Evaluation of region selective bilirubin-induced brain damage as a basis for a pharmacological treatment

The neurologic manifestations of neonatal hyperbilirubinemia in the central nervous system (CNS) exhibit high variations in the severity and appearance of motor, auditory and cognitive symptoms, which is suggestive of a still unexplained selective topography of bilirubin-induced damage. By applying the organotypic brain culture (OBC: preserving in vitro the cellular complexity, connection and architecture of the in vivo brain) technique to study hyperbilirubinemia, we mapped the regional target of bilirubin-induced damage, demonstrated a multifactorial toxic action of bilirubin, and used this information to evaluate the efficacy of drugs applicable to newborns to protect the brain. OBCs from 8-day-old rat pups showed a 2-13 fold higher sensitivity to bilirubin damage than 2-day-old preparations. The hippocampus, inferior colliculus and cerebral cortex were the only brain regions affected, presenting a mixed inflammatory-oxidative mechanism. Glutamate excitotoxicity was appreciable in only the hippocampus and inferior colliculus. Single drug treatment (indomethacin, curcumin, MgCl2) significantly improved cell viability in all regions, while the combined (cocktail) administration of the three drugs almost completely prevented damage in the most affected area (hippocampus). Our data may supports an innovative (complementary to phototherapy) approach for directly protecting the newborn brain from bilirubin neurotoxicity

Current Status of Fibroblast Growth Factor Receptor-Targeted Therapies in Breast Cancer

Breast cancer (BC) is the most common malignancy and second only to lung cancer in terms of mortality in women. Despite the incredible progress made in this field, metastatic breast cancer has a poor prognosis. In an era of personalized medicine, there is an urgent need for better knowledge of the biology leading to the disease, which can lead to the design of increasingly accurate drugs against patients’ specific molecular aberrations. Among one of the actionable targets is the fibroblast growth factor receptor (FGFR) pathway, triggered by specific ligands. The Fibroblast Growth Factor Receptors/Fibroblast Growth Factors (FGFRs/FGFs) axis offers interesting molecular targets to be pursued in clinical development. This mini-review will focus on the current knowledge of FGFR mutations, which lead to tumor formation and summarizes the state-of-the-art therapeutic strategies for targeted treatments against the FGFRs/FGFs axis in the context of BC

In Vitro Study of Mutagenesis Induced by Crocidolite-Exposed Alveolar Macrophages NR8383 in Cocultured Big Blue Rat2 Embryonic Fibroblasts

Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages. With the aim of proposing an alternative in vitro mutagenesis test, a coculture system of rat alveolar macrophages (NR8383) and transgenic Big Blue Rat2 embryonic fibroblasts was developed and tested with a crocidolite sample. Crocidolite exposure induced no detectable increase in ROS production from NR8383, contrasting with the oxidative burst that occurred following a brief exposure (1 hour) to zymosan, a known macrophage activator. In separated cocultures, crocidolite and zymosan induced different changes in the gene expressions involved in cellular inflammation in NR8383 and Big Blue. In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species. However, crocidolite exposure in separated or mixed cocultures induced no mutagenic effects whereas an increase in Big Blue mutants was detected after exposure to zymosan in mixed cocultures. NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events. The mutagenesis assay based on the coculture of NR8383 and Big Blue cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres

Synchrotron soft X-ray imaging and fluorescence microscopy reveal novel features of asbestos body morphology and composition in human lung tissues

Background: Occupational or environmental exposure to asbestos fibres is associated with pleural and parenchymal lung diseases. A histopathologic hallmark of exposure to asbestos is the presence in lung parenchyma of the so-called asbestos bodies. They are the final product of biomineralization processes resulting in deposition of endogenous iron and organic matter (mainly proteins) around the inhaled asbestos fibres. For shedding light on the formation mechanisms of asbestos bodies it is of fundamental importance to characterize at the same length scales not only their structural morphology and chemical composition but also to correlate them to the possible alterations in the local composition of the surrounding tissues. Here we report the first correlative morphological and chemical characterization of untreated paraffinated histological lung tissue samples with asbestos bodies by means of soft X-ray imaging and X-Ray Fluorescence (XRF) microscopy, which reveals new features in the elemental lateral distribution. Results: The X-ray absorption and phase contrast images and the simultaneously monitored XRF maps of tissue samples have revealed the location, distribution and elemental composition of asbestos bodies and associated nanometric structures. The observed specific morphology and differences in the local Si, Fe, O and Mg content provide distinct fingerprints characteristic for the core asbestos fibre and the ferruginous body. The highest Si content is found in the asbestos fibre, while the shell and ferruginous bodies are characterized by strongly increased content of Mg, Fe and O compared to the adjacent tissue. The XRF and SEM-EDX analyses of the extracted asbestos bodies confirmed an enhanced Mg deposition in the organic asbestos coating. Conclusions: The present report demonstrates the potential of the advanced synchrotron-based X-ray imaging and microspectroscopy techniques for studying the response of the lung tissue to the presence of asbestos fibres. The new results obtained by simultaneous structural and chemical analysis of tissue specimen have provided clear evidence that Mg, in addition to Fe, is also involved in the formation mechanisms of asbestos bodies. This is the first important step to further thorough investigations that will shed light on the physiopathological role of Mg in tissue response to the asbestos toxicity

