Pathology updates and diagnostic approaches to haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) is a complex, often under-recognised hyperinflammatory immune dysregulation syndrome arising in a diverse range of clinical scenarios and conditions. The accurate and timely diagnosis of HLH is crucial for patient survival, and usually requires a high level of clinical suspicion. The histological corollary to clinical HLH-haemophagocytosis-is neither necessary nor sufficient for the diagnosis of HLH, as it may be seen in a variety of reactive conditions and may be absent in true HLH. Nevertheless, the finding of haemophagocytosis in specific clinical situations should prompt consideration of HLH and further testing to exclude the condition. Although haemophagocytosis is traditionally described in bone marrow, identification of it in other tissues, including lymphoid, splenic, liver or neural tissue, can contribute importantly to the overall recognition of HLH. In this review we discuss the underlying pathophysiology and aetiologies of HLH, and the morphological aspects of haemophagocytosis and its associated histological findings in different tissues, and give a brief overview of diagnostic criteria and clinical evaluation.

Hepatic Stellate Cell-Specific Platelet-Derived Growth Factor Receptor-α Loss Reduces Fibrosis and Promotes Repair after Hepatocellular Injury.

Platelet-derived growth factor receptor (PDGFR)-α plays roles in cell survival, proliferation, and differentiation; however, its function in chronic liver injury sequelae, such as fibrosis, is unknown. Hepatic stellate cells (HSCs), the primary mediators of fibrosis, undergo activation, which entails differentiation to myofibroblasts, proliferation, migration, and collagen deposition, partially in response to PDGFs. To examine the role of PDGFR-α in HSCs, Lrat-Cre recombinase and Pdgfra-floxed mice were bred to generate Lrat-CrePdgfra-/- (knockout) animals, which were subjected to chronic liver injury through carbon tetrachloride treatment, bile duct ligation, and 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Although no major difference was observed after other types of liver injury, PDGFR-α loss in HSCs led to a significant albeit transient reduction in fibrosis after carbon tetrachloride injury, associated with increased HSC death and reduced migration. There was continued alleviation of hepatocellular injury in knockout mice despite ongoing carbon tetrachloride insult, associated with increased numbers of CD68 and F480 macrophages and increased clearance of damaged hepatocytes. Altogether our findings support a profibrotic role of PDGFR-α in HSCs during chronic liver injury in vivo via regulation of HSC survival and migration and affect the immune microenvironment, especially macrophages in clearing dying hepatocytes. Thus, our study provides a preclinical foundation for the future testing of therapeutic PDGFR-α inhibition in hepatic fibrosis, especially in combination with other therapies.

Platelet-Derived Growth Factor Receptor α Contributes to Human Hepatic Stellate Cell Proliferation and Migration.

Platelet-derived growth factor receptor α (PDGFRα), a tyrosine kinase receptor, is up-regulated in hepatic stellate cells (HSCs) during chronic liver injury. HSCs mediate hepatic fibrosis through their activation from a quiescent state partially in response to profibrotic growth factors. HSC activation entails enhanced expression of profibrotic genes, increase in proliferation, and increase in motility, which facilitates migration within the hepatic lobule. We show colocalization of PDGFRα in murine carbon tetrachloride, bile duct ligation, and 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine models of chronic liver injury, and investigate the role of PDGFRα on proliferation, profibrotic gene expression, and migration in primary human HSCs (HHSteCs) using the PDGFRα-specific inhibitory monoclonal antibody olaratumab. Although lacking any effects on HHSteC transdifferentiation assessed by gene expression of ACTA2, TGFB1, COL1A1, SYP1, and FN1, olaratumab specifically reduced HHSteC proliferation (AlamarBlue assay) and cell migration (transwell migration assays). Using phospho-specific antibodies, we show that olaratumab attenuates PDGFRα activation in response to PDGF-BB, and reduced phosphorylation of extracellular signal-regulated kinase 1 and 2, Elk-1, p38, Akt, focal adhesion kinase, mechanistic target of rapamycin, C10 regulator of kinase II, and C10 regulator of kinase-like, suggesting that PDGFRα contributes to mitogenesis and actin reorganization through diverse downstream effectors. Our findings support a distinct contribution of PDGFRα signaling to HSC proliferation and migration and provide evidence that inhibition of PDGFRα signaling could alter the pathogenesis of hepatic fibrosis.

PDGFRα in liver pathophysiology: emerging roles in development, regeneration, fibrosis, and cancer.

Platelet-derived growth factor receptor α (PDGFRα) is an isoform of the PDGFR family of tyrosine kinase receptors involved in cell proliferation, survival, differentiation, and growth. In this review, we highlight the role of PDGFRα and the current evidence of its expression and activities in liver development, regeneration, and pathology-including fibrosis, cirrhosis, and liver cancer. Studies elucidating PDGFRα signaling in processes ranging from profibrotic signaling, angiogenesis, and oxidative stress to epithelial-to-mesenchymal transition point toward PDGFRα as a potential therapeutic target in various hepatic pathologies, including hepatic fibrosis and liver cancer. Furthermore, PDGFRα localization and modulation during liver development and regeneration may lend insight into its potential roles in various pathologic states. We will also briefly discuss some of the current targeted treatments for PDGFRα, including multi receptor tyrosine kinase inhibitors and PDGFRα-specific inhibitors.

