This study established long‐term self‐renewing prostate cancer stem cell (PCSC) lines from the different stages of transgenic adenocarcinoma of the mouse prostate (TRAMP) progression by application ...of the neurosphere assay. It was found that TRAMP‐derived PCSCs represent a novel and valuable preclinical model for elucidating the pathogenetic mechanisms leading to prostate adenocarcinoma and for the identification of molecular mediators to be pursued as therapeutic targets.
The relevant social and economic impact of prostate adenocarcinoma, one of the leading causes of death in men, urges critical improvements in knowledge of the pathogenesis and cure of this disease. These can also be achieved by implementing in vitro and in vivo preclinical models by taking advantage of prostate cancer stem cells (PCSCs). The best‐characterized mouse model of prostate cancer is the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. TRAMP mice develop a progressive lesion called prostatic intraepithelial neoplasia that evolves into adenocarcinoma (AD) between 24 and 30 weeks of age. ADs often metastasize to lymph nodes, lung, bones, and kidneys. Eventually, approximately 5% of the mice develop an androgen‐independent neuroendocrine adenocarcinoma. Here we report the establishment of long‐term self‐renewing PCSC lines from the different stages of TRAMP progression by application of the neurosphere assay. Stage‐specific prostate cell lines were endowed with the critical features expected from malignant bona fide cancer stem cells, namely, self‐renewal, multipotency, and tumorigenicity. Notably, transcriptome analysis of stage‐specific PCSCs resulted in the generation of well‐defined, meaningful gene signatures, which identify distinct stages of human tumor progression. As such, TRAMP‐derived PCSCs represent a novel and valuable preclinical model for elucidating the pathogenetic mechanisms leading to prostate adenocarcinoma and for the identification of molecular mediators to be pursued as therapeutic targets.
Genetic analysis of Neurofibromatosis type 1 (NF1) may facilitate the identification of patients in early phases of the disease. Here, we present an overview of our diagnostic research spanning the ...last 11 years, with a focus on the description of 225 NF1 mutations, 126 of which are novel, found in a series of 607 patients (513 unrelated) in Italy. Between 2003 and 2013, 443 unrelated patients were profiled by denaturing high pressure liquid chromatography (DHPLC) analysis of 60 amplicons derived from genomic NF1 DNA and subsequent sequencing of heterozygotic PCR products. In addition, a subset of patients was studied by multiplex ligation‐dependent probe amplification (MLPA) to identify any duplications, large deletions or microdeletions present at the locus. Over the last year, 70 unrelated patients were investigated by MLPA and sequencing of 22 amplicons spanning the entire NF1 cDNA. Mutations were found in 70% of the 293 patients studied by DHPLC, thereby fulfilling the NIH criterion for the clinical diagnosis of NF1 (detection rate: 70%); furthermore, 87% of the patients studied by RNA sequencing were genetically characterized. Mutations were also found in 36 of the 159 patients not fulfilling the NIH clinical criteria. We confirmed a higher incidence of intellectual disability in patients harboring microdeletion type 1 and observed a correlation between a mild phenotype and the small deletion c.2970_2972delAAT or the missense alteration in amino acid residue 1809 (p.Arg1809Cys). These data support the use of RNA‐based methods for genetic analysis and provide novel information for improving the management of symptoms in oligosymptomatic patients.
We present an overview of our Neurofibromatosis type 1 (NF1) diagnostic research with a focus on the description of 126 novel mutations. These data support the use of RNA‐based methods for genetic analysis and provide novel information for improving the management of symptoms in oligosymptomatic patients.
