p16 INK4A drives nonalcoholic fatty liver disease phenotypes in high fat diet fed mice through biliary E2F1/FOXO1/IGF-1 signaling.

BACKGROUND AND AIMS: NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling. APPROACH AND RESULTS: 4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro. CONCLUSION: Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.

Inhibition of Secretin/Secretin Receptor Axis Ameliorates NAFLD Phenotypes.

BACKGROUND AND AIMS: Human NAFLD is characterized at early stages by hepatic steatosis, which may progress to NASH when the liver displays microvesicular steatosis, lobular inflammation, and pericellular fibrosis. The secretin (SCT)/secretin receptor (SCTR) axis promotes biliary senescence and liver fibrosis in cholestatic models through down-regulation of miR-125b signaling. We aim to evaluate the effect of disrupting biliary SCT/SCTR/miR-125b signaling on hepatic steatosis, biliary senescence, and liver fibrosis in NAFLD/NASH. APPROACH AND RESULTS: In vivo, 4-week-old male wild-type, Sct-/- and Sctr-/- mice were fed a control diet or high-fat diet (HFD) for 16 weeks. The expression of SCT/SCTR/miR-125b axis was measured in human NAFLD/NASH liver samples and HFD mouse livers by immunohistochemistry and quantitative PCR. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were evaluated in mouse liver and human NAFLD/NASH liver samples. miR-125b target lipogenesis genes in hepatocytes were screened and validated by custom RT2 Profiler PCR array and luciferase assay. Biliary SCT/SCTR expression was increased in human NAFLD/NASH samples and in livers of HFD mice, whereas the expression of miR-125b was decreased. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were observed in human NAFLD/NASH samples as well as HFD mice, which were decreased in Sct-/- and Sctr-/- HFD mice. Elovl1 is a lipogenesis gene targeted by miR-125b, and its expression was also decreased in HFD mouse hepatocytes following Sct or Sctr knockout. Bile acid profile in fecal samples have the greatest changes between wild-type mice and Sct-/- /Sctr-/- mice. CONCLUSION: The biliary SCT/SCTR/miR-125b axis promotes liver steatosis by up-regulating lipid biosynthesis gene Elovl1. Targeting the biliary SCT/SCTR/miR-125b axis may be key for ameliorating phenotypes of human NAFLD/NASH.

Mast Cells Promote Nonalcoholic Fatty Liver Disease Phenotypes and Microvesicular Steatosis in Mice Fed a Western Diet.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is simple steatosis but can develop into nonalcoholic steatohepatitis (NASH), characterized by liver inflammation, fibrosis, and microvesicular steatosis. Mast cells (MCs) infiltrate the liver during cholestasis and promote ductular reaction (DR), biliary senescence, and liver fibrosis. We aimed to determine the effects of MC depletion during NAFLD/NASH. APPROACH AND RESULTS: Wild-type (WT) and KitW-sh (MC-deficient) mice were fed a control diet (CD) or a Western diet (WD) for 16 weeks; select WT and KitW-sh WD mice received tail vein injections of MCs 2 times per week for 2 weeks prior to sacrifice. Human samples were collected from normal, NAFLD, or NASH mice. Cholangiocytes from WT WD mice and human NASH have increased insulin-like growth factor 1 expression that promotes MC migration/activation. Enhanced MC presence was noted in WT WD mice and human NASH, along with increased DR. WT WD mice had significantly increased steatosis, DR/biliary senescence, inflammation, liver fibrosis, and angiogenesis compared to WT CD mice, which was significantly reduced in KitW-sh WD mice. Loss of MCs prominently reduced microvesicular steatosis in zone 1 hepatocytes. MC injection promoted WD-induced biliary and liver damage and specifically up-regulated microvesicular steatosis in zone 1 hepatocytes. Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) expression is reduced in WT WD mice and human NASH but increased in KitW-sh WD mice. MicroRNA 144-3 prime (miR-144-3p) expression was increased in WT WD mice and human NASH but reduced in KitW-sh WD mice and was found to target ALDH1A3. CONCLUSIONS: MCs promote WD-induced biliary and liver damage and may promote microvesicular steatosis development during NAFLD progression to NASH through miR-144-3p/ALDH1A3 signaling. Inhibition of MC activation may be a therapeutic option for NAFLD/NASH treatment.

