Upregulated phospholipase D2 expression and activity is related to the metastatic properties of melanoma

The incidence rates of melanoma have increased steadily in recent decades and nearly 25% of the patients diagnosed with early-stage melanoma will eventually develop metastasis, for which there is currently no fully effective treatment. The link between phospholipases and tumors has been studied extensively, particularly in breast and colon cancers. With the aim of finding new biomarkers and therapeutic options for melanoma, the expression of different phospholipases was assessed in 17 distinct cell lines in the present study, demonstrating that phospholipase D2 (PLD2) is upregulated in metastatic melanoma as compared to normal skin melanocytes. These results were corroborated by immunofluorescence and lipase activity assays. Upregulation of PLD2 expression and increased lipase activity were observed in metastatic melanoma relative to normal skin melanocytes. So far, the implication of PLD2 activity in melanoma malignancies has remained elusive. To the best of our knowledge, the present study was the first to demonstrate that the overexpression of PLD2 enhances lipase activity, and its effect to increase the proliferation, migration and invasion capacity of melanoma cells was assessed with XTT and Transwell assays. In addition, silencing of PLD2 in melanoma cells reduced the metastatic potential of these cells. The present study provided evidence that PLD2 is involved in melanoma malignancy and in particular, in its metastatic potential, and established a basis for future studies evaluating PLD2 blockade as a therapeutic strategy to manage this condition.     

Redox activity of airborne particulate matter at different sites in the Los Angeles Basin

Epidemiologic studies have shown associations between ambient particulate matter (PM) and adverse health outcomes including increased mortality, emergency room visits, and time lost from school and work. The mechanisms of PM-related health effects are still incompletely understood, but a hypothesis under investigation is that many of the adverse health effects may derive from oxidative stress, initiated by the formation of reactive oxygen species (ROS) within affected cells. While the adverse effects from PM have historically been associated with the airborne concentration of PM and more recently fine-particle PM, we considered it relevant to develop an assay to quantitatively measure the ability of PM to catalyze ROS generation as the initial step in the induction of oxidative stress. This ability of PM could then be related to different sources, chemical composition, and physical and spatial/temporal characteristics in the ambient environment. The measurement of ROS-forming ability in relation to sources and other factors will have potential relevance to control of redox-active PM. If oxidative stress represents a relevant mechanism of toxicity from PM, the measurement of redox activity represents a first step in the elucidation of the subsequent downstream processes. We have developed an assay for PM redox activity, utilizing the reduction of oxygen by dithiothreitol which serves as an electron source. We have found that PM will catalyze the reduction of oxygen and have examined the distribution and chemical characteristics of the redox activity of PM fractions collected in different sites in the Los Angeles Basin. Samples of concentrated coarse, fine, and ultrafine PM, obtained with aerosol concentrators, were studied with regard to their chemical properties and redox activity. Redox activity was highest in the ultrafine fraction, in agreement with results indicating ultrafines were the most potent toward inducing that heme oxygenase expression and depleting intracellular glutathione, which has relevance to induction of oxidative stress. Comparison of the redox activity with chemical composition showed a reasonable correlation of redox activity with elemental carbon (r(2)=0.79), organic carbon (r(2)=0.53), and with benzo[ghi]perylene (r(2)=0.82), consistent with species typically found in mobile emission sources.     

Donor‐specific anti‐HLA antibodies are not associated with nonanastomotic biliary strictures but both are independent risk factors for graft loss after liver transplantation

Donor‐specific alloantibodies (DSA) have been associated with rejection and shorter graft survival after orthotopic liver transplantation (OLT). We examined the role of DSA in nonanastomotic biliary strictures (NAS) after OLT. Patients receiving first OLT who developed NAS (n = 68) and a control group without NAS (n = 83), with pre‐OLT and 12 months post‐OLT serum samples, were included. DSA were specified using the Luminex single antigen test. Risk factors for NAS and graft survival were analyzed. The presence of preformed DSA was not significantly different between patients with NAS and controls (P = .89). After 12 months, 26.5% of NAS patients and 16.9% of controls had generated de novo DSA (P = .15). Neither de novo class I DSA nor de novo class II DSA were associated with NAS. De novo DSA generally developed after the diagnosis of NAS. Time‐dependent regression analysis identified both NAS (aHR 8.05, CI 3.28 – 19.77, P < .01) and de novo class II DSA (aHR 2.84, CI 1.38 – 5.82, P < .01) as independent risk factors for graft loss. Preformed or de novo DSA were not associated with the development of NAS. However, NAS as well as de novo class II DSA were independent risk factors for graft loss after OLT.     

