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BACKGROUND AND AIMS: Outcomes after Kasai portoenterostomy (KPE) for biliary atresia remain highly variable for unclear reasons. As reliable early biomarkers predicting KPE outcomes are lacking, we studied the prognostic value of FGF19. APPROACH AND RESULTS: Serum and liver specimens, obtained from biliary atresia patients (N=87) at KPE or age-matched cholestatic controls (N=26) were included. Serum concentration of FGF19 and bile acids, liver mRNA expression of FGF19 , and key regulators of bile acid synthesis were related to KPE outcomes and liver histopathology. Immunohistochemistry and in situ hybridization were used for the localization of liver FGF19 expression. Serum levels (223 vs. 61 pg/mL, p <0.001) and liver mRNA expression of FGF19 were significantly increased in biliary atresia. Patients with unsuccessful KPE (419 vs. 145 pg/mL, p =0.047), and those subsequently underwent liver transplantation (410 vs. 99 pg/mL, p =0.007) had significantly increased serum, but not liver, FGF19, which localized mainly in hepatocytes. In Cox hazard modeling serum FGF19 <109 pg/mL predicted native liver survival (HR: 4.31, p <0.001) also among patients operated <60 days of age (HR: 8.77, p =0.004) or after successful KPE (HR: 6.76, p =0.01). Serum FGF19 correlated positively with increased serum primary bile acids ( R =0.41, p =0.004) and ductular reaction ( R =0.39, p =0.004). CONCLUSIONS: Increased serum FGF19 at KPE predicted inferior long-term native liver survival in biliary atresia and was associated with unsuccessful KPE, elevated serum primary bile acids, and ductular reaction.
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Atresia Biliar , Humanos , Lactente , Atresia Biliar/complicações , Portoenterostomia Hepática , Prognóstico , Ácidos e Sais Biliares , RNA Mensageiro , Resultado do Tratamento , Fatores de Crescimento de FibroblastosRESUMO
AIM: The gut-liver axis may contribute to pathophysiology of cholestatic liver disorders like biliary atresia (BA) by bacterial translocation (BT). Toll-like receptors (TLR) are pattern recognition receptors known to activate innate immunity and secretion of inflammatory cytokines. Herein, we examined BT-associated biomarkers and TLRs in relation to liver injury after successful portoenterostomy (SPE) in BA. METHODS: Serum levels of lipopolysaccharide-binding protein (LBP), CD14, LAL, TNF-α, IL-6 and FABP2 along with liver expression of TLRs (TLR1, TLR4, TLR7 and TLR9), LBP and CD14 were measured during median 4.9 (1.7-10.6) years follow-up after SPE in 45 BA patients. RESULTS: Serum LBP, CD14, TNF-α and IL-6 all increased after SPE whereas LAL and FABP-2 remained unchanged. Serum LBP correlated positively with CD14 and markers of hepatocyte injury and cholestasis, but not with Metavir fibrosis stage, transcriptional markers for fibrosis (ACTA2) or ductular reaction. Serum CD14 concentration was significantly higher in patients with portal hypertension than without. While liver expression of TLR4 and LBP remained low, TLR7 and TLR1 showed marked BA-specific increases, and TLR7 correlated with Metavir fibrosis stage and ACTA2. CONCLUSION: BT does not seem to play a significant role in liver injury after SPE in our series of BA patients.