The NF-kB family of transcription factors is up-regulated in inflammation and different cancers. Recent data described heterozygous deletions of the NF-kB Inhibitor alpha gene (NFKBIA) in about 20% ...of glioblastomas (GBM): deletions were mutually exclusive with epidermal growth factor receptor (EGFR) amplification, a frequent event in GBM. We assessed the status of NFKBIA and EGFR in 69 primary GBMs and in corresponding neurospheres (NS). NFKBIA deletion was investigated by the copy number variation assay (CNV); EGFR amplification by CNV ratio with HGF; expression of EGFR and EGFRvIII by quantitative PCR or ReverseTranscriptase PCR. Heterozygous deletions of NFKBIA were present in 3 of 69 primary GBMs and, surprisingly, in 30 of 69 NS. EGFR amplification was detected in 36 GBMs: in corresponding NS, amplification was lost in 13 cases and reduced in 23 (10 vs 47 folds in NS vs primary tumors; p < 0.001). The CNV assay was validated investigating HPRT1 on chromosome X in females and males. Results of array-CGH performed on 3 primary GBMs and 1 NS line were compatible with the CNV assay. NS cells with NFKBIA deletion had increased nuclear activity of p65 (RelA) and increased expression of the NF-kB target IL-6. In absence of EGF in the medium, EGFR amplification was more conserved and NFKBIA deletion less frequent point to a low frequency of NFKBIA deletions in GBM and suggest that EGF in the culture medium of NS may affect frequency not only of EGFR amplifications but also of NFKBIA deletions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic proportions. Given that the main target of SARS-CoV-2 are lungs leading to severe pneumonia ...with hyperactivation of the inflammatory cascade, we conducted a prospective study to assess alveolar inflammatory status in patients with moderate to severe COVID-19.
Methods
Diagnostic bronchoalveolar lavage (BAL) was performed in 33 adult patients with SARS-CoV-2 infection by real-time PCR on nasopharyngeal swab admitted to the Intensive care unit (ICU) (
n
= 28) and to the Intermediate Medicine Ward (IMW) (
n
= 5). We analyze the differential cell count, ultrastructure of cells and Interleukin (IL)6, 8 and 10 levels.
Results
ICU patients showed a marked increase in neutrophils (1.24 × 10
5
ml
− 1
, 0.85–2.07), lower lymphocyte (0.97 × 10
5
ml
− 1
, 0.024–0.34) and macrophages fractions (0.43 × 10
5
ml
− 1
, 0.34–1.62) compared to IMW patients (0.095 × 10
5
ml
− 1
, 0.05–0.73; 0.47 × 10
5
ml
− 1
, 0.28–1.01 and 2.14 × 10
5
ml
− 1
, 1.17–3.01, respectively) (
p
< 0.01). Study of ICU patients BAL by electron transmission microscopy showed viral particles inside mononuclear cells confirmed by immunostaining with anti-viral capsid and spike antibodies. IL6 and IL8 were significantly higher in ICU patients than in IMW (IL6
p
< 0.01, IL8
p
< 0.0001), and also in patients who did not survive (IL6
p
< 0.05, IL8
p
= 0.05 vs. survivors). IL10 did not show a significant variation between groups. Dividing patients by treatment received, lower BAL concentrations of IL6 were found in patients treated with steroids as compared to those treated with tocilizumab (
p
< 0.1) or antivirals (
p
< 0.05).
Conclusions
Alveolitis, associated with COVID-19, is mainly sustained by innate effectors which showed features of extensive activation. The burden of pro-inflammatory cytokines IL6 and IL8 in the broncho-alveolar environment is associated with clinical outcome.
Polychlorinated biphenyls (PCBs) are toxic chemicals, recalcitrant to degradation, bioaccumulative and persistent in the environment, causing adverse effects on ecosystems and human health. For this ...reason, the remediation of PCB-contaminated soils is a primary issue to be addressed. Phytoremediation represents a promising tool for in situ soil remediation, since the available physico-chemical technologies have strong environmental and economic impacts. Plants can extract and metabolize several xenobiotics present in the soil, but their ability to uptake and mineralize PCBs is limited due to the recalcitrance and low bioavailability of these molecules that in turn impedes an efficient remediation of PCB-contaminated soils. Besides plant degradation ability, rhizoremediation takes into account the capability of soil microbes to uptake, attack and degrade pollutants, so it can be seen as the most suitable strategy to clean-up PCB-contaminated soils. Microbes are in fact the key players of PCB degradation, performed under both aerobic and anaerobic conditions. In the rhizosphere, microbes and plants positively interact. Microorganisms can promote plant growth under stressed conditions typical of polluted soils. Moreover, in this specific niche, root exudates play a pivotal role by promoting the biphenyl catabolic pathway, responsible for microbial oxidative PCB metabolism, and by improving the overall PCB degradation performance. Besides rhizospheric microbial community, also the endophytic bacteria are involved in pollutant degradation and represent a reservoir of microbial resources to be exploited for bioremediation purposes. Here, focusing on plant-microbe beneficial interactions, we propose a review of the available results on PCB removal from soil obtained combining different plant and microbial species, mainly under simplified conditions like greenhouse experiments. Furthermore, we discuss the potentiality of “omics” approaches to identify PCB-degrading microbes, an aspect of paramount importance to design rhizoremediation strategies working efficiently under different environmental conditions, pointing out the urgency to expand research investigations to field scale.