Beta-catenin-NF-κB interactions in murine hepatocytes: a complex to die for.

UNLABELLED: Wnt/ß-catenin signaling plays an important role in hepatic homeostasis, especially in liver development, regeneration, and cancer, and loss of ß-catenin signaling is often associated with increased apoptosis. To elucidate how ß-catenin may be regulating hepatocyte survival, we investigated the susceptibility of ß-catenin conditional knockout (KO) mice and their wild-type (WT) littermates to Fas and tumor necrosis factor-α (TNF-α), two common pathways of hepatocyte apoptosis. While comparable detrimental effects from Fas activation were observed in WT and KO, a paradoxical survival benefit was observed in KO mice challenged with D-galactosamine/lipopolysaccharide. KO mice showed significantly lower morbidity and liver injury due to early, robust, and protracted activation of NF-κB in the absence of ß-catenin. Enhanced NF-κB activation in KO mice was associated with increased basal inflammation and Toll-like receptor 4 expression and lack of the p65/ß-catenin complex in hepatocytes. The p65/ß-catenin complex in WT livers underwent temporal dissociation allowing for NF-κB activation to regulate hepatocyte survival following TNF-α-induced hepatic injury. Decrease of total ß-catenin protein but not its inactivation induced p65 activity, whereas ß-catenin stabilization either chemically or due to mutations repressed it in hepatomas in a dose-dependent manner, whereas ß-catenin stabilization repressed it either chemically or due to mutations. CONCLUSION: The p65/ß-catenin complex in hepatocytes undergoes dynamic changes during TNF-α-induced hepatic injury and plays a critical role in NF-κB activation and cell survival. Modulation of ß-catenin levels is a unique mode of regulating NF-κB activity and thus may present novel opportunities in devising therapeutics in specific hepatic injuries.

Assessment of large droplet fat in frozen sections of donor liver biopsies: utility and interobserver variability of the newly described Banff method compared to a simplified Average of Fields method.

AIMS: There is great variability in the assessment and reporting of fat in frozen sections of donor liver biopsies. The Banff Working Group has proposed a novel method and definition for scoring large droplet fat (LDF) in donor liver biopsies. This study compares the Banff method with a simpler Average of Fields (AF) method and evaluates the impact of different LDF definitions. METHODS: Three pathologists assessed percentage of LDF (LDF%) in 10 donor liver biopsies using Banff and AF methods, applying the Banff LDF definition (cell distention with a single droplet larger than adjacent hepatocytes). Additionally, LDF% by the AF method was compared using two LDF definitions: Banff definition versus LDF definition 2 (single fat droplet occupying greater than half of a hepatocyte with nuclear displacement). RESULTS: Intraobserver concordance between the Banff and AF methods was similar for all three pathologists (kappa 0.76-1). Both methods exhibited 70% interobserver concordance, and there was substantial agreement (kappa 0.68) in the LDF% among the three pathologists for both methods. Comparing the two LDF definitions, results were significantly lower with the Banff definition; LDF >50% was observed in four cases with LDF definition 2 but none of the cases with the Banff definition. CONCLUSIONS: There is high interobserver and intraobserver concordance of LDF% between the Banff and AF methods. LDF% determined by the Banff definition was lower than with LDF definition 2, and needs to be validated based on graft outcome before it can be recommended for clinical use.

Genomic Analysis in the Categorization of Poorly Differentiated Primary Liver Carcinomas.

A subset of primary liver carcinomas (PLCs) cannot be classified as hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (iCCA) based on morphology and immunohistochemistry (IHC). This includes tumors with morphology suggestive of HCC but lacking hepatocellular marker expression, tumors with ambiguous morphology characterized by co-expression of hepatocellular and cholangiocytic markers, and undifferentiated pleomorphic carcinomas with no discernible line of differentiation on morphology or IHC. This study examines the role of genomic analysis in the categorization of these tumors. Genomic analysis was performed on 16 PLCs that could not be definitely classified as HCC or iCCA based on morphology and IHC using a capture-based next-generation sequencing assay (n=15) or single gene mutational analysis (n=1). Genomic alterations in TERT promoter were seen in 9/16 cases (56%) and strongly favored HCC. Genomic alterations favoring iCCA were seen in 5/16 cases (31%) and included mutations in IDH1 , PBRM1 , BAP1 , and ERBB2 , as well as FGFR2 fusion. Genomic changes were helpful in classifying 14/16 (87%) PLCs. Though not specific, these genomic alterations can provide valuable diagnostic clues in selected morphologically and immunohistochemically unclassifiable cases. Given the important differences in management between HCC and iCCA, routine use of genomic analysis in diagnostically challenging settings should be considered.