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Translocação Bacteriana , Atresia Biliar , Portoenterostomia Hepática , Receptores Toll-Like , Criança , Humanos , Atresia Biliar/cirurgia , Portoenterostomia Hepática/métodos , Receptores Toll-Like/sangue , Biomarcadores , Fator de Necrose Tumoral alfaRESUMO
Adult-type granulosa cell tumor (AGCT) is a rare ovarian malignancy characterized by slow growth and hormonal activity. The prognosis of AGCT is generally favorable, but one-third of patients with low-stage disease experience a late relapse, and over half of them die of AGCT. To identify markers that would distinguish patients at risk for relapse, we performed Lexogen QuantSeq 3' mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2 (FOXL2) mutation. We compared the transcriptomic profiles of 14 non-relapsed archival primary AGCTs (follow-up time 17-26 years after diagnosis) with 13 relapsed primary AGCTs (follow-up time 1.7-18 years) and eight relapsed tumors (follow-up time 2.8-18.9 years). Non-relapsed and relapsed primary AGCTs had similar transcriptomic profiles. In relapsed tumors three genes were differentially expressed: plasmalemma vesicle associated protein (PLVAP) was upregulated (p = 0.01), whereas argininosuccinate synthase 1 (ASS1) (p = 0.01) and perilipin 4 (PLIN4) (p = 0.02) were downregulated. PLVAP upregulation was validated using tissue microarray RNA in situ hybridization. In our patient cohort with extremely long follow-up, we observed similar gene expression patterns in both primary AGCT groups, suggesting that relapse is not driven by transcriptomic changes. These results reinforce earlier findings that molecular markers do not predict AGCT behavior or risk of relapse.
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Granulosa cell tumors (GCT) constitute only ~5% of ovarian neoplasms yet have significant consequences, as up to 80% of women with recurrent GCT will die of the disease. This study investigated the effectiveness of procaspase-activating compound 1 (PAC-1), an activator of procaspase-3, in treating adult GCT (AGCT) in combination with selected apoptosis-inducing agents. Sensitivity of the AGCT cell line KGN to these drugs, alone or in combination with PAC-1, was tested using a viability assay. Our results show a wide range in cytotoxic activity among the agents tested. Synergy with PAC-1 was most pronounced, both empirically and by mathematical modelling, when combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This combination showed rapid kinetics of apoptosis induction as determined by caspase-3 activity, and strongly synergistic killing of both KGN as well as patient samples of primary and recurrent AGCT. We have demonstrated that the novel combination of two pro-apoptotic agents, TRAIL and PAC-1, significantly amplified the induction of apoptosis in AGCT cells, warranting further investigation of this combination as a potential therapy for AGCT.
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Tumor de Células da Granulosa/tratamento farmacológico , Hidrazonas/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Piperazinas/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Benzoquinonas/administração & dosagem , Carboplatina/administração & dosagem , Caspase 3/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Feminino , Tumor de Células da Granulosa/enzimologia , Tumor de Células da Granulosa/patologia , Humanos , Técnicas In Vitro , Conceitos Matemáticos , Modelos Biológicos , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , GencitabinaRESUMO
BACKGROUND: Acute kidney injury (AKI) is common after heart surgery. Neutrophil gelatinase-associated lipocalin (NGAL) is produced in injured kidney. NGAL has been used as an early plasma biomarker for AKI in patients undergoing heart surgery. Neutrophils contain all isoforms (25-kDa, 45-kDa and 145-kDa) but the kidney produces almost exclusively the 25-kDa isoform of NGAL. We investigated first, whether there is association between NGAL and neutrophil activation, and second whether activated neutrophils are a significant source of circulating NGAL in plasma in patients undergoing cardiac surgery. METHODS: Two separate patient cohorts were studied: 1) the "kinetic cohort" (n = 29) and 2) the "FINNAKI cohort" (n = 306). As NGAL is strictly co-localized with lactoferrin in neutrophils, NGAL and lactoferrin were measured with enzyme-linked immunosorbent assay in all patients. In sixty-one patients of the "FINNAKI cohort" Western blot was used to separate NGAL isoforms according to their molecular size. Mann-Whitney U, Kruskal-Wallis H, Pearson's and Spearman's tests were used as appropriate. RESULTS: There was strong intraoperative association between NGAL and lactoferrin at all four time-points in the "kinetic cohort". In the "FINNAKI cohort", NGAL and lactoferrin concentrations correlated preoperatively (R = 0.59, p < 0.001) and at admission to the intensive care unit (R = 0.69, p < 0.001). At admission to intensive care unit, concentrations of NGAL and lactoferrin were higher in AKI than in non-AKI patients (NGAL: p < 0.001; lactoferrin: p < 0.029). In Western blot analyses, neutrophil specific 45-kDa isoform (median 41% [IQR 33.3-53.1]) and mostly neutrophil derived 145-kDa isoform (median 53.5% [IQR 44.0-64.9%]) together represented over 90% of total NGAL in plasma. Potentially kidney derived NGAL isoform (25-kDa) accounted for only 0.9% (IQR 0.3 - 3.0%) of total NGAL in plasma. There were no statistically significant differences in the distribution of NGAL isomers between AKI and non-AKI patients. CONCLUSIONS: Plasma NGAL during cardiac surgery is associated with neutrophil activation. Based on molecular size, the majority of circulating NGAL is derived from neutrophils. Neutrophil activation is a confounding factor when interpreting increased plasma NGAL in cardiac surgery.
