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Activated phosphoinositide 3-kinase (PI3Kδ) Syndrome (APDS) is an inborn error of immunity (IEI) with a variable clinical presentation, characterized by infection susceptibility and immune dysregulation that may overlaps with other Primary Immune Regulatory Disorders (PIRDs). The rarity of the disease, its recent discovery, and the multiform /multifaced clinical presentation make it difficult to establish a correct diagnosis, especially at an early stage. As a result, the true prevalence of the pathology remains unknown. There is no treatment protocol for APDS, and drug therapy is primarily focused on treating symptoms. The most common therapies include immunoglobulin replacement therapy, antimicrobial prophylaxis, and immunosuppressive drugs. Hematopoietic stem cell transplantation (HSCT) has been used in some cases, but the risk-benefit balance remains unclear. With the upcoming introduction of specific medications, such as selective inhibitors for PI3Kδ, clinicians are shifting their attention towards target therapy. This review provides a comprehensive overview of APDS with a focus on diagnostic and treatments procedures available. This review may be useful in implementing strategies for a more efficient patients' management and therapeutic interventions. Main Text. [ABSTRACT FROM AUTHOR]

Vinca sardoa (Stearn) Pignatti, known as Sardinian periwinkle, is widely diffused in Sardinia (Italy). This species contains indole alkaloids, which are known to have a great variety of biological activities. This study investigated the antileukemic activity against a B lymphoblast cell line (SUP-B15) of V. sardoa alkaloid-rich extracts obtained from plants grown in Italy, in Iglesias (Sardinia) and Rome (Latium). All the extracts showed a good capacity to induce reductions in cell proliferation of up to 50% at the tested concentrations (1–15 µg/mL). Moreover, none of the extracts showed cytotoxicity on normal cells at all the studied concentrations. [ABSTRACT FROM AUTHOR]

In recent years, immediate breast reconstruction after mastectomy surgery has steadily increased in the treatment pathway of breast cancer (BC) patients due to its potential impact on both the morpho-functional and aesthetic type of the breast and the quality of life. Although recent studies have demonstrated how recent radiotherapy techniques have allowed a reduction of adverse events related to breast reconstruction, capsular contracture (CC) remains the main complication after post-mastectomy radio-therapy (PMRT). In this study, we evaluated the association of the occurrence of CC with some clinical, histological and therapeutic parameters related to BC patients. We firstly performed bivariate statistical tests and we then evaluated the prognostic predictive power of the collected data by using machine learning techniques. Out of a sample of 59 patients referred to our institute, 28 patients (i.e., 47%) showed contracture after PMRT. As a result, only estrogen receptor status (ER) and molecular subtypes were significantly associated with the occurrence of CC after PMRT. Different machine learning models were trained on a subset of clinical features selected by a feature importance approach. Experimental results have shown that collected features have a non-negligible predictive power. The extreme gradient boosting classifier achieved an area under the curve (AUC) value of 68% and accuracy, sensitivity, and specificity values of 68%, 64%, and 74%, respectively. Such a support tool, after further suitable optimization and validation, would allow clinicians to identify the best therapeutic strategy and reconstructive timing. [ABSTRACT FROM AUTHOR]

Aging and its related disorders are important issues nowadays and the first cause of this physio-pathological condition is the overproduction of ROS. Ascorbic acid is an antioxidant mediator and its anti-aging proprieties are well known. Our previous data demonstrated that Voghera sweet pepper (VP), a distinctive type of pepper cultivated in Italy, is particularly rich in ascorbic acid. Based on these data, the anti-aging effect mediated by extracts of the edible part of VP was evaluated on an in vitro model of both young and old Normal Human Diploid Fibroblasts (NHDF). Using phase contrast microscopy, we observed that VP may help cells in the maintenance of physiological morphology during aging. Cytofluorimetric analyses revealed that VP extracts led to an increase in DNA synthesis and percentage of living cells, linked to a consequent increase in mitotic events. This hypothesis is supported by the enhancement of PCNA expression levels observed in old, treated fibroblasts, corroborating the idea that this extract could recover a young phenotype in adult fibroblasts, confirmed by the study of p16 and p53 expression levels and TEM analyses. Based on these results, we may suppose that VP can lead to the partial recovery of "young-like" phenotypes in old fibroblasts.

Background: Children with Down syndrome (DS) show a high susceptibility to recurrent infections (RI), caused by immune defects and abnormalities of the airways. Our goal was to investigate the effects of Pidotimod on RI prevention in children with DS, comparing immune and clinical parameters before (T0) and after (T1) the treatment with Pidotimod. Methods: The study was conducted at the Down syndrome outpatient Center of Bambino Gesù Children's Hospital, in Rome. We reviewed the medical records of all children with a positive history for RI and who received oral prophylaxis of Pidotimod from September 2016 to February 2017. Results: Thirty-three children met the inclusion criteria (males: 51.5%; average age: 6 years ±SD: 3). We found a significant decrease in the number of children with upper respiratory infections (82% at T0 vs 24% at T1; p = 0,0001) and with lower respiratory infections (36% at T0 vs 9% at T1; p = 0.003) after treatment with Pidotimod. We also demonstrated a significant decrease in the number of children hospitalized for respiratory infections (18% at T0 vs 3% at T1; p = 0.03). We measured T and B cells in the peripheral blood and B cell function in vitro at T0 and T1. We found that the response to CpG improved at T1. A significant increase of B cell frequency (p = 0.0009), B cell proliferation (p = 0.0278) and IgM secretion (p = 0.0478) were observed in children with DS after treatment. Conclusions: Our results provided evidence that Pidotimod may be able to prevent RI in children with Down syndrome. [ABSTRACT FROM AUTHOR]

Natural substances produced by venomous marine organisms are thought to be possible sources of useful compounds and new drugs having the potential to open new ways for pharmacology, nutrition and environmental applications. In this framework, cnidarians are very interesting being widely distributed and all are venomous organisms; so, a deep knowledge of their occurrence, morphology of venomous structures and of effects of venoms at cellular level is fundamental to evaluate the possible utilization of venomous compounds or extracts. In this research, the morphology and occurrence of nematocysts in two cnidarian species (Aurelia aurita , Velella velella), and the preliminary evaluation of the cytotoxicity of V. velella crude extract, of which cytotoxicity on cell cultures at present is unknown, were considered. The specimens were sampled in Güllük Bay, Southwestern coast of Turkey, and in the Gulf of Genova, Northwestern coast of Italy. Six nematocyst types (a-isorhiza, A-isorhiza, O-isorhiza, eurytele, polyspiras, birhopaloid) having different sizes, were observed in A. aurita , and two types (eurytele and stenotele) in V. velella. The crude extract from V. velella showed cytotoxic activity against cultured fibroblasts L929 at high doses, while inducing cell proliferation at low doses. The protein content in the extract increased remarkably after disruption of nematocysts. Image 1098 • The shape of some nematocyst types differs in A. aurita sampled in different zones. • Nematocyst number-bell diameter relationship was not observed in A. aurita. • Only eurytele and stenotele nematocyst types were observed in V. velella. • High doses of V. velella crude venom caused decrease of survival in L929 cells. • Low doses of V. velella crude venom had positive effects on L929 cells. [ABSTRACT FROM AUTHOR]