Mast Cells Induce Ductular Reaction Mimicking Liver Injury in Mice Through Mast Cell-Derived Transforming Growth Factor Beta 1 Signaling.

BACKGROUND AND AIMS: Following liver injury, mast cells (MCs) migrate into the liver and are activated in patients with cholestasis. Inhibition of MC mediators decreases ductular reaction (DR) and liver fibrosis. Transforming growth factor beta 1 (TGF-ß1) contributes to fibrosis and promotes liver disease. Our aim was to demonstrate that reintroduction of MCs induces cholestatic injury through TGF-ß1. APPROACH AND RESULTS: Wild-type, KitW-sh (MC-deficient), and multidrug resistance transporter 2/ABC transporter B family member 2 knockout mice lacking l-histidine decarboxylase were injected with vehicle or PKH26-tagged murine MCs pretreated with 0.01% dimethyl sulfoxide (DMSO) or the TGF-ß1 receptor inhibitor (TGF-ßRi), LY2109761 (10 µM) 3 days before sacrifice. Hepatic damage was assessed by hematoxylin and eosin (H&E) and serum chemistry. Injected MCs were detected in liver, spleen, and lung by immunofluorescence (IF). DR was measured by cytokeratin 19 (CK-19) immunohistochemistry and F4/80 staining coupled with real-time quantitative PCR (qPCR) for interleukin (IL)-1ß, IL-33, and F4/80; biliary senescence was evaluated by IF or qPCR for p16, p18, and p21. Fibrosis was evaluated by sirius red/fast green staining and IF for synaptophysin 9 (SYP-9), desmin, and alpha smooth muscle actin (α-SMA). TGF-ß1 secretion/expression was measured by enzyme immunoassay and qPCR. Angiogenesis was detected by IF for von Willebrand factor and vascular endothelial growth factor C qPCR. In vitro, MC-TGF-ß1 expression/secretion were measured after TGF-ßRi treatment; conditioned medium was collected. Cholangiocytes and hepatic stellate cells (HSCs) were treated with MC-conditioned medium, and biliary proliferation/senescence was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and qPCR; HSC activation evaluated for α-SMA, SYP-9, and collagen type-1a expression. MC injection recapitulates cholestatic liver injury characterized by increased DR, fibrosis/TGF-ß1 secretion, and angiogenesis. Injection of MC-TGF-ßRi reversed these parameters. In vitro, MCs induce biliary proliferation/senescence and HSC activation that was reversed with MCs lacking TGF-ß1. CONCLUSIONS: Our study demonstrates that reintroduction of MCs mimics cholestatic liver injury and that MC-derived TGF-ß1 may be a target in chronic cholestatic liver disease.

Knockout of the Tachykinin Receptor 1 in the Mdr2-/- (Abcb4-/-) Mouse Model of Primary Sclerosing Cholangitis Reduces Biliary Damage and Liver Fibrosis.

Activation of the substance P (SP)/neurokinin 1 receptor (NK1R) axis triggers biliary damage/senescence and liver fibrosis in bile duct ligated and Mdr2-/- (alias Abcb4-/-) mice through enhanced transforming growth factor-ß1 (TGF-ß1) biliary secretion. Recent evidence indicates a role for miR-31 (MIR31) in TGF-ß1-induced liver fibrosis. We aimed to define the role of the SP/NK1R/TGF-ß1/miR-31 axis in regulating biliary proliferation and liver fibrosis during cholestasis. Thus, we generated a novel model with double knockout of Mdr2-/- and NK1R-/ (alias Tacr1-/-) to further address the role of the SP/NK1R axis during chronic cholestasis. In vivo studies were performed in the following 12-week-old male mice: (i) NK1R-/-; (ii) Mdr2-/-; and (iii) NK1R-/-/Mdr2-/- (Tacr1-/-/Abcb4-/-) and their corresponding wild-type controls. Liver tissues and cholangiocytes were collected, and liver damage, changes in biliary mass/senescence, and inflammation as well as liver fibrosis were evaluated by both immunohistochemistry in liver sections and real-time PCR. miR-31 expression was measured by real-time PCR in isolated cholangiocytes. Decreased ductular reaction, liver fibrosis, biliary senescence, and biliary inflammation were observed in NK1R-/-/Mdr2-/- mice compared with Mdr2-/- mice. Elevated expression of miR-31 was observed in Mdr2-/- mice, which was reduced in NK1R-/-/Mdr2-/- mice. Targeting the SP/NK1R and/or miR-31 may be a potential approach in treating human cholangiopathies, including primary sclerosing cholangitis.