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Injúria Renal Aguda/sangue , Injúria Renal Aguda/etiologia , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Procedimentos Cirúrgicos Cardíacos/tendências , Lipocalina-2/sangue , Injúria Renal Aguda/diagnóstico , Idoso , Biomarcadores/sangue , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Biliary atresia (BA), a neonatal liver disease, is characterized by obstruction of extrahepatic bile ducts with subsequent cholestasis, inflammation, and progressive liver fibrosis. To gain insights into the pathophysiology of BA, we focused attention on GATA6, a transcription factor implicated in biliary development. Early in fetal development GATA6 expression is evident in cholangiocytes and hepatocytes, but by late gestation it is extinguished in hepatocytes. Utilizing a unique set of BA liver samples collected before and after successful portoenterostomy (PE), we found that GATA6 expression is markedly upregulated in hepatocytes of patients with BA compared with healthy and cholestatic disease controls. This upregulation is recapitulated in two murine models simulating bile duct obstruction and intrahepatic bile ductule expansion. GATA6 expression in BA livers correlates with two established negative prognostic indicators (age at PE, degree of intrahepatic bile ductule expansion) and decreases after normalization of serum bilirubin by PE. GATA6 expression in BA livers correlates with expression of known regulators of cholangiocyte differentiation ( JAGGED1, HNF1ß, and HNF6). These same genes are upregulated after enforced expression of GATA6 in human hepatocyte cell models. In conclusion, GATA6 is a novel marker and a putative driver of hepatocyte-cholangiocyte metaplasia in BA, and its expression in hepatocytes is downregulated after successful PE. NEW & NOTEWORTHY A pathological hallmark in the liver of patients with biliary atresia is ductular reaction, an expansion of new bile ductules that are thought to arise from conversion of mature hepatocytes. Here, we show that transcription factor GATA6 is a marker and potential driver of hepatocyte ductal metaplasia in biliary atresia. Hepatocyte GATA6 expression is elevated in biliary atresia, correlates with bile duct expansion, and decreases after successful portoenterostomy.
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Ductos Biliares Extra-Hepáticos/patologia , Atresia Biliar , Fator de Transcrição GATA6/metabolismo , Hepatócitos/metabolismo , Fígado/patologia , Animais , Atresia Biliar/metabolismo , Atresia Biliar/patologia , Atresia Biliar/cirurgia , Biomarcadores/metabolismo , Transdiferenciação Celular/fisiologia , Colestase/metabolismo , Modelos Animais de Doenças , Células Hep G2 , Humanos , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos , Portoenterostomia Hepática/métodosRESUMO
GATA4, a transcription factor crucial for early liver development, has been implicated in the pathophysiology of hepatoblastoma, an embryonal tumor of childhood. However, the molecular and phenotypic consequences of GATA4 expression in hepatoblastoma are not fully understood. We surveyed GATA4 expression in 24 hepatoblastomas using RNA in situ hybridization and immunohistochemistry. RNA interference was used to inhibit GATA4 in human HUH6 hepatoblastoma cells, and changes in cell migration were measured with wound healing and transwell assays. RNA microarray hybridization was performed on control and GATA4 knockdown HUH6 cells, and differentially expressed genes were validated by quantitative polymerase chain reaction or immunostaining. Plasmid transfection was used to overexpress GATA4 in primary human hepatocytes and ensuring changes in gene expression were measured by quantitative polymerase chain reaction. We found that GATA4 expression was high in most hepatoblastomas but weak or negligible in normal hepatocytes. GATA4 gene silencing impaired HUH6 cell migration. We identified 106 differentially expressed genes (72 downregulated, 34 upregulated) in knockdown versus control HUH6 cells. GATA4 silencing altered the expression of genes associated with cytoskeleton organization, cell-to-cell adhesion, and extracellular matrix dynamics (e.g. ADD3, AHNAK, DOCK8, RHOU, MSF, IGFBP1, COL4A2). These changes in gene expression reflected a more epithelial (less malignant) phenotype. Consistent with this notion, there was reduced F-actin stress fiber formation in knockdown HUH6 cells. Forced expression of GATA4 in primary human hepatocytes triggered opposite changes in the expression of genes identified by GATA4 silencing in HUH6 cells. In conclusion, GATA4 is highly expressed in most hepatoblastomas and correlates with a mesenchymal, migratory phenotype of hepatoblastoma cells.