Burkitt lymphoma is a very aggressive B-cell non-Hodgkin lymphoma. Although remarkable progress has been made in the therapeutic scenario for patients with Burkitt lymphoma, search and development of new effective anticancer agents to improve patient outcome and minimize toxicity has become an urgent issue. In this study, the antitumoral activity of Inula viscosa, a traditional herb obtained from plants collected on the Asinara Island, Italy, was evaluated in order to explore potential antineoplastic effects of its metabolites on Burkitt lymphoma. Raji human cell line was treated with increasing Inula viscosa extract concentration for cytotoxicity screening and subsequent establishment of cell cycle arrest and apoptosis. Moreover, gene expression profiles were performed to identify molecular mechanisms involved in the anticancer activities of this medical plant. The Inula viscosa extract exhibited powerful antiproliferative and cytotoxic activities on Raji cell line, showing a dose- and time-dependent decrease in cell viability, obtained by cell cycle arrest in the G2/M phase and an increase in cell apoptosis. The treatment with Inula viscosa caused downregulation of genes involved in cell cycle and proliferation (c-MYC, CCND1) and inhibition of cell apoptosis (BCL2, BCL2L1, BCL11A). The Inula viscosa extract causes strong anticancer effects on Burkitt lymphoma cell line. The molecular mechanisms underlying such antineoplastic activity are based on targeting and downregulation of genes involved in cell cycle and apoptosis. Our data suggest that Inula viscosa natural metabolites should be further exploited as potential antineoplastic agents against Burkitt lymphoma. [ABSTRACT FROM AUTHOR]

Background: Sclerosing nevus with pseudomelanomatous features (SNPFs) is a clinical and pathologic entity that mimics melanoma both clinically and histologically. The lesion is a melanocytic nevus, histologically characterized by fibrosis and a pseudomelanomatous proliferation. It is typically seen in young to middle‐aged individuals, mainly on the back, where microtrauma or inflammatory changes are more frequent. Dermoscopic description of SNPF has been reported so far in one case series. Objective: The aim of our study was to describe the dermoscopic and confocal features of SNPF. Methods: Histopathologically confirmed cases of SNPF were retrospectively collected from three referral centres in Italy. Only lesions with available clinical, dermoscopic and histopathological data were included; confocal images were also retrieved, when available. Lesions were evaluated for the presence of 12 dermoscopic and five confocal criteria previously described. Results: The study population included 93 lesions in as many patients (71 men and 22 women; median age: 38 years). Dermoscopically, we found a predominance of dark colours, in particular brown and blue, which were found in all lesions and the vast majority of the lesions (86/93; 92.5%) displayed at least one structureless area. By the combination of colours and structures, we observed that the majority of the lesions (67/92; 72%) were characterized by more than one structure and more than one colour. Confocal evaluation was performed on a subset of 24/93 lesions showing a regular architecture pattern (19/24 cases, 79%), with a predominance of the ringed pattern. The presence of focal cytologic atypia at the dermal‐epidermal junction was present in 12/24 cases (50%) with a prevalent dendritic‐shaped cell proliferation. Conclusions: The current study demonstrated that SNPF was frequently characterized, on dermoscopic examination, by more than one structure and more than one colour and on confocal microscopy by a regular ringed pattern with focal dendritic atypical cells. [ABSTRACT FROM AUTHOR]

Vitis vinifera cv Falanghina is an ancient grape variety of Southern Italy. A thorough phytochemical analysis of the Falanghina leaves was conducted to investigate its specialised metabolite content. Along with already known molecules, such as caftaric acid, quercetin-3- O -β- d -glucopyranoside, quercetin-3- O -β- d -glucuronide, kaempferol-3- O -β- d -glucopyranoside and kaempferol-3- O -β- d -glucuronide, a previously undescribed biflavonoid was identified. For this last compound, a moderate bioactivity against metastatic melanoma cells proliferation was discovered. This datum can be of some interest to researchers studying human melanoma. The high content in antioxidant glycosylated flavonoids supports the exploitation of grape vine leaves as an inexpensive source of natural products for the food industry and for both pharmaceutical and nutraceutical companies. Additionally, this study offers important insights into the plant physiology, thus prompting possible technological researches of genetic selection based on the vine adaptation to specific pedo-climatic environments. [ABSTRACT FROM AUTHOR]

Aim: Wound healing is a complex process that involves various cells and factors. Hydrogel dressings have been used widely for wound management due to their biocompatibility and water retention properties. Photocrosslinking is preferred to enhance the stability of hydrogels and avoid the release of agents. This technique does not require toxic solvents or high temperatures. In this study, the authors aimed to develop a carrageenan-based hydrogel dressing that can be crosslinked using light exposure and has the potential to accelerate wound healing. Method: The authors successfully developed a carrageenan-based hydrogel dressing that can be crosslinked using light exposure. The hydrogel dressing exhibited excellent mechanical strength, biocompatibility, and antibacterial activity. The authors evaluated the chemical and physical structure and morphology of the hydrogel dressing. The multifunctionality of the fabricated dressing was assessed by the antibacterial assay. The viability and proliferation of the fibroblasts seeded on the surface of the hydrogel were evaluated. In vitro experiments showed that the hydrogel dressing promoted cell proliferation, migration, and tissue regeneration. Results / Discussion: The results of this study suggest that the carrageenan-based hydrogel dressing crosslinked using light exposure has great potential for wound healing applications. The dressing's biocompatibility, mechanical strength, and antibacterial activity make it a promising candidate for wound management. The use of light exposure to crosslink the hydrogel dressing offers a non-invasive and convenient method for application. Conclusion: The study provides a novel approach to developing a hydrogel dressing for wound management that has the potential to accelerate wound repair. [ABSTRACT FROM AUTHOR]

Aim: Preclinical studies have shown a positive effect of dermal white adipose tissue (dWAT) on wound healing. Adipocytes can contribute to the formation of granulation tissue by differentiating into myofibroblasts and by secreting factors which induce fibroblast migration. However, an excess of adipose tissue inhibits wound closure. In this study, we investigated whether exudates from chronic wounds affect proliferation of primary adipocytes. Method: Human preadipocytes were isolated from surgically excised adipose tissue. Cells were grown and differentiated into adipocytes in 96- and 384-well plates. Wound exudates and compounds were added to preadipocytes or mature adipocytes for 3 -- 6 days. The evaluation comprised microscopic observation, metabolic activity of live cells, and staining for lipid droplets, DNA and cellular protein on fixed cells. Results / Discussion: The PPAR-gamma agonist rosiglitazone and the glucocorticoid dexamethasone proved essential for adipocyte differentiation, as demonstrated by the addition of corresponding antagonists to the induction medium. While exudates from healing wounds as well as human serum had no negative effect on adipocytes, increasing concentrations of non-healing (chronic) exudates resulted in cell damage and even toxicity. Conclusion: Chronic wound exudates damage adipocytes, thus preventing them from exerting possible beneficial effects on the healing process. Our finding may explain that tissue damage in chronic wounds frequently reaches into the adipose tissue. [ABSTRACT FROM AUTHOR]

Recently, much attention has been devoted to urban air pollution because epidemiological studies have reported health impacts related to particulate matter (PM). PM10 and PM2.5 were collected during different seasons in Torino, a northern Italian city, and were characterised by inorganic chemical species (secondary particulates and bio-available iron). The biological effects of aqueous and organic solvent PM extracts on human epithelial lung A549 were evaluated, and the effects on cell proliferation and lactate dehydrogenase (LDH) release were assayed. The average PM10 concentration during the sampling period was 47.9 ± 18.0 μg/m; the secondary particles accounted for 49 % ± 9 % of the PM10 total mass, and the bio-available iron concentration was 0.067 ± 0.045 μg/m. The PM2.5/PM10 ratio in Torino ranged from 0.47 to 0.90 and was higher in cold months than in warm months. The PM10 and PM2.5 extracts inhibited cell proliferation and induced LDH release in a dose-dependent manner with a seasonal trend. The PM10 extract had a stronger effect on LDH release, whereas the PM2.5 extract more strongly inhibited cell proliferation. No significant differences were observed in the effects induced by the two extracts, and no significant correlations were found between the biological effects and the PM components evaluated in this study, thus emphasising the importance of the entire mixture in inducing a cytotoxic response. [ABSTRACT FROM AUTHOR]