Correction to: Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Secretin/secretin receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.

Primary biliary cholangitis (PBC) primarily targets cholangiocytes and is characterized by liver fibrosis and biliary proliferation. Activation of the secretin (Sct)/secretin receptor (SR) axis, expressed only by cholangiocytes, increases biliary proliferation, liver fibrosis, and bicarbonate secretion. We evaluated the effectiveness of SR antagonist treatment for early-stage PBC. Male and female dominant-negative TGF-ß receptor II (dnTGF-ßRII) (model of PBC) and wild-type mice at 12 wk of age were treated with saline or the SR antagonist, Sec 5-27, for 1 wk. dnTGF-ßRII mice expressed features of early-stage PBC along with enhanced Sct/SR axis activation and Sct secretion. dnTGF-ßRII mice had increased biliary proliferation or senescence, inflammation, and liver fibrosis. In dnTGF-ßRII mice, there was increased microRNA-125b/TGF-ß1/TGF-ß receptor 1/VEGF-A signaling. Human early-stage PBC patients had an increase in hepatobiliary Sct and SR expression and serum Sct levels. Increased biliary Sct/SR signaling promotes biliary and hepatic damage during early-stage PBC.-Kennedy, L., Francis, H., Invernizzi, P., Venter, J., Wu, N., Carbone, M., Gershwin, M. E., Bernuzzi, F., Franchitto, A., Alvaro, D., Marzioni, M., Onori, P., Gaudio, E., Sybenga, A., Fabris, L., Meng, F., Glaser, S., Alpini, G. Secretin/secretin receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.

Diagnosing Osteomyelitis: A Histology Guide for Pathologists.

Histopathologic examination of bone specimens coupled with bone culture is considered the gold standard for the diagnosis of osteomyelitis (OM). Despite this, studies have demonstrated interpathologist agreement in the diagnosis of OM as low as 30%, largely stemming from a lack of specific definitions and diagnostic criteria. Review of the literature has provided insight into the lifecycle of OM, illustrating the histologic progression of OM phases from acute to chronic, and provides support for defining subcategories of OM. Using an algorithmic histopathologic tool consisting of 15 criteria, each with an associated score, we defined 5 categories of OM: (1) acute OM, (2) acute and chronic OM, (3) chronic OM, (4) chronic active OM, and (5) chronic inactive OM. We reviewed 462 microscopic slides from 263 patients with suspected OM, and for each slide, we determined an algorithm-derived diagnosis, which was then used to calculate a total histopathologic load score (Jupiter score). Algorithm-derived diagnoses recapitulated original clinical diagnoses and diagnosed cases as OM that had not been originally diagnoses. These novel cases were more likely to have subsequent clinical complications. Finally, pathologic load scores were assessed for association with the category of OM.

Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes.