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Movimento Celular , Fator de Transcrição GATA4/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hepatoblastoma/patologia , Neoplasias Hepáticas/patologia , Mesoderma/patologia , Adolescente , Adulto , Estudos de Casos e Controles , Proliferação de Células , Criança , Pré-Escolar , Feminino , Fator de Transcrição GATA4/antagonistas & inibidores , Fator de Transcrição GATA4/genética , Hepatoblastoma/genética , Humanos , Lactente , Neoplasias Hepáticas/genética , Masculino , Mesoderma/metabolismo , Pessoa de Meia-Idade , Prognóstico , RNA Interferente Pequeno/genética , Adulto JovemRESUMO
Testicular Leydig cells produce androgens essential for proper male reproductive development and fertility. Here, we describe a new Leydig cell ablation model based on Cre/Lox recombination of mouse Gata4 and Gata6, two genes implicated in the transcriptional regulation of steroidogenesis. The testicular interstitium of adult Gata4flox/flox ; Gata6flox/flox mice was injected with adenoviral vectors encoding Cre + GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-GFP). The vectors efficiently and selectively transduced Leydig cells, as evidenced by GFP reporter expression. Three days after Ad-Cre-IRES-GFP injection, expression of androgen biosynthetic genes (Hsd3b1, Cyp17a1 and Hsd17b3) was reduced, whereas expression of another Leydig cell marker, Insl3, was unchanged. Six days after Ad-Cre-IRES-GFP treatment, the testicular interstitium was devoid of Leydig cells, and there was a concomitant loss of all Leydig cell markers. Chromatin condensation, nuclear fragmentation, mitochondrial swelling, and other ultrastructural changes were evident in the degenerating Leydig cells. Liquid chromatography-tandem mass spectrometry demonstrated reduced levels of androstenedione and testosterone in testes from mice injected with Ad-Cre-IRES-GFP. Late effects of treatment included testicular atrophy, infertility and the accumulation of lymphoid cells in the testicular interstitium. We conclude that adenoviral-mediated gene delivery is an expeditious way to probe Leydig cell function in vivo Our findings reinforce the notion that GATA factors are key regulators of steroidogenesis and testicular somatic cell survival.Free Finnish abstract: A Finnish translation of this abstract is freely available at http://www.reproduction-online.org/content/154/4/455/suppl/DC2.
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Adenoviridae/genética , Fator de Transcrição GATA4/metabolismo , Fator de Transcrição GATA6/metabolismo , Vetores Genéticos , Células Intersticiais do Testículo/metabolismo , Transdução Genética , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Sobrevivência Celular , Feminino , Fertilidade , Fator de Transcrição GATA4/deficiência , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA6/deficiência , Fator de Transcrição GATA6/genética , Genótipo , Hormônios Esteroides Gonadais/biossíntese , Insulina/genética , Insulina/metabolismo , Integrases/genética , Células Intersticiais do Testículo/ultraestrutura , Masculino , Camundongos Knockout , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Fenótipo , Gravidez , Progesterona Redutase/genética , Progesterona Redutase/metabolismo , Proteínas/genética , Proteínas/metabolismo , Transdução de Sinais , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroide Isomerases/genética , Esteroide Isomerases/metabolismo , Fatores de TempoRESUMO
Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.