Abstract: Human type II alveolar cells (A549) were exposed to aqueous- and organic-solvent PM10 extracts to evaluate their effects on cell proliferation, proinflammatory cytokine release and cytotoxicity (assayed by lactate dehydrogenase, LDH, activity). PM10 samples collected in Torino (northwest Italy) were analysed for inorganic chemical species (bioavailable iron and secondary particulates) and endotoxins, which are potentially inflammatory promoters in human airways. During the sampling period, PM10 concentration was 55.4±39.1μg/m3, secondary particles constituted 42±9% of the PM10 total mass, and bioavailable iron concentration was 0.078±0.095μg/m3. PM10 inhibits cell proliferation and induces both IL-6 and LDH release in a dose- and time-dependent manner, with a seasonal trend. The different effects of aqueous and organic PM10 extracts demonstrate the importance of particle composition for the induction of cytotoxic effects on A549 cells. A first comparison between the biological effects induced by PM10 extracts and PM10 components was performed. [Copyright &y& Elsevier]

Abstract: Mature apples of ‘Fuji’ and ‘Fuji’ sports sourced from around the world were found to possess clumps of multicellular, branched callus hairs in the outer 17mm of the outer cortex. The clumps of callus hairs were particularly well developed in samples of ‘Fuji Suprema’ from Brazil, moderately developed in ‘Fuji KIKU’ from Italy and in ‘Fuji’ from South Africa, Chile, New Zealand and the USA, and were consistently least developed in ‘Fuji’ from China. These previously unreported callus hairs grow in the intercellular air spaces between the parenchyma cells and also in larger air lacunae in the apple flesh. In both of these locations they have the potential to reduce the efficiency of gas exchange into and out of the fruit during storage. Initial observations suggest that the callus hairs within a clump do not continue to develop after harvest. In ripe apples, the cells of the callus hairs contain chlorophyll and starch and under UV light the contents of the vacuoles autofluoresce an intense blue colour. The outer surface of each callus hair cell is covered with characteristic globular protuberances which attach the hairs to each other and to the surrounding parenchyma. After a period of storage, many of the clumps of callus hairs remain packed with starch granules even though starch has been metabolised from the surrounding parenchyma. [Copyright &y& Elsevier]

Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells. Cultures were treated with different concentrations of substances illegally used in cattle (17β-estradiol, clenbuterol and boldione). Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to determine the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest concentrations. The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17β-estradiol treated cells PCNA expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could have a role in the early stage of pathogenesis of granulosa cell tumour in cattle. [ABSTRACT FROM AUTHOR]

Abstract: Workshop participants agreed that genotoxicity tests in mammalian cells in vitro produce a remarkably high and unacceptable occurrence of irrelevant positive results (e.g. when compared with rodent carcinogenicity). As reported in several recent reviews, the rate of irrelevant positives (i.e. low specificity) for some studies using in vitro methods (when compared to this “gold standard”) means that an increased number of test articles are subjected to additional in vivo genotoxicity testing, in many cases before, e.g. the efficacy (in the case of pharmaceuticals) of the compound has been evaluated. If in vitro tests were more predictive for in vivo genotoxicity and carcinogenicity (i.e. fewer false positives) then there would be a significant reduction in the number of animals used. Beyond animal (or human) carcinogenicity as the “gold standard”, it is acknowledged that genotoxicity tests provide much information about cellular behaviour, cell division processes and cellular fate to a (geno)toxic insult. Since the disease impact of these effects is seldom known, and a verification of relevant toxicity is normally also the subject of (sub)chronic animal studies, the prediction of in vivo relevant results from in vitro genotoxicity tests is also important for aspects that may not have a direct impact on carcinogenesis as the ultimate endpoint of concern. In order to address the high rate of in vitro false positive results, a 2-day workshop was held at the European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy in April 2006. More than 20 genotoxicity experts from academia, government and industry were invited to review data from the currently available cell systems, to discuss whether there exist cells and test systems that have a reduced tendency to false positive results, to review potential modifications to existing protocols and cell systems that might result in improved specificity, and to review the performance of some new test systems that show promise of improved specificity without sacrificing sensitivity. It was concluded that better guidance on the likely mechanisms resulting in positive results that are not biologically relevant for human health, and how to obtain evidence for those mechanisms, is needed both for practitioners and regulatory reviewers. Participants discussed the fact that cell lines commonly used for genotoxicity testing have a number of deficiencies that may contribute to the high false positive rate. These include, amongst others, lack of normal metabolism leading to reliance on exogenous metabolic activation systems (e.g. Aroclor-induced rat S9), impaired p53 function and altered DNA repair capability. The high concentrations of test chemicals (i.e. 10mM or 5000μg/ml, unless precluded by solubility or excessive toxicity) and the high levels of cytotoxicity currently required in mammalian cell genotoxicity tests were discussed as further potential sources of false positive results. Even if the goal is to detect carcinogens with short in vitro tests under more or less acute conditions, it does not seem logical to exceed the capabilities of cellular metabolic turnover, activation and defence processes. The concept of “promiscuous activation” was discussed. For numerous mutagens, the decisive in vivo enzymes are missing in vitro. However, if the substrate concentration is increased sufficiently, some other enzymes (that are unimportant in vivo) may take over the activation—leading to the same or a different active metabolite. Since we often do not use the right enzyme systems for positive controls in vitro, we have to rely on their promiscuous activation, i.e. to use excessive concentrations to get an empirical correlation between genotoxicity and carcinogenicity. A thorough review of published and industry data is urgently needed to determine whether the currently required limit concentration of 10mM or 5000μg/ml, and high levels of cytotoxicity, are necessary for the detection of in vivo genotoxins and DNA-reactive, mutagenic carcinogens. In addition, various measures of cytotoxicity are currently allowable under OECD test guidelines, but there are few comparative data on whether different measures would result in different maximum concentrations for testing. A detailed comparison of cytotoxicity assessment strategies is needed. An assessment of whether test endpoints can be selected that are not intrinsically associated with cytotoxicity, and therefore are less susceptible to artefacts produced by cytotoxicity, should also be undertaken. There was agreement amongst the workshop participants that cell systems which are p53 and DNA-repair proficient, and have defined Phase 1 and Phase 2 metabolism, covering a broad set of enzyme forms, and used within the context of appropriately set limits of concentration and cytotoxicity, offer the best hope for reduced false positives. Whilst there is some evidence that human lymphocytes are less susceptible to false positives than the current rodent cell lines, other cell systems based on HepG2, TK6 and MCL-5 cells, as well as 3D skin models based on primary human keratinocytes also show some promise. Other human cell lines such as HepaRG, and human stem cells (the target for carcinogenicity) have not been used for genotoxicity investigations and should be considered for evaluation. Genetic engineering is also a valuable tool to incorporate missing enzyme systems into target cells. A collaborative research programme is needed to identify, further develop and evaluate new cell systems with appropriate sensitivity but improved specificity. In order to review current data for selection of appropriate top concentrations, measures and levels of cytotoxicity, metabolism, and to be able to improve existing or validate new assay systems, the participants called for the establishment of an expert group to identify the in vivo genotoxins and DNA-reactive, mutagenic carcinogens that we expect our in vitro genotoxicity assays to detect as well as the non-genotoxins and non-carcinogens we expect them not to detect. [Copyright &y& Elsevier]

