Characterization of tumor-derived mesenchymal stem cells potentially differentiating into cancer-associated fibroblasts in lung cancer

Purpose: The goal of this study was to understand if mesenchymal stem cells isolated from lung tumor tissue (T-MSCs) may differentiate into cancer associated fibroblasts (CAFs), that promote neoplastic progression, angiogenesis and metastasis in the epithelial solid tumors, mimicking the tumor microenvironmental influence. Methods: MSCs were been obtained from healthy (Control, C-MSCs) and tumor (T-MSCs) tissue of one patient who underwent a lobectomy for a lung adenocarcinoma pT1bN0. Isolated cells were characterized for the presence of molecular markers (identified by routine diagnostic characterization in differentiated tumoral cells), stemness properties, and CAF-related markers expression. Subsequently, cells were co-cultured with a lung adenocarcinoma cell line (A549 cells) to evaluate the effects on proliferation, oncogene expression and IL6 secretion. Results: C- and T-MSCs did not present EGFR mutations unlike tumor tissue and showed a stem-like immunophenotype, characterized by the ability to differentiate towards osteo-, chondro- and adipogenic lineages. The expression of markers referred to CAFs (alfa-SMA, HI-1alfa, MMP11, VEGF, CXCL12, TGF-Beta1, TGF-BetaRII, IL6, TNFalfa) was significantly higher in T-MSCs than in C-MSCs. The co-cultures with A549 cells led to the over-expression of selected oncogenes and to the increase of IL6 secretion in T-MSCs but not in C-MSCs. Conclusions: MSCs isolated from tumor tissue displayed distinct properties compared to MSCs isolated from healthy tissue, suggesting T-MSCs differentiation towards a CAF-related phenotype under the influence of the tumoral microenvironment

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The 2015 World Health Organization Classification of lung tumors: new entities since the 2004 Classification.

In the last few years different new pulmonary neoplastic lesions have been recognised and some of them, namely NUT carcinoma, PEComatous tumors, pneumocytic adenomyoepithelioma, pulmonary myxoid sarcoma, myoepithelial tumors/carcinomas entered in the last 2015-WHO classification of lung tumors. In addition angiomatoid fibrous histiocytoma and ciliated muconodular papillary tumor have been morphologically and genetically characterized albeit not yet included in the 2015-WHO classification.In the present paper we summarised the clinical, morphological, immunohistochemical and molecular features of these new entities. The knowledge of key histologic and molecular characteristics may help pathologists in achieving a correct diagnosis thus leading to an adequate therapeutic approach.

Characterization of tumor-derived mesenchymal stem cells potentially differentiating into cancer-associated fibroblasts in lung cancer.

PURPOSE: The goal of this study was to understand if mesenchymal stem cells isolated from lung tumor tissue (T-MSCs) may differentiate into cancer associated fibroblasts (CAFs), that promote neoplastic progression, angiogenesis and metastasis in the epithelial solid tumors, mimicking the tumor microenvironmental influence. METHODS: MSCs were been obtained from healthy (Control, C-MSCs) and tumor (T-MSCs) tissue of one patient who underwent a lobectomy for a lung adenocarcinoma pT1bN0. Isolated cells were characterized for the presence of molecular markers (identified by routine diagnostic characterization in differentiated tumoral cells), stemness properties, and CAF-related markers expression. Subsequently, cells were co-cultured with a lung adenocarcinoma cell line (A549 cells) to evaluate the effects on proliferation, oncogene expression and IL6 secretion. RESULTS: C- and T-MSCs did not present EGFR mutations unlike tumor tissue and showed a stem-like immunophenotype, characterized by the ability to differentiate towards osteo-, chondro- and adipogenic lineages. The expression of markers referred to CAFs (α-SMA, HI-1α, MMP11, VEGF, CXCL12, TGF-ß1, TGF-ßRII, IL6, TNFα) was significantly higher in T-MSCs than in C-MSCs. The co-cultures with A549 cells led to the over-expression of selected oncogenes and to the increase of IL6 secretion in T-MSCs but not in C-MSCs. CONCLUSIONS: MSCs isolated from tumor tissue displayed distinct properties compared to MSCs isolated from healthy tissue, suggesting T-MSCs differentiation towards a CAF-related phenotype under the influence of the tumoral microenvironment.