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Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição GATA4/biossíntese , Hepatoblastoma/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina , Fator de Transcrição GATA4/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hepatoblastoma/genética , Hepatoblastoma/patologia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Interferente PequenoRESUMO
Targeted treatments are needed for advanced adult-type granulosa cell tumors (AGCTs). We set out to assess tumor tissue and circulating levels of TNF-related apoptosis-inducing ligand (TRAIL), a promising anti-cancer cytokine, in patients affected by AGCT. We analyzed tissue expression of TRAIL in 127 AGCTs using immunohistochemistry or RT-PCR. Soluble TRAIL was measured by means of ELISA from 141 AGCT patient serum samples, as well as the conditioned media of 15 AGCT patient-derived primary cell cultures, and the KGN cell line. Tissue and serum TRAIL levels were analyzed in relationship with clinical parameters, and serum estradiol, FSH, and LH levels. We found that AGCT samples expressed TRAIL mRNA and protein at levels comparable to normal granulosa cells. AGCT cells did not release soluble TRAIL. TRAIL protein levels were decreased in tumors over 10 cm in diameter (p = 0.04). Consistently, circulating TRAIL levels correlated negatively to tumor dimension (p = 0.01). Circulating TRAIL levels negatively associated with serum estradiol levels. In multiple regression analysis, tumor size was an independent factor contributing to the decreased levels of soluble TRAIL in AGCT patients. AGCTs associate with significantly decreased tumor tissue and serum TRAIL levels in patients with a large tumor mass. These findings encourage further study of agonistic TRAIL treatments in patients with advanced or recurrent AGCT.
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Regulação Neoplásica da Expressão Gênica , Tumor de Células da Granulosa/genética , Neoplasias Ovarianas/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Estradiol/sangue , Feminino , Imunofluorescência , Tumor de Células da Granulosa/sangue , Tumor de Células da Granulosa/metabolismo , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF/sangue , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Células Tumorais Cultivadas , Adulto JovemRESUMO
DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging owing to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of cytosine-guanine dinucleotide islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared with adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development.
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Neoplasias das Glândulas Suprarrenais/genética , Metilação de DNA , Microdissecção e Captura a Laser , Análise de Sequência de DNA , Sulfitos , Neoplasias das Glândulas Suprarrenais/etiologia , Animais , Castração , Humanos , Camundongos , Reação em Cadeia da PolimeraseRESUMO
Hepatoblastoma (HB) is the most common malignant liver tumor among children. To gain insight into the pathobiology of HB, we performed RNA sequence analysis on 5 patient-derived xenograft lines (HB-243, HB-279, HB-282, HB-284, HB-295) and 1 immortalized cell line (HUH6). Using cultured hepatocytes as a control, we found 2,868 genes that were differentially expressed in all of the HB lines on mRNA level. The most upregulated genes were ODAM, TRIM71, and IGDCC3, and the most downregulated were SAA1, SAA2, and NNMT. Protein-protein interaction analysis identified ubiquitination as a key pathway dysregulated in HB. UBE2C, encoding an E2 ubiquitin ligase often overexpressed in cancer cells, was markedly upregulated in 5 of the 6 HB cell lines. Validation studies confirmed UBE2C immunostaining in 20 of 25 HB tumor specimens versus 1 of 6 normal liver samples. The silencing of UBE2C in two HB cell models resulted in decreased cell viability. RNA sequencing analysis showed alterations in cell cycle regulation after UBE2C knockdown. UBE2C expression in HB correlated with inferior patient survival. We conclude that UBE2C may hold prognostic utility in HB and that the ubiquitin pathway is a potential therapeutic target in this tumor.