In Japan, most cows are fed indoors, so cow manure must be carefully treated and used to manage soil fertility. The objective of this study was to compare the net greenhouse gas (GHG) balance (NGHGB) of Italian ryegrass - corn (maize) double-cropping fields receiving farmyard manure (FYM), slurry, or methane fermentation digestion liquid (MFDL). FYM, Slurry, MFDL, mineral fertilizer only (Fert.), and no-N control (Cont.) plots were set up in a randomized block design (n = 3). FYM, slurry, or MFDL was applied to meet the K requirement for forage production, and then mineral fertilizers were supplemented to meet the N and P requirements. From September 2017 to September 2020, C inputs as manure and crop residue, heterotrophic respiration (R H ), and emissions of methane (CH 4 ) and nitrous oxide (N 2 O) from soil were determined. The similarity of the total yields in FYM, Slurry, MFDL, and Fert. plots reflected judicious fertility management. However, the residue-C input of Italian ryegrass was 38% greater in FYM plots than in the other plots. Manure-C input decreased in the order of FYM > Slurry > MFDL plots. R H was greater in FYM and Slurry plots than in MFDL plots. Net ecosystem C balance (NECB) ([residue-C + manure-C] - [R H -C + CH 4 -C]) decreased in the order of FYM > Slurry > MFDL plots. N 2 O emission was greater in Slurry and MFDL plots than in FYM plots. Consequently, NGHGB ([CH 4 and N 2 O emissions] - NECB) in terms of CO 2 equivalent decreased in the order of MFDL > Slurry > FYM plots, so FYM application contributed most to GHG mitigation. Yield-scaled NGHGB was smallest in FYM plots owing to the synergy of the greatest residue-C and manure-C inputs, less N 2 O emission, and the achievement of a high enough yield, reflecting judicious fertility management based on manure and mineral fertilizer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author. Published by Elsevier B.V. All rights reserved.)

Updates in classification of gastro-entero-pancreatic neuroendocrine neoplasms better reflect the biological characteristics of these tumours. In the present study, we analysed the characteristics of neuroendocrine tumours that could aid in a more precise stratification of risk groups. In addition, we have highlighted the importance of grade (re)assessment based on investigation of secondary tumour lesions. Two hundred and sixty-four cases of neuroendocrine tumours of gastro-entero-pancreatic origin from three centres were included in the study. Tumour morphology, mitotic count and Ki67 labelling index were evaluated in specimens of primary tumours, lymph node metastases and distant metastases. These variables were correlated with overall survival (OS) and relapse-free survival (RFS). Tumour stage, number of affected lymph nodes, presence of tumour deposits and synchronous/metachronous metastases were tested as possible prognostic features. Mitotic count, Ki-67 labelling index, primary tumour site, tumour stage, presence of tumour deposits and two or more affected lymph nodes were significant predictors of OS and RFS. At the same time, mitotic count and Ki-67 labelling index can be addressed as continuous variables determining prognosis. We observed a very high correlation between the measures of proliferative activity in primary and secondary tumour foci. The presence of isolated tumour deposits was identified as an important determinant of both RFS and OS for pancreatic (hazard ratio [HR] = 7.61, 95% confidence interval [CI] = 3.96-14.6, P < 0.0001 for RFS; HR = 3.28, 95% CI = 1.56-6.87, P = 0.0017 for OS) and ileal/jejunal neuroendocrine tumours (HR = 1.98, 95% CI = 1.25-3.13, P = 0.0036 for RFS and HR 2.59, 95% CI = 1.27-5.26, P = 0.009 for OS). The present study identifies the presence of mesenterial tumour deposits as an important prognostic factor for gastro-entero-pancreatic neuroendocrine tumours, provides evidence that proliferative parameters need to be treated as continuous variables and further supports the importance of grade determination in all available tumour foci., (© 2021 British Society for Neuroendocrinology.)

Abstract: An 18-year-old female lion (Panthera leo) was referred to the Department of Animal Pathology of the University of Turin (Italy). At necropsy, multiple nodular, 4–20-mm, confluent white firm nodules were scattered throughout the pleural surfaces of the thoracic wall and of the lungs. Histological lesions were represented by proliferations of papillary structures lined by cuboidal basophilic mesothelial cells with large, oval nuclei and abundant granular eosinophilic cytoplasm. Immunohistochemistry revealed immunoreactivity for pancytokeratin and vimentin. None of the cells expressed calretinin antigen. Asbestos fibers and asbestos bodies were not detected respectively by light microscopy and by Scanning Electron Microscope–Energy Dispersive Spectrometer investigations. On the contrary, chrysotile asbestos were identified in samples from shelter material. Histological and immunohistochemical findings were consistent with the diagnosis of an epithelial malignant mesothelioma. To our best knowledge, this is the first report of a pleural mesothelioma in a lion. [Copyright &y& Elsevier]

The article offers information on the Second Hippo Tumor Suppressor Pathway Workshop that was held on November 2-5, 2010 at Chigi Palace in Ariccia, Rome, Italy. The workshop discusses the role of drosophila, which has Hippo (Hpo) kinase cascade, in the regulation of cell proliferation and apoptosis. The workshop also explains the association of Hpo pathway with ovarian and liver cancer.

The ethno-pharmaceutical use of the edible fixed oil produced from lentisk (Pistacia lentiscus) berries covers a long tradition in several Mediterranean regions. Many of the health-promoting properties of lentisk berry oil (LBO) have been associated with the content of polar (poly)phenolic compounds. However, the polar fraction (methanol 80%, v/v) of LBO (LBO-pf) remains poorly and inadequately characterized. We assessed the phytochemical composition (fatty acids, phytosterols and polyphenols) of cold-pressed LBO produced in Cilento (Campania region, Italy) over four years of production (2015-2018). Main phenolic compounds present in LBO-pf were identified and semi-quantified combining ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and HPLC with diode array detection. Phenolic compounds, also responsible for oil stability and antioxidant properties, are relatively abundant in LBO, compared to other edible oils. LBO-pf induced clear dose-dependent effects on the growth of HT-29 cell line derived from human colorectal adenocarcinoma, as evidenced by the cell cycle arrest. Our data support the health-promoting properties of cold-pressed LBO, which is obtained with good yield from spontaneous plants growing in semiarid regions.

Background: During the coronavirus disease-2019 (COVID-19) pandemic, Italian hospitals faced the most daunting challenges of their recent history, and only essential therapeutic interventions were feasible. From March to April 2020, the Laboratory of Advanced Cellular Therapies (Vicenza, Italy) received requests to treat a patient with severe COVID-19 and a patient with acute graft-versus-host disease with umbilical cord-derived mesenchymal stromal cells (UC-MSCs). Access to clinics was restricted due to the risk of contagion. Transport of UC-MSCs in liquid nitrogen was unmanageable, leaving shipment in dry ice as the only option., Methods: We assessed effects of the transition from liquid nitrogen to dry ice on cell viability; apoptosis; phenotype; proliferation; immunomodulation; and clonogenesis; and validated dry ice-based transport of UC-MSCs to clinics., Results: Our results showed no differences in cell functionality related to the two storage conditions, and demonstrated the preservation of immunomodulatory and clonogenic potentials in dry ice. UC-MSCs were successfully delivered to points-of-care, enabling favourable clinical outcomes., Conclusions: This experience underscores the flexibility of a public cell factory in its adaptation of the logistics of an advanced therapy medicinal product during a public health crisis. Alternative supply chains should be evaluated for other cell products to guarantee delivery during catastrophes.