A Case–Control Study by ddPCR of ALU 260/111 and LINE-1 266/97 Copy Number Ratio in Circulating Cell-Free DNA in Plasma Revealed LINE-1 266/97 as a Potential Biomarker for Early Breast Cancer Detection

Background: In Western countries, breast cancer (BC) is the most common cancer in women. Early detection has a positive impact on survival, quality of life, and public health costs. Mammography screening programs have increased early detection rates, but new approaches to more personalized surveillance could further improve diagnosis. Circulating cell-free DNA (cfDNA) in blood could provide a potential tool for early diagnosis by analyzing cfDNA quantity, circulating tumor DNA mutations, or cfDNA integrity (cfDI). Methods: Plasma was obtained from the blood of 106 breast cancer patients (cases) and 103 healthy women (controls). Digital droplet PCR was used for the determination of ALU 260/111 bp and LINE-1 266/97 bp copy number ratio and cfDI. cfDNA abundance was calculated using copies of the EEF1A2 gene. The accuracy of biomarker discrimination was analyzed with receiver operating characteristic curve (ROC). Sensitivity analyses were performed to account for age as a potential confounder. Results: Cases had significantly lower ALU 260/111 or LINE-1 266/97 copy number ratios (median; ALU 260/111 = 0.08, LINE-1 266/97 = 0.20), compared with control (median; ALU 260/111 = 0.10, LINE-1 266/97 = 0.28) (p < 0.001). ROC analysis showed that copy number ratio discriminated cases from controls (area under the curve, AUC = 0.69, 95% CI: 0.62–0.76 for ALU and 0.80, 95% CI: 0.73–0.86 for LINE-1). ROC from cfDI confirmed the better diagnostic performance of LINE-1 compared with ALU. Conclusions: Analysis of LINE-1 266/97 copy number ratio or cfDI by ddPCR appears to be a useful noninvasive test that could aid in early BC detection. Further studies in a large cohort are needed to validate the biomarker

A vivens ex vivo study on the synergistic effect of electrolysis and freezing on the cell nucleus

Freezing-cryosurgery, and electrolysis-electrochemical therapy (EChT), are two important minimally invasive surgery tissue ablation technologies. Despite major advantages they also have some disadvantages. Cryosurgery cannot induce cell death at high subzero freezing temperatures and requires multiple freeze thaw cycles, while EChT requires high concentrations of electrolytic products-which makes it a lengthy procedure. Based on the observation that freezing increases the concentration of solutes (including products of electrolysis) in the frozen region and permeabilizes the cell membrane to these products, this study examines the hypothesis that there could be a synergistic effect between freezing and electrolysis in their use together for tissue ablation. Using an animal model we refer to as vivens ex vivo, which may be of value in reducing the use of animals for experiments, combined with a Hematoxylin stain of the nucleus, we show that there are clinically relevant protocols in which the cell nucleus appears intact when electrolysis and freezing are used separately but is affected by certain combinations of electrolysis and freezing. \ua9 2015 Lugnani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Tumour infiltrating lymphocytes and immune-related genes as predictors of outcome in pancreatic adenocarcinoma

Background: We investigated the correlation between pancreatic ductal adenocarcinoma patient progno- sis and the presence of tumour infiltrating lymphocytes and expression of 521 immune sys- tem genes. Methods: Intratumoural CD3+, CD8+, and CD20+ lymphocytes were examined by immunohistochem- istry in 12 PDAC patients with different outcomes who underwent pancreaticoduodenect- omy. The results were correlated with gene expression profile using the digital multiplexed NanoString nCounter analysis system (NanoString Technologies, Seattle, WA, USA). Results: Twenty immune system genes were significantly differentially expressed in patients with a good prognosis relative to patients with a worse prognosis: TLR2 and TLR7 (Toll-like recep- tor superfamily); CD4, CD37, FOXP3, PTPRC (B cell and T cell signalling); IRF5, IRF8, STAT1, TFE3 (transcription factors); ANP32B, CCND3 (cell cycle); BTK (B cell develop- ment); TNF, TNFRF1A (TNF superfamily); HCK (leukocyte function); C1QA (complement system); BAX, PNMA1 (apoptosis); IKBKE (NF\u3baB pathway). Differential expression was more than twice log 2 for TLR7, TNF, C1QA, FOXP3, and CD37. Discussion: Tumour infiltrating lymphocytes were present at higher levels in samples from patients with better prognosis. Our findings indicate that tumour infiltrating lymphocyte levels and expres- sion level of the immune system genes listed above influence pancreatic ductal adenocarcinoma prognosis. This information could be used to improve selection of best responders to immune inhibitors