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Background: In response to hypoxia, tumor cells undergo transcriptional reprogramming including upregulation of carbonic anhydrase (CA) IX, a metalloenzyme that maintains acid-base balance. CAIX overexpression has been shown to correlate with poor prognosis in various cancers, but the role of this CA isoform in hepatoblastoma (HB) has not been examined. Methods: We surveyed the expression of CAIX in HB specimens and assessed the impact of SLC-0111, a CAIX inhibitor, on cultured HB cells in normoxic and hypoxic conditions. Results: CAIX immunoreactivity was detected in 15 out of 21 archival pathology HB specimens. The CAIX-positive cells clustered in the middle of viable tumor tissue or next to necrotic areas. Tissue expression of CAIX mRNA was associated with metastasis and poor clinical outcome of HB. Hypoxia induced a striking upregulation of CAIX mRNA and protein in three HB cell models: the immortalized human HB cell line HUH6 and patient xenograft-derived lines HB-295 and HB-303. Administration of SLC-0111 abrogated the hypoxia-induced upregulation of CAIX and decreased HB cell viability, both in monolayer and spheroid cultures. In addition, SLC-0111 reduced HB cell motility in a wound healing assay. Transcriptomic changes triggered by SLC-0111 administration differed under normoxic vs. hypoxic conditions, although SLC-0111 elicited upregulation of several tumor suppressor genes under both conditions. Conclusion: Hypoxia induces CAIX expression in HB cells, and the CAIX inhibitor SLC-0111 has in vitro activity against these malignant cells.
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BACKGROUND: We aimed to quantify ductular reaction (DR) in biliary atresia using a neural network in relation to underlying pathophysiology and prognosis. METHODS: Image-processing neural network model was applied to 259 cytokeratin-7-stained native liver biopsies of patients with biliary atresia and 43 controls. The model quantified total proportional DR (DR%) composed of portal biliary epithelium (BE%) and parenchymal intermediate hepatocytes (PIH%). The results were related to clinical data, Sirius Red-quantified liver fibrosis, serum biomarkers, and bile acids. RESULTS: In total, 2 biliary atresia biopsies were obtained preoperatively, 116 at Kasai portoenterostomy (KPE) and 141 during post-KPE follow-up. DR% (8.3% vs. 5.9%, p=0.045) and PIH% (1.3% vs. 0.6%, p=0.004) were increased at KPE in patients remaining cholestatic postoperatively. After KPE, patients with subsequent liver transplantation or death showed an increase in DR% (7.9%-9.9%, p = 0.04) and PIH% (1.6%-2.4%, p = 0.009), whereas patients with native liver survival (NLS) showed decreasing BE% (5.5%-3.0%, p = 0.03) and persistently low PIH% (0.9% vs. 1.3%, p = 0.11). In Cox regression, high DR predicted inferior NLS both at KPE [DR% (HR = 1.05, p = 0.01), BE% (HR = 1.05, p = 0.03), and PIH% (HR = 1.13, p = 0.005)] and during follow-up [DR% (HR = 1.08, p<0.0001), BE% (HR = 1.58, p = 0.001), and PIH% (HR = 1.04, p = 0.008)]. DR% correlated with Sirius red-quantified liver fibrosis at KPE (R = 0.47, p<0.0001) and follow-up (R = 0.27, p = 0.004). A close association between DR% and serum bile acids was observed at follow-up (R = 0.61, p<0.001). Liver fibrosis was not prognostic for NLS at KPE (HR = 1.00, p = 0.96) or follow-up (HR = 1.01, p = 0.29). CONCLUSIONS: DR predicted NLS in different disease stages before transplantation while associating with serum bile acids after KPE.
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Atresia Biliar , Aprendizado Profundo , Humanos , Atresia Biliar/diagnóstico , Atresia Biliar/cirurgia , Prognóstico , Portoenterostomia Hepática/métodos , Cirrose Hepática/diagnóstico , Cirrose Hepática/cirurgia , Cirrose Hepática/complicações , Ácidos e Sais BiliaresRESUMO
Portoenterostomy (PE) has remained as the generally accepted first line surgical treatment for biliary atresia (BA) for over 50 years. Currently, close to half of BA patients survive beyond 10 years with their native livers, and most of them reach adulthood without liver transplantation (LT). Despite normalization of serum bilirubin by PE, ductular reaction and portal fibrosis persist in the native liver. The chronic cholangiopathy progresses to cirrhosis, complications of portal hypertension, recurrent cholangitis or hepatobiliary tumors necessitating LT later in life. Other common related health problems include impaired bone health, neuromotor development and quality of life. Only few high-quality trials are available for evidence-based guidance of post-PE adjuvant medical therapy or management of the disease complications. Better understanding of the pathophysiological mechanisms connecting native liver injury to clinical outcomes is critical for development of accurate follow-up tools and novel therapies designed to improve native liver function and survival.