Over the past decades, the occurrence, distribution and intensity of harmful algal blooms involving the dinoflagellate Alexandrium pacificum have increased in marine coastal areas disturbed by anthropogenic inputs. This invasive species produces saxitoxin, which causes the paralytic shellfish poisoning syndrome in humans upon consumption of contaminated seafood. Blooms of A. pacificum have been reported in metal-contaminated coastal ecosystems, suggesting some ability of these microorganisms to adapt to and/or resist in metal stress conditions. This study seeks to characterize the modifications in membrane proteomes (by 2-D electrophoresis coupled to LC-MS/MS), cell growth and morphometry (measured with an inverted microscope), in response to metal stress (addition of Zn 2+ , Pb 2+ , Cu 2+ and Cd 2+ ), in two Mediterranean A. pacificum strains: SG C10-3 and TAR C5-4F, respectively isolated from the Santa Giusta Lagoon (Sardinia, Italy) and from the Tarragona seaport (Spain), both metal-contaminated ecosystems. In the SG C10-3 cultures grown in a metal cocktail, cell growth was significantly delayed, and cell size increased (22% of 37.5 μm cells after 25 days of growth). Conversely, no substantial change was observed for cell growth or cell size in the TAR C5-4F cultures grown in a metal cocktail (P > 0.10), thus indicating intraspecific variability in the responses of A. pacificum strains to metal contamination. Regardless of the conditions tested, the total number of proteins constituting the membrane proteome was significantly higher for TAR C5-4F than for SG C10-3, which may help TAR C5-4F to thrive better in contaminated conditions. For both strains, the total number of proteins constituting the membrane proteomes was significantly lower in response to metal stress (29% decrease in the SG C10-3 proteome: 82 ± 12 proteins for controls, and 58 ± 12 in metal-contaminated cultures; 17% decrease in the TAR C5-4F proteome: 101 ± 8 proteins for controls, and 84 ± 5 in metal-contaminated cultures). Moreover, regardless of the strain, proteins with significantly modified expression in response to stress were mainly down-regulated (representing 45% of the proteome for SG C10-3 and 38% for TAR C5-4F), clearly showing the harmful effects of the metals. Protein down-regulation may affect cell transport (actin and phospholipid scramblase in SG C10-3), photosynthesis (RUBISCO in SG C10-3, light-harvesting protein in TAR C5-4F, and high-CO 2 -inducing periplasmic protein in both strains), and finally energy metabolism (ATP synthase in both strains). However, other modifications in protein expression may confer to these A. pacificum strains a capacity for adaptation and/or resistance to metal stress conditions, for example by (i) limiting the metal entry through the plasma membrane of the SG C10-3 cells (via the down-regulation of scramblase) and/or (ii) reducing the oxidative stress generated by metals in SG C10-3 and TAR C5-4F cells (due to down-regulation of ATP-synthase)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Wound-healing is a dynamic skin reparative process that results in a sequence of events, including inflammation, proliferation, and migration of different cell types as fibroblasts. Fibroblasts play a crucial role in repairing processes, from the late inflammatory phase until the fully final epithelization of the injured tissue. Within this context, identifying tools able to implement cell proliferation and migration could improve tissue regeneration. Recently, plants species from all over the world are coming out as novel tools for therapeutic applications thanks to their phytochemicals, which have antioxidant properties and can promote wound healing. In this paper, we aimed at investigating antioxidant activity of waste extracts from different medicinal plants, endemic of the Mediterranean area, on fibroblast proliferation and wound healing. We determined the amount of total phenols and anti-oxidant activity by ABTS assay. We then evaluated the cytotoxicity of the compounds and the proliferative capabilities of fibroblasts by scratch assay. Our results showed that waste extracts retain antioxidant and regenerative properties, inducing tissue re-establishment after environmental stress exposure. Taken together, our findings suggest that waste material could be used in the future also in combinations to stimulate wound healing processes and antioxidant responses in damaged skin., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The pathogenesis of endometriosis is unknown, but some evidence supports a genetic predisposition. The purpose of this study was to evaluate the recent literature on the genetic characterization of women affected by endometriosis and to evaluate the influence of polymorphisms of the wingless-type mammalian mouse tumour virus integration site family member 4 (WNT4), vezatin (VEZT), and follicle stimulating hormone beta polypeptide (FSHB) genes, already known to be involved in molecular mechanisms associated with the proliferation and development of endometriotic lesions in the Sardinian population. Materials and Methods: In order to provide a comprehensive and systematic tool for those approaching the genetics of endometriosis, the most cited review, observational, cohort and case-control studies that have evaluated the genetics of endometriosis in the last 20 years were collected. Moreover, 72 women were recruited for a molecular biology analysis of whole-blood samples-41 patients affected by symptomatic endometriosis and 31 controls. The molecular typing of three single nucleotide polymorphisms (SNPs) was evaluated in patients and controls: rs7521902, rs10859871 and rs11031006, mapped respectively in the WNT4, VEZT and FSHB genes. In this work, the frequency of alleles, genotypes and haplotypes of these SNPs in Sardinian women is described. Results: From the initial search, a total of 73 articles were chosen. An analysis of the literature showed that in endometriosis pathogenesis, the contribution of genetics has been well supported by many studies. The frequency of genotypes observed in the groups of the study population of 72 women was globally coherent with the law of the Hardy-Weinberg equilibrium. For the SNP rs11031006 (FSHB), the endometriosis group did not show an increase in genotypic or allelic frequency due to this polymorphism compared to the control group (p = 0.9999, odds ratio (OR) = 0.000, 95% confidence interval (CI), 0.000-15.000 and p = 0.731, OR = 1639, 95% CI, 0.39-683, respectively, for the heterozygous genotype and the polymorphic minor allele). For the SNP rs10859871 (VEZT), we found a significant difference in the frequency of the homozygous genotype in the control group compared to the affected women (p = 0.0111, OR = 0.0602, 95% CI, 0.005-0.501). For the SNP rs7521902 (WNT4), no increase in genotypic or allelic frequency between the two groups was shown (p = 0.3088, OR = 0.4133, 95% CI, 0.10-1.8 and p = 0.3297, OR = 2257, 95% CI, 0.55-914, respectively, for the heterozygous genotype and the polymorphic minor allele). Conclusion: An analysis of recent publications on the genetics of endometriosis showed a discrepancy in the results obtained in different populations. In the Sardinian population, the results obtained do not show a significant association between the investigated variants of the genes and a greater risk of developing endometriosis, although several other studies in the literature have shown the opposite. Anyway, the data underline the importance of evaluating genetic variants in different populations. In fact, in different ethnic groups, it is possible that specific risk alleles could act differently in the pathogenesis of the disease.