Epithelioid Mesothelioma Patients with Very Long Survival Display Defects in DNA Repair

Aim: DNA repair has an important role in malignant pleural mesothelioma (MPM) tumorigenesis and progression. Prognostic/predictive biomarkers for better management of MPM patients are needed. In the present manuscript, we analyzed the expression of more than 700 genes in a cohort of MPM patients to possibly find biomarkers correlated with survival. Methods: A total of 54 MPM patients, all with epithelioid histology, whose survival follow-up and formalin-fixed paraffin-embedded tumors were available, were included in the study. Gene expression profiles were evaluated using a Nanostring platform analyzing 760 genes involved in different cellular pathways. The percentages of proliferating tumor cells positive for RAD51 and BRCA1 foci were evaluated using an immunofluorescence assay, as a readout of homologous recombination repair status. Results: Patient median survival time was 16.9 months, and based on this value, they were classified as long and short survivors (LS/SS) with, respectively, an overall survival ≥ and <16.9 months as well as very long and very short survivors (VLS/VSS) with an overall survival ≥ than 33.8 and < than 8.45 months. A down-regulation in the DNA damage/repair expression score was observed in LS and VLS as compared to SS and VSS. These findings were validated by the lower number of both RAD51 and BRCA1-positive tumor cells in VLS as compared to VSS. Conclusions: The down-regulation of DNA repair signature in VLS was functionally validated by a lower % of RAD51 and BRCA1-positive tumor cells. If these data can be corroborated in a prospective trial, an easy, cost-effective test could be routinely used to better manage treatment in MPM patients

Mast Cells in Peritoneal Fluid From Women With Endometriosis and Their Possible Role in Modulating Sperm Function

Endometriosis is a local pelvic inflammatory process, frequently associated with infertility, with altered function of immune-related cells in the peritoneal environment. Mast cells are known to be key players of the immune system and have been recently involved in endometriosis and in infertility, with their mediators directly suppressing sperm motility. In this study, we evaluated the mast cell population and their mediators in the peritoneal fluid of infertile patients with endometriosis and their impact on human sperm motility. Peritoneal fluids, collected by laparoscopy from 11 infertile patients with endometriosis and 9 fertile controls were evaluated for the presence of mast cells, tryptase levels and their effect on sperm motility. Furthermore, an in vitro model of mast cells-sperm interaction in peritoneal fluid was set up, using LAD2 cell line as a mast cell model, and analyzed from a functional as well as a morphological point of view. Mast cell peritoneal fluid population and its main mediator, tryptase, is more represented in endometriosis confirming an involvement of these cells in this disease. Anyway it appears unlikely that tryptase enriched peritoneal fluid, which fails to inhibit sperm motility, could contribute to endometriosis associated infertility. Despite of this, sperm interaction with the mast cell surface (LAD2) induced a significantly mast cell-degranulation response in the peritoneal fluid from endometriosis which could directly modulate sperm function other than motility. This evidence lead us to suppose that there is, between these elements, an interrelationship which deserves further studies

CD99 Expression and Prognostic Impact in Glioblastoma: A Single-Center Cohort Study

Glioblastoma is the most frequent and aggressive brain tumor in adults. This study aims to evaluate the expression and prognostic impact of CD99, a membrane glycoprotein involved in cellular migration and invasion. In a cohort of patients with glioblastoma treated with surgery, radiotherapy and temozolomide, we retrospectively analyzed tumor expression of CD99 by immunohistochemistry (IHC) and by quantitative real-time polymerase chain reaction (qRT-PCR) for both the wild type (CD99wt) and the truncated (CD99sh) isoforms. The impact on overall survival (OS) was assessed with the Kaplan-Meier method and log-rank test and by multivariable Cox regression. Forty-six patients with glioblastoma entered this study. Immunohistochemical expression of CD99 was present in 83%. Only the CD99wt isoform was detected by qRT-PCR and was significantly correlated with CD99 expression evaluated by IHC (rho = 0.309, p = 0.037). CD99 expression was not associated with OS, regardless of the assessment methodology used (p = 0.61 for qRT-PCR and p = 0.73 for IHC). In an exploratory analysis of The Cancer Genome Atlas, casuistry of glioblastomas CD99 expression was not associated with OS nor with progression-free survival. This study confirms a high expression of CD99 in glioblastoma but does not show any significant impact on survival. Further preclinical studies are needed to define its role as a therapeutic target in glioblastoma