α-Calcitonin gene-related peptide (α-CGRP) is a 37-amino acid neuropeptide involved in several pathophysiological processes. α-CGRP is involved in the regulation of cholangiocyte proliferation during cholestasis. In this study, we aimed to evaluate if α-CGRP regulates bile duct ligation (BDL)-induced liver fibrosis by using a α-CGRP knockout (α-CGRP-/-) mouse model. α-CGRP-/- and wild-type (WT) mice were subjected to sham surgery or BDL for 7 days. Then, liver fibrosis and cellular senescence as well as the expression of kinase such as p38 and C-Jun N-terminal protein kinase (JNK) in mitogen-activated protein kinases (MAPK) signaling pathway were evaluated in total liver, together with measurement of cellular senescence in cholangiocytes or hepatic stellate cells (HSCs). There was enhanced hepatic expression of Calca (coding α-CGRP) and the CGRP receptor components (CRLR, RAMP-1 and RCP) in BDL and in both WT α-CGRP-/- and BDL α-CGRP-/- mice, respectively. Moreover, there was increased CGRP serum levels and hepatic mRNA expression of CALCA and CGRP receptor components in late-stage PSC samples compared to healthy control samples. Depletion of α-CGRP reduced liver injury and fibrosis in BDL mice that was associated with enhanced cellular senescence of hepatic stellate cells and reduced senescence of cholangiocytes as well as decreased activation of p38 and JNK MAPK signaling pathway. Cholangiocyte supernatant from BDL α-CGRP-/- mice inhibited the activation and increased cellular senescence of cultured human HSCs (HHSCs) compared to HHSCs stimulated with BDL cholangiocyte supernatant. Taken together, endogenous α-CGRP promoted BDL-induced cholestatic liver fibrosis through differential changes in senescence of HSCs and cholangiocytes and activation of p38 and JNK signaling. Modulation of α-CGRP/CGRP receptor signaling may be key for the management of biliary senescence and liver fibrosis in cholangiopathies.

Knockout of l-Histidine Decarboxylase Prevents Cholangiocyte Damage and Hepatic Fibrosis in Mice Subjected to High-Fat Diet Feeding via Disrupted Histamine/Leptin Signaling.

Feeding a high-fat diet (HFD) coupled with sugar, mimicking a Western diet, causes fatty liver disease in mice. Histamine induces biliary proliferation and fibrosis and regulates leptin signaling. Wild-type (WT) and l-histidine decarboxylase (Hdc-/-) mice were fed a control diet or an HFD coupled with a high fructose corn syrup equivalent. Hematoxylin and eosin and Oil Red O staining were performed to determine steatosis. Biliary mass and cholangiocyte proliferation were evaluated by immunohistochemistry. Senescence and fibrosis were measured by quantitative PCR and immunohistochemistry. Hepatic stellate cell activation was detected by immunofluorescence. Histamine and leptin levels were measured by enzyme immunoassay. Leptin receptor (Ob-R) was evaluated by quantitative PCR. The HDC/histamine/histamine receptor axis, ductular reaction, and biliary senescence were evaluated in patients with nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, or end-stage liver disease. Hdc-/- HFD mice had increased steatosis compared with WT HFD mice. WT HFD mice had increased biliary mass, biliary proliferation, senescence, fibrosis, and hepatic stellate cell activation, which were reduced in Hdc-/- HFD mice. In Hdc-/- HFD mice, serum leptin levels increased, whereas biliary Ob-R expression decreased. Nonalcoholic steatohepatitis patients had increased HDC/histamine/histamine receptor signaling. Hdc-/- HFD mice are susceptible to obesity via dysregulated leptin/Ob-R signaling, whereas the lack of HDC protects from HFD-induced fibrosis and cholangiocyte damage. HDC/histamine/leptin signaling may be important in managing obesity-induced biliary damage.

Ursodeoxycholate inhibits mast cell activation and reverses biliary injury and fibrosis in Mdr2-/- mice and human primary sclerosing cholangitis.

Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. MCs infiltrate Mdr2-/- mice liver (model of primary sclerosing cholangitis (PSC)). MC-derived histamine increases inflammation, hepatic stellate cell (HSC) activation and fibrosis. The objective was to determine the effects of UDCA treatment on MC infiltration, biliary damage, inflammation and fibrosis in Mdr2-/- mice and human PSC. Wild-type and Mdr2-/- mice were fed bile acid control diet or UDCA (0.5% wt/wt). Human samples were collected from control and PSC patients treated with placebo or UDCA (15 mg/kg/BW). MC infiltration was measured by immunhistochemistry and quantitative polymerase chain reaction (qPCR) for c-Kit, chymase, and tryptase. The HDC/histamine/histamine receptor (HR)-axis was evaluated by EIA and qPCR. Intrahepatic bile duct mass (IBDM) and biliary proliferation was evaluated by CK-19 and Ki-67 staining. Fibrosis was detected by immunostaining and qPCR for fibrotic markers. Inflammatory components were measured by qPCR. HSC activation was measured by SYP-9 staining. Inflammation was detected by qPCR for CD68. In vitro, MCs were treated with UDCA (40 µM) prior to HA secretion evaluation and coculturing with cholangiocytes or HSCs. BrDU incorporation and fibrosis by qPCR was performed. UDCA reduced MC number, the HDC/histamine/HR-axis, IBDM, HSC activation, inflammation, and fibrosis in Mdr2-/- mice and PSC patients. In vitro, UDCA decreases MC-histamine release, which was restored by blocking ASBT and FXRß. Proliferation and fibrosis decreased after treatment with UDCA-treated MCs. We conclude that UDCA acts on MCs reducing histamine levels and decreases the inflammatory/hyperplastic/fibrotic reaction seen in PSC. Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. Following liver injury like primary sclerosing cholangitis in mice and humans, MCs infiltrate. MC-derived histamine increases biliary damage, fibrosis, and inflammation. UDCA treatment decreases these parameters via reduced MC activation.

A Pilot Validation Study Comparing Fluorescence-Imitating Brightfield Imaging, A Slide-Free Imaging Method, With Standard Formalin-Fixed, Paraffin-Embedded Hematoxylin-Eosin-Stained Tissue Section Histology for Primary Surgical Pathology Diagnosis.

CONTEXT.­: Digital pathology using whole slide images has been recently approved to support primary diagnosis in clinical surgical pathology practices. Here we describe a novel imaging method, fluorescence-imitating brightfield imaging, that can capture the surface of fresh tissue without requiring prior fixation, paraffin embedding, tissue sectioning, or staining. OBJECTIVE.­: To compare the ability of pathologists to evaluate direct-to-digital images with standard pathology preparations. DESIGN.­: One hundred surgical pathology samples were obtained. Samples were first digitally imaged, then processed for standard histologic examination on 4-µm hematoxylin-eosin-stained sections and digitally scanned. The resulting digital images from both digital and standard scan sets were viewed by each of 4 reading pathologists. The data set consisted of 100 reference diagnoses and 800 study pathologist reads. Each study read was compared to the reference diagnosis, and also compared to that reader's diagnosis across both modalities. RESULTS.­: The overall agreement rate, across 800 reads, was 97.9%. This consisted of 400 digital reads at 97.0% versus reference and 400 standard reads versus reference at 98.8%. Minor discordances (defined as alternative diagnoses without clinical treatment or outcome implications) were 6.1% overall, 7.2% for digital, and 5.0% for standard. CONCLUSIONS.­: Pathologists can provide accurate diagnoses from fluorescence-imitating brightfield imaging slide-free images. Concordance and discordance rates are similar to published rates for comparisons of whole slide imaging to standard light microscopy of glass slides for primary diagnosis. It may be possible, therefore, to develop a slide-free, nondestructive approach for primary pathology diagnosis.

Placental Maternal Vascular Malperfusion is Associated with Prepregnancy and Early Pregnancy Maternal Cardiovascular and Thrombotic Profiles.