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Atresia Biliar , Transplante de Fígado , Adulto , Atresia Biliar/complicações , Atresia Biliar/diagnóstico , Atresia Biliar/cirurgia , Humanos , Fígado/patologia , Fígado/cirurgia , Portoenterostomia Hepática/efeitos adversos , Qualidade de VidaRESUMO
Biliary atresia (BA) is a chronic neonatal cholangiopathy characterized by fibroinflammatory bile duct damage. Reliable biomarkers for predicting native liver survival (NLS) following portoenterostomy (PE) surgery are lacking. Herein we explore the utility of 22 preidentified profibrotic molecules closely connected to ductular reaction (DR) and prevailing after successful PE (SPE), in predicting PE outcomes and liver injury. We used qPCR and immunohistochemistry in a BA cohort including liver samples obtained at PE (n = 53) and during postoperative follow-up after SPE (n = 25). Of the 13 genes over-expressed in relation to cholestatic age-matched controls at PE, only secretin receptor (SCTR) expression predicted cumulative 5-year NLS and clearance of jaundice. Patients in the highest SCTR expression tertile showed 34-55% lower NLS than other groups at 1-5 years after PE (P = 0.006-0.04 for each year). SCTR expression was also significantly lower [42 (24-63) vs 75 (39-107) fold, P = 0.015] among those who normalized their serum bilirubin after PE. Liver SCTR expression localized in cholangiocytes and correlated positively with liver fibrosis, DR, and transcriptional markers of fibrosis (ACTA2) and cholangiocytes (KRT7, KRT19) both at PE and after SPE. SCTR is a promising prognostic marker for PE outcomes and associates with liver injury in BA.
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Atresia Biliar , Receptores dos Hormônios Gastrointestinais , Atresia Biliar/metabolismo , Biomarcadores/metabolismo , Humanos , Lactente , Recém-Nascido , Fígado/metabolismo , Fígado/cirurgia , Portoenterostomia Hepática , Receptores Acoplados a Proteínas G , Receptores dos Hormônios Gastrointestinais/genética , Resultado do TratamentoRESUMO
The neuropilins NRP1 and NRP2 are multifunctional glycoproteins that have been implicated in several cancer-related processes including cell survival, migration, and invasion in various tumor types. Here, we examine the role of neuropilins in hepatoblastoma (HB), the most common pediatric liver malignancy. Using a combination of immunohistochemistry, RNA analysis and western blotting, we observed high level expression of NRP1 and NRP2 in 19 of 20 HB specimens and in a majority of human HB cell lines (HUH6 and five cell lines established from patient-derived xenografts) studied but not in normal hepatocytes. Silencing of NRP2 expression in HUH6 and HB-282 HB cells resulted in decreased cell viability, impaired cytoskeleton remodeling, and reduced cell motility, suggesting that NRP2 contributes to the malignant phenotype. We propose that neuropilins warrant further investigation as biomarkers of HB and potential therapeutic targets.