Seminal cell-free DNA levels measured by PicoGreen fluorochrome are associated with sperm fertility criteria.

Previous investigations suggested that elevated cell-free DNA (cfDNA) can indicate non-healthy states. However, the potential association between cfDNA seminal plasma levels and fertility sperm parameters has not yet been determined. Therefore, the present study evaluated the association between seminal cfDNA levels and sperm fertility criteria to determine the use of seminal cfDNA quantification. An in vivo protocol quantified cfDNA levels of semen samples obtained from 163 male patients using fluorescent PicoGreen dye staining. To confirm if semen cfDNA quantification is realistic, an in vitro complementary test was performed using three or four semen samples. The fresh sperm samples were exposed to paraquat that generates high levels of superoxide anion causing oxidative stress and cell mortality. The results showed significant association between dsDNA levels and several sperm fertility parameters, such as low viability and alterations of motility and morphology. The in vitro analysis confirmed the association between dsDNA levels and sperm viability. Together, these results suggest that dsDNA levels could be an important biomarker to test sperm fertility.

An unusual thoracic localizations of Erdheim- Chester disease: A case report.

Erdheim- Chester disease is a rare non- Langerhans cell histiocytosis that usually involves the bones, heart, central nervous system, retroperitoneum, eyes, kidneys, skin and adrenals. Lungs are affected in up to one-half cases; at CT scan various patterns are described: interstitial disease, consolidations, micronodules and microcysts, with or without pleural involvement. We presented a case of a 59 year-old man with unusual intrathoracic manifestation of Erdheim- Chester disease. Singularities of our report are the lonely thoracic involvement at the onset of the disease and a histiocytic lesion in the posterior mediastinum.

The effects of rosuvastatin on lipid-lowering, inflammatory, antioxidant and fibrinolytics blood biomarkers are influenced by Val16Ala superoxide dismutase manganese-dependent gene polymorphism.

Rosuvastatin is a cholesterol-lowering drug that also attenuates the inflammatory process and oxidative stress via the reduction of superoxide anion production. Superoxide anions are metabolized by manganese-dependent superoxide dismutase (MnSOD or SOD2) in the mitochondria. In humans, there is a gene polymorphism where a change of alanine (Ala) to valine (Val) occurs at the 16th amino acid (Ala16Val-SOD2). The VV genotype has been associated with the risk of developing several metabolic diseases, such as hypercholesterolemia. Thus, to further explore this phenomenon, this study investigated the influence of the Val16Ala-SOD2 polymorphism on the lipid profile and inflammatory and fibrinolytic biomarkers of 122 hypercholesterolemic patients undergoing the first pharmacological cholesterol-lowering therapy who were treated with 20 mg rosuvastatin for 120 days. The findings indicate that the VV patients who present a low-efficiency SOD2 enzyme exhibit an attenuated response to rosuvastatin compared with the A-allele patients. The effect of rosuvastatin on inflammatory and fibrinolytic biomarkers was also less intense in the VV patients. These results suggest some pharmacogenetic effects of Val16Ala-SOD2 in hypercholesterolemia treatment.

Quercetin ameliorates polychlorinated biphenyls-induced testicular DNA damage in rats.

Polychlorinated biphenyls (PCBs) are a group of environmental contaminants widely reported to cause gonadal toxicity in both humans and animals. This study investigated the amelioratory role of quercetin in PCBs-induced DNA damage in male Wistar rats. Polychlorinated biphenyls were administered intraperitoneally at a dose of 2 mg kg(-1) alone or in combination with quercetin (orally) at 50 mg kg(-1) for 25 days. Quercetin modulation of PCBs-induced gonadal toxicity was evaluated using selected oxidative stress indices, comet assay, measurement of DNA concentration and histology of the testes. Administration of PCBs alone caused a significant (P < 0.05) depletion in the total thiol level in testes of treated rats. Conversely, the levels of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) production were markedly elevated in testes of PCBs-treated rats compared with control. Further, PCBs exposure produced statistically significant increases in DNA tail migration, degraded double-stranded DNA (dsDNA) concentration and histological alterations of testes of the treated rats compared to control. Quercetin cotreatment significantly improved the testicular antioxidant status, decreased DNA fragmentation and restored the testicular histology, thus demonstrating the protective effect of quercetin in PCBs-treated rats.