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•Rhizoremediation is the most promising biotechnology for PCB-polluted soils.•Root exudates can induce the biphenyl catabolic pathway enhancing degradation ability.•Better knowledge on in situ phyto-rhizoremediation of PCB-polluted soils is needed.•“Omics” can unravel key players and metabolisms for remediation of PCB-polluted sites.
Polychlorinated biphenyls (PCBs) are persistent organic pollutants widely produced and used in many countries until the increasing concern about their environmental risk lead to their ban in the ...1980s. Although their emissions decreased, PCBs are nowadays still present in the environment and can be reemitted from reservoir compartments such as contaminated soils. In the last two decades, there has been a growing interest in bioremediation technologies that use plants and microorganisms (i.e. rhizoremediation) to degrade organic chemicals in contaminated sites. Different studies have been conducted to investigate the potential of plant-microbe interactions in the remediation of organic chemical contaminated soils. They range from short-term and laboratory/greenhouse experiments to long-term and field trials and, when correctly set up, they could provide useful data such as PCB rhizoremediation half-lives in soil. Such type of data are important input parameters for multimedia fate models that aim to estimate the time requested to achieve regulatory thresholds in a PCB contaminated site, allowing to draw up its remediation plan. This review focuses on the main factors influencing PCB fate, persistence and bioavailability in soil including PCB mixture congener composition, soil organic carbon forms, microorganism activity, plant species and soil conditions. Furthermore, it provides an estimate of rhizoremediation half-lives of the ten PCB families starting from the results of literature rhizoremediation experiments. Finally, guidance to perform appropriate experiments to obtain comparable, accurate and useful data for fate estimation is proposed.
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•PCB bioavailability influences their persistence in soil.•Plant-microbe beneficial interactions could decrease PCB persistence in soil.•PCB half-lives in soil reported in the literature range from ~1 to >20years.•PCB rhizoremediation half-lives in soil range from ~50days to ~11years.•Comparable rhizoremediation experiments are needed to obtain robust data.
Chronic lung allograft dysfunction (CLAD) and interstitial lung disease associated with collagen tissue diseases (CTD-ILD) are two end-stage lung disorders in which different chronic triggers induce ...activation of myo-/fibroblasts (LFs). Everolimus, an mTOR inhibitor, can be adopted as a potential strategy for CLAD and CTD-ILD, however it exerts important side effects. This study aims to exploit nanomedicine to reduce everolimus side effects encapsulating it inside liposomes targeted against LFs, expressing a high rate of CD44. PEGylated liposomes were modified with high molecular weight hyaluronic acid and loaded with everolimus (PEG-LIP(ev)-HA400kDa). Liposomes were tested by in vitro experiments using LFs derived from broncholveolar lavage (BAL) of patients affected by CLAD and CTD-ILD, and on alveolar macrophages (AM) and lymphocytes isolated, respectively, from BAL and peripheral blood. PEG-LIP-HA400kDa demonstrated to be specific for LFs, but not for CD44-negative cells, and after loading everolimus, PEG-LIP(ev)-HA400kDa were able to arrest cell cycle arrest and to decrease phospho-mTOR level. PEG-LIP(ev)-HA400kDa showed anti-inflammatory effect on immune cells. This study opens the possibility to use everolimus in lung fibrotic diseases, demonstrating that our lipids-based vehicles can vehicle everolimus inside cells exerting the same drug molecular effect, not only in LFs, but also in immune cells.