CRIPTO promotes an aggressive tumour phenotype and resistance to treatment in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre‐loxP‐controlled lentiviral vectors expressing CRIPTO in cell line‐derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft‐derived ex vivo tumour slices. CRIPTO‐overexpressing patient‐derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial‐to‐mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2‐CRIPTO cells formed tumours when injected into immune‐compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High‐level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO‐expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Estradiol Is a Critical Mediator of Macrophage-Nerve Cross Talk in Peritoneal Endometriosis

Endometriosis occurs in approximately 10% of women and is associated with persistent pelvic pain. It is defined by the presence of endometrial tissue (lesions) outside the uterus, most commonly on the peritoneum. Peripheral neuroinflammation, a process characterized by the infiltration of nerve fibers and macrophages into lesions, plays a pivotal role in endometriosis-associated pain. Our objective was to determine the role of estradiol (E2) in regulating the interaction between macrophages and nerves in peritoneal endometriosis. By using human tissues and a mouse model of endometriosis, we demonstrate that macrophages in lesions recovered from women and mice are immunopositive for estrogen receptor β, with up to 20% being estrogen receptor α positive. In mice, treatment with E2 increased the number of macrophages in lesions as well as concentrations of mRNAs encoded by Csf1, Nt3, and the tyrosine kinase neurotrophin receptor, TrkB. By using in vitro models, we determined that the treatment of rat dorsal root ganglia neurons with E2 increased mRNA concentrations of the chemokine C-C motif ligand 2 that stimulated migration of colony-stimulating factor 1–differentiated macrophages. Conversely, incubation of colony-stimulating factor 1 macrophages with E2 increased concentrations of brain-derived neurotrophic factor and neurotrophin 3, which stimulated neurite outgrowth from ganglia explants. In summary, we demonstrate a key role for E2 in stimulating macrophage-nerve interactions, providing novel evidence that endometriosis is an estrogen-dependent neuroinflammatory disorder.Endometriosis affects 10% of reproductive age women and is associated with persistent pelvic pain.¹ It is defined by the presence of endometrial-like tissue (lesions) found outside the uterus, most commonly on the peritoneum. The mechanisms underlying endometriosis-associated pain are poorly understood, but it has been postulated that estrogen-dependent neuroinflammation may be involved.² Notably, the presence of endometrial tissue fragments on the peritoneum elicits an immune response, including recruitment of macrophages,³ blood vessels, and nerve fibers into the resultant lesions.^4,5 Within the lesions, CD68⁺ macrophages have been detected in close association with nerve fibers.⁶Studies investigating macrophage activation and recruitment have revealed that endometriosis-associated macrophages exhibit a phenotype consistent with the alternative end of the macrophage activation spectrum.^7,8 In a mouse model of endometriosis that included cell transfer of polarized macrophages, Bacci et al⁷ reported that mice injected with proinflammatory macrophages [macrophage (interferon γ)] developed microscopic lesions, but those injected with alternatively activated macrophages [macrophage (IL-4)] developed larger lesions with a well-developed vasculature. Our studies in a mouse model of endometriosis have revealed that macrophages resident in peritoneal lesions can originate from both the peritoneum and the endometrium.⁹Sensory C, sensory Aδ, cholinergic, and adrenergic nerve fibers have been identified within lesions,^10,11 with greater nerve fiber density in areas that exhibit high macrophage density.⁶ Studies in zebrafish have shown that macrophages will migrate toward damaged peripheral nerves,¹² consistent with a role for neuron-derived factors in immune-nerve cross talk.Endometriosis lesions have an estrogen-rich microenvironment associated with enhanced expression of biosynthetic enzymes, including aromatase.¹³ It is well established that estrogen action can be mediated by estrogen receptors α (ERα) and β (ERβ), both widely expressed are the human endometrium.¹⁴ Notably, a proportion of the soma of afferent nerve fibers innervating the uterus and peritoneum is reported to express one or both ERs.¹⁵ Although some studies have analyzed the expression of ERs in macrophages isolated from the peritoneal fluid of women with endometriosis,^16,17 expression of ERs in lesion-resident macrophages has not yet been determined.Our objective was to determine whether estradiol (E2) plays a role in the regulation of macrophage-nerve cross talk in endometriosis by exploring both the expression of ERs in human tissue samples and the impact of E2 on nerves and macrophages using in vitro and in vivo models.