Association between advanced oxidation protein products and 5-year mortality risk among amazon riparian elderly population.

Proteins are important targets of several modifications caused by oxidative stress, leading to structural changes and consequently partial or total loss of their functions. The oxidized proteins include advanced oxidation protein products (AOPP) derived from oxidation-modified albumin, as well as fibrinogen and lipoproteins. An increase in AOPP levels indicates an oxidative stress state and the presence of coexisting inflammation. Several investigations have also suggested an association between high AOPP levels and aging-related diseases. However, the link between elevated AOPP levels and elderly mortality risk has not yet been investigated. Here, we report on a 5-year longitudinal study that investigated the potential association between AOPP levels and mortality using a population-based representative sample of riparian elders living in Brazilian Amazon region (Maués-AM). Age, sex, socioeconomic and cultural conditions, chronic morbidities, polypharmacy, and previous morbidities were also tested as potential confounders. The AOPP levels were measured in 540 (84.78%) individuals, all of whom were followed over a 5-year period in order to establish the mortality rate. Within this study period, 74 (13.7%) elders died and 466 (86.3%) survived. The AOPP levels were higher among the elders who died within the 5-year period (46.27 ± 40.6 mmol/L) compared with those who survived (36.79 ± 20.84 mmol/L) (p = 0.002). The analysis confirmed the link between high AOPP levels and mortality risk, independent of other intervenient factors. These results suggest that elevated AOPP levels could be used to predict mortality risk in elderly patients.

Phylogeography and demographic history of two widespread Indo-Pacific mudskippers (Gobiidae: Periophthalmus).

This study provides a first description of the phylogeographic patterns and evolutionary history of two species of the mudskipper genus Periophthalmus. These amphibious gobies are distributed throughout the whole Indo-Pacific region and Atlantic coast of Africa, in peritidal habitats of soft-bottom coastal ecosystems. Three sequence datasets of two widely distributed species, Periophthalmus argentilineatus and P. kalolo, were obtained by amplifying and sequencing two mtDNA markers (D-loop and 16S rDNA) and the nDNA rag1 region. The three datasets were then used to perform phylogeographic, demographic and population genetic analyses. Our results indicate that tectonic events and past climatic oscillations strongly contributed to shape present genetic differentiation, phylogeographic and demographic patterns. We found support for the monophyly of P. kalolo, and only shallow genetic differentiation between East-African and Indo-Malayan populations of this species. However, our collections of the morphospecies P. argentilineatus include three molecularly distinct lineages, one of them more closely related to P. kalolo. The presence of Miocenic timings for the most recent common ancestors of some of these morphologically similar clades, suggests the presence of strong stabilising selection in mudskippers' habitats. At population level, demographic analyses and palaeoecological records of mangrove ecosystems suggest that Pleistocene bottlenecks and expansion plus secondary contact events of the studied species were associated with recurrent sea transgressions during interglacials, and sea regressions or stable regimes during glacials, respectively.

In vitro effects of Ala16Val manganese superoxide dismutase gene polymorphism on human white blood cells exposed to methylmercury.