The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in ...disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs that correlates with neutrophils count; moreover, the analysis of lung tissues of COVID-19 deceased patients showed a subset of alveolar reactive pneumocytes with a co-expression of epithelial marker and a mesenchymal marker, confirming the induction of EMT mechanism after severe SARS-CoV2 infection. By airway
in vitro
models, cultivating A549 or 16HBE at air-liquid interface, adding alveolar macrophages (AM), neutrophils and SARS-CoV2, we demonstrated that to trigger a complete EMT expression pattern are necessary the induction of NETosis by SARS-CoV2 and the secretion of AM factors (TGF-β, IL8 and IL1β). All our results highlight the possible mechanism that can induce lung fibrosis after SARS-CoV2 infection.
Bronchiolitis Obliterans Syndrome seriously reduces long-term survival of lung transplanted patients. Up to now there is no effective therapy once BOS is established. Nanomedicine introduces the ...possibility to administer drugs locally into lungs increasing drug accumulation in alveola reducing side effects. Imatinib was loaded in gold nanoparticles (GNP) functionalized with antibody against CD44 (GNP-HCIm). Lung fibroblasts (LFs) were derived from bronchoalveolar lavage of BOS patients. GNP-HCIm cytotoxicity was evaluated by MTT assay, apoptosis/necrosis and phosphorylated-cAbl (cAbl-p). Heterotopic tracheal transplantation (HTT) mouse model was used to evaluate the effect of local GNP-HCIm administration by Alzet pump. GNP-HCIm decreased LFs viability compared to Imatinib (44.4 ± 1.8% vs. 91.8 ± 3.2%, p < 0.001), inducing higher apoptosis (22.68 ± 4.3% vs. 6.43 ± 0.29; p < 0.001) and necrosis (18.65 ± 5.19%; p < 0.01). GNP-HCIm reduced cAbl-p (0.41 GNP-HCIm, 0.24 Imatinib vs. to control; p < 0.001). GNP-HCIm in HTT mouse model by Alzet pump significantly reduced tracheal lumen obliteration (p < 0.05), decreasing apoptosis (p < 0.05) and TGF-β-positive signal (p < 0.05) in surrounding tissue. GNP-HCIm treatment significantly reduced lymphocytic and neutrophil infiltration and mast cells degranulation (p < 0.05). Encapsulation of Imatinib into targeted nanoparticles could be considered a new option to inhibit the onset of allograft rejection acting on BOS specific features.
This paper describes the results of a rhizoremediation greenhouse experiment planned to select the best plant species and soil management for the bioremediation of weathered polychlorinated biphenyls ...(PCBs). We evaluated the ability of different plant species to stimulate activity and diversity of the soil microbial community leading to the reduction of PCB concentrations in a heavily contaminated soil (at mg kg-1 dw level), of the national priority site for remediation (SIN) “Brescia-Caffaro” in Italy. Biostimulation was determined in large size (6kg) pots, to reflect semi-field conditions with a soil/root volume ratio larger than in most rhizoremediation experiments present in the literature. In total, 10 treatments were tested in triplicates comparing 7 plant species (grass and trees) and 5 soil/cultivation conditions (i.e., only one plant species, plant consociation, redox cycle, compost or ammonium thiosulfate addition) with the appropriate unplanted controls. After 18months of biostimulation the overall reduction of total PCBs varied between 14 and 20%. Microbial analysis revealed a shift in the microbial community structure over time and showed that all the planted treatments significantly enhanced microbial hydrolytic activity and the abundance of bacterial populations, including potential PCB degraders, in the soil surrounding plant roots. The plant species most effective in reducing the contaminant concentrations were Festuca arundinacea cultivated adding compost or in consociation with Cucurbita pepo ssp. pepo and Medicago sativa cultivated with Rhizobium spp. and mycorrhizal fungi; they reduced total PCB concentrations of about 20% and showed the significant depletion of a high number of PCB congeners (29, 37 and 23, respectively, out of the 79 measured). Our results suggest that these plant species are particularly efficient in increasing soil PCB bioavailability and in stimulating microbial degradation. They could be used in field rhizoremediation strategies to enhance the natural attenuation process and reduce PCB levels in historically contaminated sites.
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•Plants stimulated microbial diversity abundance and activity in root surrounding soil.•Best degrader treatment was tall fescue with compost also increasing bacterial abundance.•Depletion of both low and high chlorinated PCB was observed.•Tall fescue and pumpkin treatments reduced about 40 PCB congeners.•Overall total PCB reduction was about 20% in realistic semi-field conditions.
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