In Italy a particularly valuable chestnut is "Marrone di Roccadaspide", a protected geographical indication (PGI) product, deriving from a Castanea sativa cultivar, typical of Salerno province in Campania region. As chestnut industrial processes yield a large amount of shell by-products, in this study the possibility to retrain this waste food as potential source of bioactives was investigated. The ability of "Marrone di Roccadaspide" shell MeOH extract to modulate the pro-inflammatory transcriptional factor NF-κB after LPS stimulation, along with the antioxidant activity by a cell-based in vitro test, were evaluated. To correlate the NF-κB inhibition (67.67% at 5 μg/mL) and the strong antioxidant activity to the chemical composition, an analytical approach based on LC-ESI/LTQOrbitrap/MS/MS n along with NMR characterization of isolated compounds was developed. The identification of hydrolysable and condensed tannins, along with flavonoids, phenol glucosides, ellagic acid derivatives, and triterpenoids was accomplished. The most representative compounds were quantitatively analyzed by LC-ESI/QTrap/MS/MS, showing bartogenic acid as the compound occurring in the highest amount (103.08 mg/100 g shells). With the aim to explore the possibility to employ chestnut shells as suitable source of bioactives for the preparation of functional ingredients, the chemical composition and the antioxidant activity of "eco-friendly" extracts (EtOH and EtOH:H 2 O 7:3) was finally evaluated, showing a high superimposability of the EtOH:H 2 O (7:3) extract to the MeOH extract., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 4% and desperately needs novel effective therapeutics. Integrin αvβ6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed that high levels of β6 mRNA correlated strongly with significantly poorer survival (n = 491 cases, p = 3.17 × 10 -8 ). In two separate cohorts, we showed that over 80% of PDACs expressed αvβ6 protein and that paired metastases retained αvβ6 expression. In vitro, integrin αvβ6 promoted PDAC cell growth, survival, migration, and invasion. Treatment of both αvβ6-positive human PDAC xenografts and transgenic mice bearing αvβ6-positive PDAC with the αvβ6 blocking antibody 264RAD, combined with gemcitabine, significantly reduced tumour growth (p < 0.0001) and increased survival (log-rank test, p < 0.05). Antibody therapy was associated with suppression of tumour cell activity (suppression of pErk growth signals, increased apoptosis seen as activated caspase-3) and suppression of the pro-tumourigenic microenvironment (suppression of TGFβ signalling, fewer αSMA-positive myofibroblasts, decreased blood vessel density). These data show that αvβ6 promotes PDAC growth through both tumour cell and tumour microenvironment mechanisms and represents a valuable target for PDAC therapy. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Benign terminal deoxynucleotidyl transferase (TdT)-positive cells have been documented in a variety of nonhematopoietic tissues. Scant data are, however, available on their presence in nonneoplastic lymph nodes. This study is aimed to (1) characterize the presence/distribution of benign TdT-positive cells in pediatric and adult reactive lymph nodes and (2) define the phenotype and nature of such elements. This retrospective study considered 141 reactive lymph nodes from pediatric and adult patients without history of neoplastic disease. TdT-positive cells were characterized by immunohistochemical and morphometric analyses, and their presence was correlated with the clinical-pathological features. The nature of TdT-positive cells was investigated by (1) double immunostaining for early lymphoid cell markers and (2) assessment of TdT expression in fetal lymph nodes. Sparse TdT-positive cells were documented in all pediatric cases and in most (76%) adult lymph nodes. TdT-positive cell density was higher in children than adults (15.9/mm 2 versus 8.6/mm 2 ; P < .05). TdT positivity did not correlate with any clinical or histological parameter, and double immunostaining disclosed a phenotype compatible with early lymphoid precursors (positivity for CD34 and CD10, and variable expression of CD7). A very high TdT-positive cell density (802.4/mm 2 ) was reported in all fetal lymph nodes. In conclusion, TdT-positive cells are a common finding in pediatric and adult lymph nodes. The interstitial distribution and low number of such cells allow for the differential diagnosis with precursor lymphoid neoplasms. The high density in fetal lymph nodes and the phenotype of such cells suggest their belonging to an immature lymphoid subset gradually decreasing with age., (Copyright © 2018 Elsevier Inc. All rights reserved.)

CD4 + regulatory T cells have been intensively studied during aging, but little is still known about age-related changes of other regulatory T cell subsets. It was, therefore, the goal of the present study to analyze CD8 + human leukocyte antigen-antigen D related (HLADR) + T cells in old age, a cell population reported to have suppressive activity and to be connected to specific genetic variants. We demonstrate a strong increase in the number of CD8 + HLADR + T cells with age in a cohort of female Sardinians as well as in elderly male and female persons from Austria. We also show that CD8 + HLADR + T cells lack classical activation molecules, such as CD69 and CD25, but contain increased numbers of checkpoint inhibitory molecules, such as cytotoxic T lymphocyte-associated antigen 4, T cell immunoglobulin and mucin protein-3, LAG-3, and PD-1, when compared with their HLADR - counterparts. They also have the capacity to inhibit the proliferation of autologous peripheral blood mononuclear cells. This suppressive activity is, however, decreased when CD8 + HLADR + T cells from elderly persons are analyzed. In accordance with this finding, CD8 + HLADR + T cells from persons of old age contain lower percentages of checkpoint inhibitory molecules than young controls. We conclude that in spite of high abundance of a CD8 + regulatory T cell subset in old age its expression of checkpoint inhibitory molecules and its suppressive function on a per cell basis are reduced. Reduction of suppressive capacity may support uncontrolled subclinical inflammatory processes referred to as "inflamm-aging."

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) is a rare disorder which can be an incidental finding in imaging tests performed during the investigation of another condition, or is the final diagnosis in patients evaluated for chronic obstructive complaints. To explore the possible association between specific histopathology features and the mode of clinical presentation, we retrieved the clinical, functional, radiological, and pathological data of all 13 patients diagnosed with DIPNECH at our Institution over a 14-year period (2000-2014). As compared to patients with incidental disease (6/13, 46 %), patients with symptomatic disease were younger [mean (SD): 57.7 vs. 68.7 years, p = 0.046], were more likely to have mosaic attenuation (100 vs. 0 %, p = 0.001) and small multiple nodules (100 vs. 17 %, p = 0.005) at CT, and showed a significantly higher number of foci of linear neuroendocrine proliferation [median (IQR): 28 (13-37) vs. 6 (5-13), p = 0.018] and of tumorlets [median (IQR): 10 (8-20) vs. 1 (1-1), p = 0.002] at histology. Incidental disease was found in association with pulmonary adenocarcinoma in five out of six patients (83.3 %). The results of our study provide preliminary evidence that symptomatic patients with DIPNECH represent a specific subset characterized by younger age and a higher burden of foci of neuroendocrine proliferation.

Aim: To evaluate the optimal time interval from definitive surgery to commencing chemotherapy in early breast cancer (EBC)., Patients and Methods: The relationship between time to initiation of adjuvant chemotherapy (TTC), calculated in weeks, and disease-free (DFS) or overall survival (OS), was assessed in 921 EBC patients with rapidly proliferating tumours (thymidine labelling index >3% or G3 or Ki67 >20%), randomised in a phase III clinical trial (NCT01031030) to receive chemotherapy with or without anthracyclines (epirubicin→cyclophosphamide, methotrexate and fluorouracil (CMF) versus CMF→epirubicin versus CMF). DFS, OS and 95% confidence intervals (95% confidence interval (CI)) were calculated by the Kaplan-Meier method. Multivariate Cox analysis was performed in relation with nodal involvement, oestrogen receptor and human epidermal growth factor receptor 2 (HER2) status, Ki67 value, type of adjuvant chemotherapy, menopausal status and tumour size., Results: At a median follow-up of 105 months (range 2-188), a prolonged TTC resulted in a significant increase in the risk of relapse: hazard ratio (HR) 1.15 (95% CI 1.02-1.30, p=0.019). Using a backward elimination procedure, TTC, tumour size and nodal involvement remained significantly associated with DFS. A time-dependent receiver-operating characteristic (ROC) curve analysis was subsequently utilised to evaluate the best cut-off for TTC, identifying 7 weeks as the best threshold for longer OS (p=0.043): 8-year OS 88% (95% CI 85-90) for patients with a TTC <7 weeks and 78% (95% CI 68-87) for the other group., Conclusions: Our results confirm that a shorter TTC may reduce relapses and possibly also improve clinical outcome in patients with highly proliferating EBC., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Oxford classification of Immunoglobulin A Nephropathy (IgAN) identifies four pathological features as predictors of renal outcome (MEST-score): mesangial proliferation (M); endocapillary proliferation (E); segmental glomerulosclerosis (S); tubular atrophy/interstitial fibrosis (T). In particular extracapillary proliferation (Ex) was not considered as an independent histological variable predicting renal outcome. Recently the VALIGA study provided a validation of the Oxford classification in a large European cohort of IgAN patients and re-stated that Ex is not associated with a worse renal prognosis. We propose a retrospective study to evaluate the predictive value of the MEST-score in a multi-centre, single region group of patients from central Italy and in addition, to investigate Ex as a marker predicting renal outcome., Methods: One hundred and seven patients were enrolled in this study. Clinical data of each patient were available at diagnosis and follow-up. The median age at diagnosis was 36.7 years; 72% of the patients were males. Histological parameters were those included in the MEST-score of the Oxford classification; in addition, Ex was also assessed., Results: Multiple linear regression models for survey were used. Statistical analysis showed a correlation between the progression of renal decline, in terms of estimated glomerular filtration rate (slope eGFR), and M, S, T. Differently from Oxford and VALIGA studies, no correlation was found with E, while Ex correlated with a decline of eGFR., Conclusions: Our results suggest that Ex represents an additional independent variable associated with a faster decline of renal function in IgAN., (© 2015 Asian Pacific Society of Nephrology.)