Environmental contamination by methylmercury (MeHg) is an enormous public health problem in world regions such as Amazonia. MeHg toxic effects seem to be influenced by environmental and genetic factors. However, few studies have evaluated the genetic influences of MeHg toxicity in humans. Therefore, the aim of this study was to evaluate the genetic influence of Ala16Val manganese superoxide dismutase gene polymorphism (Ala16Val-MnSOD) on the cytotoxic effects of in vitro human leukocytes exposed to MeHg. Subjects were selected from 100 individuals aged 26.4 ± 7.3 years genotyped to Ala16Val-MnSOD polymorphism (AA = 6, VV = 6, and AV = 12) to perform in vitro testing using white blood cells (WBCs). Reactive oxygen species production was measured using 2',7'-dichlorofluorescein diacetate fluorimetric assay, and cell viability was measured using MTT assay on WBC samples from the same subjects that were both exposed and not exposed to MeHg (2.5 µM for 6 h). The results showed that AA- and VV-WBCs exposed to MeHg did not display increased reactive oxygen species levels compared to those in cells that were not exposed. However, AV-leukocytes exposed to MeHg displayed increased ROS levels. Cellular viability comparison among genotypes exposed to MeHg showed that the viability of AA-WBCs was lower than that of VV-WBC, with mean values of 3.46 ± 0.13 and 3.08 ± 0.77 (standard error), respectively (P = 0.033), whereas heterozygous cells (AV) displayed intermediate values. This difference was likely due to the higher basal H2O2 production of AA-WBCs compared to that of other genotypes. These results suggest that the Ala16Val-MnSOD polymorphism has toxicogenetic effects in human cells exposed to MeHg.

The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside.

The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine=12.240 mg/g, theobromine=6.733 mg/g and total catechins=4.336 mg/g. Cells were exposed to 10 µM SNP during a 6 h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20 mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5 mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels.

Is there a role for prostate tumour overexpressed-1 in the diagnosis of HGPIN and of prostatic adenocarcinoma? A comparison with alpha-methylacyl CoA racemase.

Prostate Tumour Overexpressed-1 (PTOV1) was recently identified as a novel gene and protein during a differential display screening for genes overexpressed in prostate cancer (PCa). Alpha-Methyl-CoA racemase (AMACR) mRNA was identified as being overexpressed in PCa. PTOV1 and racemase were immunohistochemically evaluated in PCa, high-grade prostatic intraepithelial neoplasia (HGPIN), atrophy and normal-looking epithelium (NEp) in 20 radical prostatectomies (RPs) with pT2a Gleason score 6 prostate cancer with the aim of analyzing the differences in marker expression between PTOV1 and AMACR. The level of expression of PTOV1 and AMACR increased from NEp and atrophy through HGPIN, away from and adjacent to prostate cancer, to PCa. With the ROC curve analysis the overall accuracy in distinguishing PCa vs HGPIN away from and adjacent to cancer was higher for AMACR than for PTOV1. In conclusion, AMACR can be considered a more accurate marker than PTOV1 in the identification of HGPIN and of PCa. However, PTOV1 may aid in the diagnosis of PCa, at least to supplement AMACR as another positive marker of carcinoma and to potentially increase diagnostic accuracy.

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2010-31 January 2011.

This article documents the addition of 238 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alytes dickhilleni, Arapaima gigas, Austropotamobius italicus, Blumeria graminis f. sp. tritici, Cobitis lutheri, Dendroctonus ponderosae, Glossina morsitans morsitans, Haplophilus subterraneus, Kirengeshoma palmata, Lysimachia japonica, Macrolophus pygmaeus, Microtus cabrerae, Mytilus galloprovincialis, Pallisentis (Neosentis) celatus, Pulmonaria officinalis, Salminus franciscanus, Thais chocolata and Zootoca vivipara. These loci were cross-tested on the following species: Acanthina monodon, Alytes cisternasii, Alytes maurus, Alytes muletensis, Alytes obstetricans almogavarii, Alytes obstetricans boscai, Alytes obstetricans obstetricans, Alytes obstetricans pertinax, Cambarellus montezumae, Cambarellus zempoalensis, Chorus giganteus, Cobitis tetralineata, Glossina fuscipes fuscipes, Glossina pallidipes, Lysimachia japonica var. japonica, Lysimachia japonica var. minutissima, Orconectes virilis, Pacifastacus leniusculus, Procambarus clarkii, Salminus brasiliensis and Salminus hilarii.

Immunohistochemical expression of prostate stem cell antigen in cystoprostatectomies with incidental prostate cancer.