Characteristics of maternal vascular malperfusion (MVM) are frequently observed in placentas from pregnancies impacted by preeclampsia, intrauterine growth restriction, preterm labor, and intrauterine fetal demise. We sought to evaluate the associations of features of MVM with subclinical measures of cardiovascular health and coagulation potential in healthy young women. Sixty-three healthy young women were recruited and assessed prior to pregnancy on cycle day 9 ± 4, at gestational age 90 ± 6 of early pregnancy, and gestational age 216 ± 5 of late pregnancy. Women were assessed for plasma volume, blood pressure, response to volume loading, cardiac output, and uterine hemodynamics. Platelet-poor plasma was collected to assess thrombin generation on a subset of 33 women at all time points. Following delivery, placentas were collected and analyzed for evidence of MVM. Thrombin generation (TG) was evaluated in the presence of tissue factor (TF) with and without recombinant soluble thrombomodulin (TM). For each, we compared TG lagtime, peak level, and endogenous thrombin potential (ETP). Comparisons were made between dichotomized presence and absence of each individual feature of MVM and cardiovascular and coagulation features. Mean ± standard deviation are presented. Women were 31 ± 4 years of age, body mass index of 24 ± 5 kg/m2, 86% white race, and 80% nulliparous. MVM occurred in 70% of placentas, with infarcts and agglutination (44%), decidual arteriopathy (40%), accelerated villous maturation (32%), placental hypoplasia (29%), and distal villous hypoplasia (17%) documented. Decidual arteriopathy and distal villous hypoplasia were associated with prepregnancy maternal physiology, including decreased plasma volume and subclinical cardiovascular variations. All assessed MVM characteristics had identifiable early pregnancy physiologic characteristics consistent with altered cardiovascular function and decreased uterine response to pregnancy when compared with women who did and did not develop MVM. Accelerated villous maturation was the only MVM feature to differ by thrombin generation parameters in early pregnancy. Thrombin generation potential and blood pressure were elevated in late pregnancy in women who developed decidual arteriopathy. Prepregnancy health status and adaptation to pregnancy play important roles in pregnancy outcomes. Both cardiovascular health and thrombin generation potential may influence early placentation. Longitudinal assessment of subclinical maternal factors may allow for better understanding of the etiologies of MVM lesions, as well as allow for identification of a timeline of the origins of placental pathologies.

Prolonged Administration of Melatonin Ameliorates Liver Phenotypes in Cholestatic Murine Model.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is characterized by biliary senescence and hepatic fibrosis. Melatonin exerts its effects by interacting with Melatonin receptor 1 and 2 (MT1/MT2) melatonin receptors. Short-term (1 wk) melatonin treatment reduces a ductular reaction and liver fibrosis in bile duct-ligated rats by down-regulation of MT1 and clock genes, and in multidrug resistance gene 2 knockout (Mdr2-/-) mice by decreased miR200b-dependent angiogenesis. We aimed to evaluate the long-term effects of melatonin on liver phenotype that may be mediated by changes in MT1/clock genes/miR200b/maspin/glutathione-S transferase (GST) signaling. METHODS: Male wild-type and Mdr2-/- mice had access to drinking water with/without melatonin for 3 months. Liver damage, biliary proliferation/senescence, liver fibrosis, peribiliary inflammation, and angiogenesis were measured by staining in liver sections, and by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay in liver samples. We confirmed a link between MT1/clock genes/miR200b/maspin/GST/angiogenesis signaling by Ingenuity Pathway Analysis software and measured liver phenotypes and the aforementioned signaling pathway in liver samples from the mouse groups, healthy controls, and PSC patients and immortalized human PSC cholangiocytes. RESULTS: Chronic administration of melatonin to Mdr2-/- mice ameliorates liver phenotypes, which were associated with decreased MT1 and clock gene expression. CONCLUSIONS: Melatonin improves liver histology and restores the circadian rhythm by interaction with MT1 through decreased angiogenesis and increased maspin/GST activity.

Health disparities among tennessee pediatric renal tumor patients.

