Little is known on the mechanism of liver injury mediated by bile acids following Kasai portoenterostomy (KPE) in biliary atresia (BA). We sought to quantify individual serum bile acids, 7-alpha-hydroxy-4-cholesten-3-one (C4), and fibroblast growth factor 19 (FGF19) after KPE and to evaluate their prognostic utility and transcriptomic regulation. Serum (n=244) and liver specimens (n=105) prospectively obtained from BA patients (n=54) after KPE were included. Bile acids were analyzed using mass spectrometry, gene expression with quantitative PCR, and histopathology using a neural network model. Following KPE, serum bile acids correlated positively with biochemical liver injury, pediatric end-stage liver disease score, liver stiffness, histological ductular reaction, and liver fibrosis. Bile acids were higher among patients who developed portal hypertension (79.6 vs. 11.9 μmol/l, p<0.0001), esophageal varices (91.6 vs. 16.2 μmol/l, p<0.0001), or required liver transplantation (LT, 115.3 vs. 22.0 μmol/l, p<0.0001) during follow up; bile acids predicted these outcomes in time-dependent regression models. Accumulation of conjugated bile acids, cholic acid, and taurine conjugates predicted LT risk while associating with histological liver injury. Serum C4 (0.04 vs. 0.00 μmol/l, p=0.04) and liver CYP7A1 were higher in native liver survivors than in LT patients (fold-change 16.9 vs. 7.0, p=0.02). Primary bile acids correlated negatively with C4 (R=-0.38, p<0.001) and CYP7A1 (R=-0.49, p=0.01). Unlike in native liver survivors (R=-0.19, p=0.66), serum FGF19 correlated with liver FGF19 (R=0.59, p=0.04) without an inverse association with serum primary bile acids in LT patients (R=0.26, p=0.08). Accumulation and altered composition of serum bile acids predicted progressive liver disease and poorer transplant-free survival following KPE. Poor prognosis was associated with low bile acid synthesis and aberrantly increased liver FGF19. Biliary atresia (BA), a fibro-obliterating biliary disease of infants, remains the most common indication for pediatric liver transplantation caused by rapid progression of liver injury. To identify predictive biomarkers of disease progression and to elucidate the pathophysiology of BA liver injury, we profiled serum bile acids and studied their liver metabolism after Kasai portoenterostomy. Accumulation and altered composition of circulating bile acids predicted progression of liver disease and need for liver transplantation. Patients with poor prognosis showed low bile acid synthesis and abnormal liver expression of fibroblast growth factor 19.
Late lung development is a period of alveolar and microvascular formation, which is pivotal in ensuring sufficient and effective gas exchange. Defects in late lung development manifest in premature infants as a chronic lung disease named bronchopulmonary dysplasia (BPD). Numerous studies demonstrated the therapeutic properties of exogenous bone marrow and umbilical cord-derived mesenchymal stromal cells (MSCs) in experimental BPD. However, very little is known regarding the regenerative capacity of resident lung MSCs (L-MSCs) during normal development and in BPD. In this study we aimed to characterize the L-MSC population in homeostasis and upon injury. We used single-cell RNA sequencing (scRNA-seq) to profile in situ Ly6a+ L-MSCs in the lungs of normal and O2-exposed neonatal mice (a well-established model to mimic BPD) at 3 developmental timepoints (postnatal days 3, 7, and 14). Hyperoxia exposure increased the number and altered the expression profile of L-MSCs, particularly by increasing the expression of multiple pro-inflammatory, pro-fibrotic, and anti-angiogenic genes. In order to identify potential changes induced in the L-MSCs transcriptome by storage and culture, we profiled 15 000 Ly6a+ L-MSCs after in vitro culture. We observed great differences in expression profiles of in situ and cultured L-MSCs, particularly those derived from healthy lungs. Additionally, we have identified the location of Ly6a+/Col14a1+ L-MSCs in the developing lung and propose Serpinf1 as a novel, culture-stable marker of L-MSCs. Finally, cell communication analysis suggests inflammatory signals from immune and endothelial cells as main drivers of hyperoxia-induced changes in L-MSCs transcriptome.
Abstract Endothelial cells (ECs) form a tissue-specific barrier for disseminating cancer cells in distant organs. However, the molecular regulation of the ECs in the metastatic niche remains unclear. Here, we analyze using scRNA-Seq, the transcriptional reprogramming of lung ECs six hours after the arrival of melanoma cells in mouse lungs. We discover a reactive capillary EC cluster (rCap) that increases from general capillary ECs in response to infiltrating cancer cells. rCap is enriched for angiogenic and inflammatory pathways and is also found in human lung datasets. The JAK-STAT activated oncogenic Pim3 kinase is a marker of rCap, being upregulated in spontaneous metastasis models. Notably, PIM inhibition increases vascular leakage and metastatic colonization and impairs the EC barrier by decreasing the junctional cadherin-5 and catenins α, β and δ. These results highlight the pulmonary endothelium’s plasticity and its protection by PIM3, which may impair the efficacy of PIM inhibitors in cancer therapies.