High expression of prostate stem cell antigen (PSCA) has been shown to be associated with adverse prognostic features in clinically-diagnosed prostate cancer. The aim of this study is to analyze PSCA expression in cystoprostatectomies with incidental prostate carcinoma (PCa). PSCA expression was evaluated immunohistochemically in normal-looking epithelium (NEp), high-grade prostatic intraepithelial neoplasia (HGPIN) and pT2a Gleason score 6 acinar adenocarcinoma. The evaluation was carried out on 20 cystoprostatectomies (CyPs) with incidental PCa from men with bladder urothelial carcinoma (UC), and 20 radical prostatectomies (RPs) with hormonally untreated PCa from men with clinically detected PCa. Ki-67 was also investigated. The percentages of PSCA positive cells in HGPIN were significantly higher than in NEp (NEp: CyP, mean 2.92%+/-standard deviation 6.26%; RP, 3.5%+/-6.46%. HGPIN: CyP, 13.67%+/-12.78%; RP, 14.67%+/-11.34%) (p<0.001). The proportions of positive cells in PCa were greater than in HGPIN (CyP, 20.25%+/-15.96%; RP, 22.58%+/-13.67%) (p<0.001). For Ki-67 labeling, the proportions of positive nuclei in the CyPs significantly increased from NEp through HGPIN to PCa. A similar trend was seen in the RPs. In the CyPs the percentages of PSCA and Ki67 positive cells were lower than in the RPs, the differences between the CyP and RP compartments being not statistically significant. Our findings suggest that PSCA is a marker associated with neoplastic transformation of prostate cells, both in CyPs and RPs. However, there are no significant differences between CyPs with incidental prostate carcinoma and RPs with clinically diagnosed cancer.

Polymorphic microsatellite loci in the widespread amphidromous goby Sicyopterus lagocephalus and cross-genus amplification among Sicydiinae.

Microsatellite DNA markers were isolated in an amphidromous goby (Sicyopterus lagocephalus) from a partial genomic library enriched for AC repeats. Eight microsatellites were highly polymorphic with six to 33 alleles per locus and expected heterozygosities ranging from 0.53 to 0.97. Cross-species amplifications were performed within the sub-family Sicydiinae by genotyping individuals from two species of the genus Cotylopus. Some of these loci were successfully amplified and showed polymorphism in the second genus.

Immunohistochemical evaluation of global DNA methylation and histone acetylation in papillary urothelial neoplasm of low malignant potential.

A preceding study has shown that karyometry detected subvisual differences in chromatin organization status between non-recurrent and recurrent papillary urothelial neoplasm of low malignant potential (PUNLMP). The status of chromatin organization depends on epigenetic events, such as DNA methylation and histone acetylation. The aim of this study is to explore global DNA methylation and global histone acetylation in non-recurrent and recurrent PUNLMP. 5-methylcytosine (5MeC) and acetylated histone H3 lysine 9 (AcH3K9) were investigated by immunohistochemistry (IHC) in 20 PUNLMP cases (10 non-recurrent and 10 recurrent), in 5 cases of normal urothelium (NU) and in 5 cases of muscle invasive pT2 urothelial carcinoma (UC). For global DNA methylation, the mean percentage of positive nuclei in the cells adjacent to the stroma increased from NU (79%) through non-recurrent and recurrent PUNLMP (86% and 93%, respectively) to UC (97%). The percentages of positive nuclei in the intermediate cell layers and in the superficial cells in the four groups were similar to those adjacent to the stroma. The proportion of nuclei with weak-to-moderate intensity was far greater than that of those strongly stained and increased steadily from NU to UC. For global histone acetylation, the mean percentage of positive nuclei was highest in non-recurrent PUNLMP (i.e. 90%) and lowest in recurrent PUNLMP (i.e. 81%). In NU and UC the mean percentages of positive nuclei were 84% and 86%, respectively. The percentage of positive nuclei decreased from the cell layer adjacent to the stroma to the superficial cell layer. The proportion of nuclei with weak-to-moderate intensity was slightly greater than that of those strongly stained. In comparison with global DNA methylation, the proportion of strongly stained nuclei was much higher. In conclusion, there are differences in global DNA methylation and histone acetylation patterns between non-recurrent and recurrent PUNLMP. Further studies are needed to elucidate the complex interplay between chromatin structure, its modifications and recurrence of PUNLMP.