BACKGROUND/PURPOSE: Wilms tumor (WT) poses a cancer health disparity to black children globally, which has not been evaluated thoroughly for other pediatric renal cancers. We aimed to characterize health disparities among Tennessee children treated for any renal cancer. METHODS: The Tennessee Cancer Registry (TCR) was queried for patients ≤18 years having any renal cancer (n = 160). To clarify treatment and outcomes, we performed a retrospective cohort study of pediatric renal cancer patients in our institutional cancer registry (ICR; n = 121). Diagnoses in both registries included WT, Sarcoma/Other, and Renal Cell Carcinoma. Wilcoxon/Pearson, Kaplan-Meier, and logistic regression were completed. RESULTS: In both registries, WT comprised the most common renal cancer and youngest median age. Sarcoma was intermediate in frequency and age, and RCC was least common, having the oldest age (p < 0.001). In the TCR, black patients comprised 26% of all patients, presented more commonly with distant disease than white patients (37% v. 16%; p = 0.021), and showed worse overall survival (73% v. 89%; p = 0.018), while the ICR showed similar survival between race groups (92% v. 93%, p = 0.868). Sarcoma and metastases were independent predictors of death in both registries (p ≤ 0.002). CONCLUSIONS: Black children in Tennessee presented with more advanced disease and experienced worse survival when combining all renal cancer types, particularly RCC and Sarcoma. When treated at a comprehensive pediatric cancer center, these survival disparities appear diminished. TYPE OF STUDY: Prognostic study. LEVEL OF EVIDENCE: Level II (retrospective cohort).

Pinealectomy or light exposure exacerbates biliary damage and liver fibrosis in cholestatic rats through decreased melatonin synthesis.

Melatonin, a neuroendocrine hormone synthesized by the pineal gland and cholangiocytes, decreases biliary hyperplasia and liver fibrosis during cholestasis-induced biliary injury via melatonin-dependent autocrine signaling through increased biliary arylalkylamine N-acetyltransferase (AANAT) expression and melatonin secretion, downregulation of miR-200b and specific circadian clock genes. Melatonin synthesis is decreased by pinealectomy (PINX) or chronic exposure to light. We evaluated the effect of PINX or prolonged light exposure on melatonin-dependent modulation of biliary damage/ductular reaction/liver fibrosis. Studies were performed in male rats with/without BDL for 1 week with 12:12 h dark/light cycles, continuous light or after 1 week of PINX. The expression of AANAT and melatonin levels in serum and cholangiocyte supernatant were increased in BDL rats, while decreased in BDL rats following PINX or continuous light exposure. BDL-induced increase in serum chemistry, ductular reaction, liver fibrosis, inflammation, angiogenesis and ROS generation were significantly enhanced by PINX or light exposure. Concomitant with enhanced liver fibrosis, we observed increased biliary senescence and enhanced clock genes and miR-200b expression in total liver and cholangiocytes. In vitro, the expression of AANAT, clock genes and miR-200b was increased in PSC human cholangiocyte cell lines (hPSCL). The proliferation and activation of HHStecs (human hepatic stellate cell lines) were increased after stimulating with BDL cholangiocyte supernatant and further enhanced when stimulated with BDL rats following PINX or continuous light exposure cholangiocyte supernatant via intracellular ROS generation. Conclusion: Melatonin plays an important role in the protection of liver against cholestasis-induced damage and ductular reaction.

Knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes in the Mdr2-/- mouse model of primary sclerosing cholangitis (PSC).

BACKGROUND: Cholangiocytes are the target cells of cholangiopathies including primary sclerosing cholangitis (PSC). Vimentin is an intermediate filament protein that has been found in various types of mesenchymal cells. The aim of this study is to evaluate the role of vimentin in the progression of biliary damage/liver fibrosis and whether there is a mesenchymal phenotype of cholangiocytes in the Mdr2-/- model of PSC. METHODS: In vivo studies were performed in 12 wk. Mdr2-/- male mice with or without vimentin Vivo-Morpholino treatment and their corresponding control groups. Liver specimens from human PSC patients, human intrahepatic biliary epithelial cells (HIBEpiC) and human hepatic stellate cell lines (HHSteCs) were used to measure changes in epithelial-to-mesenchymal transition (EMT). FINDINGS: There was increased mesenchymal phenotype of cholangiocytes in Mdr2-/- mice, which was reduced by treatment of vimentin Vivo-Morpholino. Concomitant with reduced vimentin expression, there was decreased liver damage, ductular reaction, biliary senescence, liver fibrosis and TGF-ß1 secretion in Mdr2-/- mice treated with vimentin Vivo-Morpholino. Human PSC patients and derived cell lines had increased expression of vimentin and other mesenchymal markers compared to healthy controls and HIBEpiC, respectively. In vitro silencing of vimentin in HIBEpiC suppressed TGF-ß1-induced EMT and fibrotic reaction. HHSteCs had decreased fibrotic reaction and increased cellular senescence after stimulation with cholangiocyte supernatant with reduced vimentin levels. INTERPRETATION: Our study demonstrated that knockdown of vimentin reduces mesenchymal phenotype of cholangiocytes, which leads to decreased biliary senescence and liver fibrosis. Inhibition of vimentin may be a key therapeutic target in the treatment of cholangiopathies including PSC. FUND: National Institutes of Health (NIH) awards, VA Merit awards.