Partial pneumonectomy of telomerase null mice carrying shortened telomeres initiates cell growth arrest resulting in a limited compensatory growth response.

Telomerase mutations and significantly shortened chromosomal telomeres have recently been implicated in human lung pathologies. Natural telomere shortening is an inevitable consequence of aging, which is also a risk factor for development of lung disease. However, the impact of shortened telomeres and telomerase dysfunction on the ability of lung cells to respond to significant challenge is still largely unknown. We have previously shown that lungs of late generation, telomerase null B6.Cg-Terc(tm1Rdp) mice feature alveolar simplification and chronic stress signaling at baseline, a phenocopy of aged lung. To determine the role telomerase plays when the lung is challenged, B6.Cg-Terc(tm1Rdp) mice carrying shortened telomeres and wild-type controls were subjected to partial pneumonectomy. We found that telomerase activity was strongly induced in alveolar epithelial type 2 cells (AEC2) of the remaining lung immediately following surgery. Eighty-six percent of wild-type animals survived the procedure and exhibited a burst of early compensatory growth marked by upregulation of proliferation, stress response, and DNA repair pathways in AEC2. In B6.Cg-Terc(tm1Rdp) mice carrying shortened telomeres, response to pneumonectomy was characterized by decreased survival, diminished compensatory lung growth, attenuated distal lung progenitor cell response, persistent DNA damage, and cell growth arrest. Overall, survival correlated strongly with telomere length. We conclude that functional telomerase and properly maintained telomeres play key roles in both long-term survival and the early phase of compensatory lung growth following partial pneumonectomy.

Elimination of Wnt Secretion From Stellate Cells Is Dispensable for Zonation and Development of Liver Fibrosis Following Hepatobiliary Injury.

Alterations in the Wnt signaling pathway including those impacting hepatic stellate cells (HSCs) have been implicated in liver fibrosis. In the current study, we first examined the expression of Wnt genes in human HSC (HHSCs) after treatment with a profibrogenic factor TGF-ß1. Next, we generated HSC-specific Wntless (Wls) knockout (KO) using the Lrat-cre and Wls-floxed mice. KO and littermate controls (CON) were characterized for any basal phenotype and subjected to two liver fibrosis protocols. In vitro, TGF-ß1 induced expression of Wnt2, 5a and 9a while decreasing Wnt2b, 3a, 4, and 11 in HHSC. In vivo, KO and CON mice were born at normal Mendelian ratio and lacked any overt phenotype. Loss of Wnt secretion from HSCs had no effect on liver weight and did not impact ß-catenin activation in the pericentral hepatocytes. After 7 days of bile duct ligation (BDL), KO and CON showed comparable levels of serum alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total and direct bilirubin. Comparable histology, Sirius red staining, and immunohistochemistry for α-SMA, desmin, Ki-67, F4/80, and CD45 indicated similar proliferation, inflammation, and portal fibrosis in both groups. Biweekly administration of carbon tetrachloride for 4 or 8 weeks also led to comparable serum biochemistry, inflammation, and fibrosis in KO and CON. Specific Wnt genes were altered in HHSCs in response to TGF-ß1; however, eliminating Wnt secretion from HSC did not impact basal ß-catenin activation in normal liver nor did it alter the injury-repair response during development of liver fibrosis.

Phosphatase-dependent regulation of epithelial mitogen-activated protein kinase responses to toxin-induced membrane pores.

Diverse bacterial species produce pore-forming toxins (PFT) that can puncture eukaryotic cell membranes. Host cells respond to sublytic concentrations of PFT through conserved intracellular signaling pathways, including activation of mitogen-activated protein kinases (MAPK), which are critical to cell survival. Here we demonstrate that in respiratory epithelial cells p38 and JNK MAPK were phosphorylated within 30 min of exposure to pneumolysin, the PFT from Streptococcus pneumoniae. This activation was tightly regulated, and dephosphorylation of both MAPK occurred within 60 min following exposure. Pretreatment of epithelial cells with inhibitors of cellular phosphatases, including sodium orthovanadate, calyculin A, and okadaic acid, prolonged and intensified MAPK activation. Specific inhibition of MAPK phosphatase-1 did not affect the kinetics of MAPK activation in PFT-exposed epithelial cells, but siRNA-mediated knockdown of serine/threonine phosphatases PP1 and PP2A were potent inhibitors of MAPK dephosphorylation. These results indicate an important role for PP1 and PP2A in termination of epithelial responses to PFT and only a minor contribution of dual-specificity phosphatases, such as MAPK phosphatase-1, which are the major regulators of MAPK signals in other cell types. Epithelial regulation of MAPK signaling in response to membrane disruption involves distinct pathways and may require different strategies for therapeutic interventions.