Aims and Background: The aim of this retrospective multicenter study was to evaluate the impact of progesterone receptor (PgR) loss on locoregional recurrence in patients with estrogen receptor (ER)-positive primary breast cancer and ER-positive locoregional recurrence., Patients and Methods: Eight Italian oncology centers collected data from consecutive patients with ER-positive breast cancer and a subsequent ER-positive locoregional recurrence., Results: Data were available for 265 patients diagnosed with breast cancer between 1990 and 2009. Median metastasis-free survival was 111 months in patients with PgR-positive primary tumors and locoregional recurrence (PgRpos), 38 months in patients with PgR-negative primary tumors and locoregional recurrence (PgRneg), and 63 months in patients with PgR-positive primary tumors and PgR-negative locoregional recurrence (PgRloss). In multivariate analysis, PgR status was independently associated with metastasis-free survival, with a hazard ratio of 2.84 (95% CI 1.34-6.00) for PgRneg compared with PgRpos, and 2.93 (95% CI: 1.51-5.70) for PgRloss compared with PgRpos., Conclusions: PgR absence was found to be a negative prognostic factor in breast cancer patients with ER-positive locoregional recurrence. Thus, PgR status could be a biological marker in ER-positive recurrent breast cancer.

Objective: To test the immunohistochemical staining pattern of some mismatch repair (MMR) system proteins in endometriotic tissue (ET) and eutopic endometrium., Methods: This was a retrospective study conducted at the Pathology and Obstetrics and Gynecology Departments of the Udine University Hospital. We analyzed 528 samples obtained from 246 patients affected by endometriosis and 71 samples from 71 patients with normal endometrium. A tissue microarray model was used to analyze the immunohistochemical expression of MMR system proteins., Results: Significant loss of MMR proteins was found in the stromal component of ETs. We found MSH2 to be expressed at a higher level than any other MMR system proteins in eutopic endometrium and ETs, to be significantly correlated to Ki-67 expression in both stromal and glandular components of ETs, and to be expressed at a significantly higher level in ETs than in eutopic endometrium. When considering the subgroup of endometriosis with high recurrence rate and glandular cytoplasmic staining for aurora A kinase, we found MMR proteins expressed at a significantly higher level in these ETs than in other ETs and eutopic endometrium of unaffected women., Conclusions: We found significant loss of MMR proteins (known to be associated with microsatellite instability) in the stromal component of ETs. The group of ETs with glandular cytoplasmic staining for aurora A kinase had higher MMR protein expression, suggesting an increased activity of this system. Our result suggests a novel role of increased MSH2 expression in cellular proliferation of endometriosis.

Background: Information on malignant pancreatic neuroendocrine neoplasms (pNENs) is mostly from retrospective studies in highly selected patients. The aim of this prospective, multicentre study was to assess treatment and outcomes of malignant pNENs in clinical practice., Patients and Methods: Consecutive patients with newly diagnosed, histologically-proven pNENs were included and followed-up for 2 years. Tumours were defined as malignant when nodal or distant metastases were present or invasion of extrapancreatic structures/organs was evident., Results: A total of 140 patients with malignant pNENs were included. Ninety-eight patients (70.0%) underwent a surgical resection (76 radical and 22 palliative). Other non-surgical treatments were used in 101 patients (72.1%): somatostatin analogues (n = 63), chemotherapy (n = 30), ablative treatments (n = 15) and peptide-receptor radionuclide therapy (n = 14). No relationship was observed between the 2010 WHO classification and type of treatment. A surgical resection was more often performed in incidentally detected tumours located in the pancreas body tail. Two-year progression-free survival was 63.8%: 82% after a radical resection, 44% after a palliative resection and 41% without a resection. A radical resection and Ki67 proliferative index >5% and >10% were the only significant prognostic determinants in multivariate analysis., Conclusions: A radical resection is the cornerstone treatment of malignant pNENs and represents, together with Ki67 assessment, the most powerful prognostic factor for 2-year outcomes., (© 2013 International Hepato-Pancreato-Biliary Association.)

Purpose: Biological markers are crucial factors in order to differentiate female breast cancers and to determine the right therapy. This study aims at evaluating whether testing for biomarkers for female breast cancer has similar frequency and characteristics across and within countries., Methods: Population-based cancer registries of the Association for cancer registration and epidemiology in Romance language countries (GRELL) were asked to complete a questionnaire on biomarkers testing. The data collected referred to invasive female breast cancer cases diagnosed between 2004 and 2009. The investigation focused on 1) the overexpression and amplification of the human epidermal growth factor receptor 2 oncogene (HER2); 2) the expression of oestrogen (ER) and progesterone (PgR) receptors; and 3) the proliferation index (PI). Weighted percentages, the heterogeneity among and within countries, and the correlation between responses and calendar years were evaluated. The study was based on 19,644 breast cancers., Results: Overall, 85.9% of the cases were tested for HER2, 91.8% for both ER and PgR, and 74.1% for proliferative markers. For HER2 and ER-PgR, the frequency of testing increased from 2004 to 2009. Testing varied among countries (HER2 from 82.0% to 95.9%, ER-PgR from 89.3% to 98.9%, PI from 10% to 92%) and also within the same country (e.g. HER2 in Italy from 51% to 99%) as well as within single cancer registries. The most relevant differences were in the scores for positive/negative/not clearly defined HER2 (e.g. HER2 was defined positive if IHC 3+ in 21/33 registries), and in the cut-off of positive cells for ER/PgR (from >0% to >30%) and PI positivity (from >0% to >20%)., Conclusions: Biological markers are widely tested in the Romance language countries; however, the parameters defining their positivity may vary, raising concerns about homogeneity in breast cancer classification and treatment., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The microalgal biomass applications strongly depend on cell composition and the production of low cost products such as biofuels appears to be economically convenient only in conjunction with wastewater treatment. As a preliminary study, in view of the development of a wastewater treatment pilot plant for nutrient removal and algal biomass production, a biological wastewater system was carried out on a laboratory scale growing a newly isolated freshwater algal strain, Desmodesmus communis, and a natural consortium of microalgae in effluents generated by a local wastewater reclamation facility. Batch cultures were operated by using D. communis under different growth conditions to better understand the effects of CO₂, nutrient concentration and light intensity on the biomass productivity and biochemical composition. The results were compared with those obtained using a natural algal consortium. D. communis showed a great vitality in the wastewater effluents with a biomass productivity of 0.138-0.227 g L⁻¹ d⁻¹ in the primary effluent enriched with CO₂, higher biomass productivity compared with the one achieved by the algal consortium (0.078 g L⁻¹ d⁻¹). D. communis cultures reached also a better nutrient removal efficiency compared with the algal consortium culture, with almost 100% for ammonia and phosphorous at any N/P ratio characterizing the wastewater nutrient composition. Biomass composition was richer in polysaccharides and total fatty acids as the ammonia concentration in the water decreased. In view of a future application of this algal biomass, due to the low total fatty acids content of 1.4-9.3 wt% and the high C/N ratio of 7.6-39.3, anaerobic digestion appeared to be the most appropriate biofuel conversion process., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In 2005, in accordance with recommendations made by the European Medicines Agency, the Italian Drug Agency ordered withdrawal of the hexavalent Hexavac(®) vaccine (Sanofi Pasteur MSD) from the market. Concerns had been raised about the low immunogenicity of the hepatitis B virus component of the vaccine, assessed by measurement of serum antibody levels, and its potential consequences on long-term protection against hepatitis B infection. We evaluated memory T cell response to establish whether there are differences in the protective mechanisms among children who had received either Hexavac(®) or Infanrix-hexa(®) (GlaxoSmithKline) as their primary vaccination. Immunological memory was determined by measuring the ability of T cells to proliferate and secrete IFNγ by ELISA and intracellular cytokines (IFNγ and IL-2) when cultured with hepatitis B surface antigen (HBsAg). The different memory subsets of T cells were also measured. The results indicate that, although they generate different serum antibody levels, both vaccines are efficient in generating T recall responses in vitro five years after the primary vaccination. The less immunogenic Hexavac(®) vaccine induces a strong T antigen response, as indicated by increased blast proliferation and the enhanced presence of memory subsets after HBsAg recall stimulation. These findings suggest that cellular immune response should be considered alongside serological markers as a surrogate of protection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Drug eluting stent (DES) use has improved clinical outcome after percutaneous coronary interventions. However, DES-treated patients may have a higher risk of stent thrombosis, mainly due to uncorrect stent deployment or lack of reendothelialization. Thus, the availabilily of different approaches with comparable efficacy to DES, but higher safety, especially in bleeding-prone patients, have to be investigated., Study Design: The EREMUS is a multicenter open-label prospective randomized three-arm clinical trial that will investigate the efficacy of a paclitaxel coated balloon + an endothelial progenitor capture stent for the treatment of native coronary lesions, and compare it to a DES strategy, or the sole endothelial progenitor capture stent. An angiographic follow-up with optical coherence tomography analysis will be scheduled 9 months after index procedure. Noninferiority regarding the primary endpoint (late luminal loss) between study group and DES is hypothesized. All patients will undergo clinical follow-up until 24 months from index hospitalization., Conclusions: The EREMUS trial will determine whether a composite approach with a paclitaxel coated balloon + an endothelial progenitor capture stent in coronary lesions at medium-to-high risk of restenosis will achieve similar results compared to DES regarding inhibition of intrastent proliferation; complete stent strut reendothelialization, a safety issue, will also be investigated., (Copyright © 2011 Wiley-Liss, Inc.)