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BACKGROUND: Acute Kidney Injury (AKI) is a common clinical complication. Plasma/serum neutrophil gelatinase-associated lipocalin (NGAL) has been proposed as a rapid marker of AKI. However, NGAL is not kidney-specific. It exists in three isoforms (monomeric, homo-dimeric and hetero-dimeric). Only the monomeric isoform is produced by renal tubular cells and plasma NGAL levels are confounded by the release of all NGAL isoforms from neutrophils. Our aim was to investigate whether NGAL is released into blood from injured renal tubules. METHODS: Kidney transplantation (n = 28) served as a clinical model of renal ischaemic injury. We used ELISA to measure NGAL concentrations at 2 minutes after kidney graft reperfusion in simultaneously taken samples of renal arterial and renal venous blood. Trans-renal gradients (venous-arterial) of NGAL were calculated. We performed Western blotting to distinguish between renal and non-renal NGAL isoforms. Liver-type fatty acid binding protein (LFABP) and heart-type fatty acid binding protein (HFABP) served as positive controls of proximal and distal tubular damage. RESULTS: Significant renal release of LFABP [trans-renal gradient 8.4 (1.7-30.0) ng/ml, p = 0.005] and HFABP [trans-renal gradient 3.7 (1.1-5.0) ng/ml, p = 0.003] at 2 minutes after renal graft reperfusion indicated proximal and distal tubular damage. NGAL concentrations were comparable in renal venous and renal arterial blood. Thus, there was no trans-renal gradient of NGAL. Western blotting revealed that the renal NGAL isoform represented only 6% of the total NGAL in renal venous blood. CONCLUSIONS: Ischaemic proximal and distal tubular damage occurs in kidney transplantation without concomitant NGAL washout from the kidney graft into blood. Plasma/serum NGAL levels are confounded by the release of NGAL from neutrophils. Present results do not support the interpretation that increase in plasma NGAL is caused by release from the renal tubules.
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Successful portoenterostomy (SPE) improves the short-term outcome of patients with biliary atresia (BA) by relieving cholestasis and extending survival with native liver. Despite SPE, hepatic fibrosis progresses in most patients, leading to cirrhosis and a deterioration of liver function. The goal of this study was to characterize the effects of SPE on the BA liver transcriptome. We used messenger RNA sequencing to analyze global gene-expression patterns in liver biopsies obtained at the time of portoenterostomy (n = 13) and 1 year after SPE (n = 8). Biopsies from pediatric (n = 2) and adult (n = 2) organ donors and other neonatal cholestatic conditions (n = 5) served as controls. SPE was accompanied by attenuation of inflammation and concomitant up-regulation of key extracellular matrix (ECM) genes. Highly overexpressed genes promoting biliary fibrosis and bile duct integrity, such as integrin subunit beta 6 and previously unreported laminin subunit alpha 3, emerged as candidates to control liver fibrosis after SPE. At a cellular level, the relative abundance of activated hepatic stellate cells and liver macrophages decreased following SPE, whereas portal fibroblasts (PFs) and cholangiocytes persisted. Conclusion: The attenuation of inflammation following SPE coincides with emergence of an ECM molecular fingerprint, a set of profibrotic molecules mechanistically connected to biliary fibrosis. The persistence of activated PFs and cholangiocytes after SPE suggests a central role for these cell types in the progression of biliary fibrosis.
RESUMO
Interleukin (IL)-8 (CXCL8), a chemokine involved in neutrophil recruitment, has been implicated in ductular reaction and liver fibrogenesis. We studied liver and serum IL-8 expression in a large biliary atresia (BA) cohort and explored its prognostic and pathophysiological potential. IL-8 expression was assessed in liver utilizing quantitative polymerase chain reaction (qPCR), immunohistochemistry and in situ hybridization and in serum using an enzyme-linked immunosorbent assay, among 115 BA patients, 10 disease controls and 68 normal controls. Results were correlated to portoenterostomy (PE) outcomes, biochemical and histological liver injury, transcriptional markers of fibrosis and cholangiocytes, and expression of other related cytokines. IL-8 was markedly overexpressed in liver and serum of BA patients at PE (n = 88) and in serum samples obtained during postoperative follow-up (n = 40). IL-8 expression in the liver was predominantly in cholangiocytes within areas of ductular reaction. Liver IL-8 mRNA expression correlated positively with its serum concentration, bile ductular proliferation, Metavir fibrosis stage, and transcriptional markers of activated myofibroblasts (ACTA2) and cholangiocytes (KRT19). Taken together, IL-8 may mediate liver injury in BA by promoting ductular reaction and associated liver fibrogenesis. Prognostic value of serum IL-8 to predict native liver survival was limited and confined to the postoperative period after PE.