Inmunoglobulina/genética , Mucosa Intestinal/inmunología , Intestinos/inmunología , Intestinos/patología , Masculino , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Receptores CCR10/genética , Sindecano-1/metabolismo , Trasplante Homólogo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Recurrent rejection shortens graft survival after intestinal transplantation (ITx) in children, most of whom also experience early acute cellular rejection (rejectors). To elucidate mechanisms common to early and recurrent rejection, we used a test cohort of 20 recipients to test the hypothesis that candidate peripheral blood leukocyte genes that trigger rejection episodes would be evident late after ITx during quiescent periods in genome-wide gene expression analysis and would achieve quantitative real-time PCR replication pre-ITx (another quiescent period) and in the early post-ITx period during first rejection episodes. Eight genes were significantly up-regulated among rejectors in the late post-ITx and pre-ITx periods, compared with nonrejectors: TBX21, CCL5, GNLY, SLAMF7, TGFBR3, NKG7, SYNE1, and GK5. Only CCL5 was also up-regulated in the early post-ITx period. Among resting peripheral blood leukocyte subsets in randomly sampled nonrejectors, CD14(+) monocytes expressed the CCL5 protein maximally. Compared with nonrejectors, rejectors demonstrated higher counts of both circulating CCL5(+)CD14(+) monocytes and intragraft CD14(+) monocyte-derived macrophages in immunohistochemistry of postperfusion and early post-ITx biopsies from the test and an independent replication cohort. Donor-specific alloreactivity measured with CD154(+) T-cytotoxic memory cells correlated with the CCL5 gene and intragraft CD14(+) monocyte-derived macrophages at graft reperfusion and early post-ITx. CCL5 gene up-regulation and CD14(+) macrophages likely prime cellular ITx rejection. Infiltration of reperfused intestine allografts with CD14(+) macrophages may predict rejection events.