Significance of green granules in neutrophils and monocytes.

Intracytoplasmic green granules in neutrophils have been the subject of conjecture and discussion. Sometimes nicknamed "death cells," these granules are often associated with severe liver disease and have been said to predict acute bad outcomes in severely ill patients. Some recommend that the laboratory community report these granules to treating clinicians as an indication of poor prognosis. We report two patients presenting with secondary liver disease who were found to have blue-green neutrophilic inclusions on the peripheral blood smear. One patient died, while the other patient recovered. We propose that this morphologic finding is likely transient and is related to liver disease and that recovery is possible. Additionally, prognosis in intensive care units continues to be determined by a number of variables, such as age, comorbidities, and severity of illness.

The management of incidental findings of reduction mammoplasty specimens.

Reduction mammoplasty is one of the most commonly performed procedures in plastic surgery. Occasionally, there are findings reported by pathologists that are unfamiliar to the treating surgeon. The aim of the present study was to determine the types of pathologies encountered in reduction mammoplasty specimens. From this list of diagnoses, a best practice guideline for management will be organized to better assist plastic surgeons in the management of patients with incidental findings on pathology reports. A total of 441 pathology reports from patients who underwent bilateral or unilateral reduction mammoplasty in the past three years were identified. A list of 21 different pathologies was generated from the pathology reports, along with supplemental data from recent texts and articles. Occult carcinomas were encountered in two cases (0.45%) and high-risk lesions were found in three cases (0.68%) at the authors' institution. An algorithm was then constructed to organize the pathologies according to risk of malignancy and assign them to a management guideline. There are many different lesions encountered incidentally in reduction mammoplasty specimens that may or may not confer some cancer risk. It is important for plastic surgeons to know which lesions need closer follow-up to provide the best care for their patients.

Les réductions mammaires font partie des interventions les plus exécutées en chirurgie plastique. Il arrive que le médecin traitant ne soit pas familier avec les observations du pathologiste. La présente étude visait à déterminer le type de pathologies observées dans des prélèvements de réductions mammaires. À partir de cette liste de diagnostics, des directives de pratiques exemplaires de prise en charge sont exposées pour mieux aider le plasticien à prendre en charge les patients présentant des observations fortuites dans le rapport de pathologie. Au total, les chercheurs ont trouvé 441 rapports de pathologie de patients qui ont subi une réduction mammaire bilatérale ou unilatérale depuis trois ans. Ils ont dressé une liste de 21 pathologies différentes à partir des rapports de pathologie, de même que d'autres, tirées de textes et articles récents. Dans l'établissement des auteurs, on a observé deux cas de carcinomes occultes (0,45 %) et trois cas de lésions à haut risque (0,68 %). Un algorithme a ensuite été construit pour classer les pathologies d'après le risque de malignité et leur accoler une directive de prise en charge. Il existe de nombreuses lésions observées fortuitement dans les échantillons de réductions mammaires qui peuvent ou non s'associer à un risque de cancer. Il est important pour le plasticien de savoir quelles lésions doivent faire l'objet d'un suivi plus étroit pour dispenser les meilleurs soins aux patients.