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.
Phosphatidylinositol (PI) is the precursor of many important signaling molecules in eukaryotic cells and, most probably, PI also has important functions in cellular membranes. However, these functions are poorly understood, which is largely due to that i) only few PI species with specific acyl chains are available commercially and ii) there are no simple methods to synthesize such species. Here, we present a simple biochemical protocol to synthesize a variety of labeled or unlabeled PI species from corresponding commercially available phosphatidylcholines. The protocol can be carried out in a single vial in a two-step process which employs three enzymatic reactions mediated by i) commercial phospholipase D from Streptomyces chromofuscus, ii) CDP-diacylglycerol synthase overexpressed in E. coli and iii) PI synthase of Arabidopsis thaliana ectopically expressed in E. coli The PI product is readily purified from the reaction mixture by liquid chromatography since E. coli does not contain endogenous PI or other coeluting lipids. The method allows one to synthesize and purify labeled or unlabeled PI species in 1 or 2 days.Typically, 40-60% of (unsaturated) PC was converted to PI albeit the final yield of PI was less (25-35%) due to losses upon purification.
Endometritis is one of the major causes of infertility in mares. Escherichia coli and β-haemolytic streptococci are among the bacterial species most frequently isolated from the equine uterus. Some bacteria such as β-hemolytic streptococci, can persist in dormant forms and cause prolonged, latent or recurrent infections. Dormant bacteria may be present despite negative bacterial cultures, and they are resistant to antimicrobial treatment due to their resting metabolic state. The purpose of this study was to study formalin-fixed paraffin-embedded equine endometrial biopsies for the presence and localization of E. coli-bacteria, with a chromogenic RNAscope®-method for detection of E. coli-related 16S ribosomal RNA. Hematoxylin-eosin-stained endometrial biopsies were evaluated to determine the level of inflammation and degeneration. During estrus, samples were taken for endometrial culture and cytology with a double-guarded uterine swab. The samples included eight samples with moderate to severe endometrial inflammation detected in endometrial histopathology, and growth of E. coli in bacterial culture, six samples with moderate to severe endometrial inflammation but negative bacterial culture, and five samples with no endometrial pathology (grade I endometrial biopsy, negative endometrial culture and cytology) serving as controls. Positive and negative control probes were included in the RNA in situ hybridization, and results were confirmed with a fluorescence detection method (fluorescence in situ hybridization). Only unspecific signals of limited size and frequency of occurrence were detected in all samples, with random localization in the endometrium. No samples contained rod-shaped signals corresponding to bacterial findings. In conclusion, there was no evidence of bacterial invasion in the endometrium regardless of the inflammatory status of the biopsy or previous bacterial culture results. According to these findings on a small number of samples, invasion of E. coli is not a common finding in the lamina propria of mares, but these bacteria may also evade detection due to localized foci of infections, or supra-epithelial localization under the cover of biofilm. These bacteria and biofilm covering the epithelium may also be lost during formalin-fixation and processing.
ABSTRACT Late lung development is a period of alveolar and microvascular formation, which is pivotal in ensuring sufficient and effective gas exchange. Defects in late lung development manifest in premature infants as a chronic lung disease named bronchopulmonary dysplasia (BPD). Numerous studies demonstrated the therapeutic properties of exogenous bone marrow and umbilical cord-derived mesenchymal stromal cells (MSCs) in experimental BPD. However, very little is known regarding the regenerative capacity of resident lung MSCs (L-MSCs) during normal development and in BPD. In this study we aimed to characterize the L-MSC population in homeostasis and upon injury. We used single-cell RNA sequencing (scRNA-seq) to profile in situ Ly6a + L-MSCs in the lungs of normal and O 2 -exposed neonatal mice (a well-established model to mimic BPD) at three developmental timepoints (postnatal days 3, 7 and 14). Hyperoxia exposure increased the number, and altered the expression profile of L-MSCs, particularly by increasing the expression of multiple pro-inflammatory, pro-fibrotic, and anti-angiogenic genes. In order to identify potential changes induced in the L-MSCs transcriptome by storage and culture, we profiled 15,000 Ly6a + L-MSCs after in vitro culture. We observed great differences in expression profiles of in situ and cultured L-MSCs, particularly those derived from healthy lungs. Additionally, we have identified the location of L-MSCs in the developing lung and propose Serpinf1 as a novel, culture-stable marker of L-MSCs. Finally, cell communication analysis suggests inflammatory signals from immune and endothelial cells as main drivers of hyperoxia-induced changes in L-MSCs transcriptome.