The association between levels of circulating endothelial progenitor cells (EPCs) and heart transplant recipients (HTX) with cardiac allograft vasculopathy (CAV) is under debate. The chemokine receptor CXCR4 plays an important role in the mobilization of progenitor cells and is implicated in pathological conditions, including cardiovascular disease. This study aims to evaluate the association between EPCs and CXCR4-positive cells in HTX patients. Peripheral blood mononuclear cells (PBMCs) from 34 HTX patients and 25 control participants were analyzed by flow cytometry for CXCR4-positive cells and EPCs. Endothelial progenitor cells were defined by the expression of a range of hematopoietic and endothelial lineage markers in different combinations. The ability to form endothelial cell colonies in vitro was also assessed by colony-forming unit (CFU) assay. Phenotypic analysis of EPCs by flow cytometry revealed similar levels in HTX patients compared to controls. In addition, no difference was observed between levels of EPCs or CFU number in patients with and without CAV. By contrast, CFU assay revealed a reduced number of CFUs in HTX patients compared to controls (3.3% ± 0.95 and 13.3% ± 4.5%, respectively, P = 0.014). Likewise, levels of CXCR4-positive cells were significantly reduced (15.9 ± 1.4 in patients vs 24.8 ± 3.3% in controls, P < 0.01), negatively correlated with Framingham risk score (rho = -0.4, P = 0.02) and the number of risk factors (rho = -0.3, P = 0.049). Levels of CXCR4-positive cells were also correlated with CFU number (r = 0.65, P = 0.0005). These findings further develop our understanding of the role of EPCs and endothelial CFUs in cardiovascular disease, in addition to highlighting the potential importance of CXCR4 in heart transplantation.

Pancreatic endocrine tumors are rare diseases and devising a clinically effective prognostic stratification of patients is a major clinical challenge. This study aimed at assessing whether the tumor-node-metastasis (TNM)-based staging and proliferative activity-based grading recently proposed by the European NeuroEndocrine Tumors Society (ENETS) have clinical value. TNM was applied to 274 patients with histologically diagnosed pancreatic endocrine tumors operated from 1991 to 2005, with last follow-up at December 2007. According to World Health Organization (WHO) classification, 246 were well-differentiated neoplasms (51 benign, 56 uncertain behavior, 139 carcinomas) and 28 poorly differentiated carcinomas. Grading was based on Ki67 immunohistochemistry. Survival analysis not only ascertained the prognostic value of the TNM system but also highlighted that in the absence of nodal and distant metastasis, infiltration and tumor dimensions over 4 cm had prognostic significance. T parameters were then appropriately modified to reflect this weakness. The 5-year survival for modified TNM stages I, II, III and IV were 100, 93, 65 and 35%, respectively. Multivariate analysis identified TNM stages as independent predictors of death, in which stages II, III and IV showed a risk of death of 7, 29 and 58 times higher than stage I tumors (P<0.0001). Ki67-based grading resulted an independent predictor of survival with cut-offs at 5 and 20%. In conclusion, WHO classification assigns clinically significant diagnostic categories to pancreatic endocrine tumors that need prognostic stratification by applying a staging system. The ENETS-TNM provides the best option, but it requires some modifications to be fully functional. The modified TNM described in this study ameliorates the clinical applicability and prediction of outcome of the ENETS-TNM; it (i) assigns a risk of death proportional to the stage at the time of diagnosis, and (ii) allows a clinically based staging of patients, as the T parameters as modified permit their clinical-radiological recognition. Ki67-based grading discerns prognosis of patients with same stage diseases.

Much study has been dedicated to the understanding of the mechanisms leading to the progression of renal injury and to the development of strategies to limit this progression or possibly induce tissue regeneration. Among several identified mechanisms, the role of angiotensin II is widely recognized. Moreover, the progression of glomerular damage is characterized by capillary loss, reduction of the proliferative response, and production of antiangiogenic factors. Several lines of evidence support the potential effect of therapeutic startegies aimed at interfering with angiotensin II or stimulating angiogenesis in order to reduce the progression of renal injury. Recent work has underlined the potential of strategies involving the use of stem cells. Different populations of stem cells have been identified in the adult kidney. During renal injury, stem cells derived from the bone marrow that migrate through the circulation to the kidney may contribute to tissue repair. The regenerative potential of stem cells could be exploited by administration of ex vivo expanded stem cell populations or by the development of techniques to expand and differentiate local stem cells.

In human prostate cancer, Arginase 2 (ARG2) and nitric oxide synthase (NOS) are concomitantly expressed by tumor cells, and induce tumor immune escape via peroxynitrite-dependent Tyrosine nitrosylation. Since there were no data regarding this immune suppressive mechanism in other tumor types, and an evaluation of its clinical relevance in human tumors had still to be provided, we have investigated presence and clinical relevance of ARG2 and NOS expression in lung cancer. No evidence of NOS expression was found, no significant NOS enzymatic activity was detected. Instead, ARG2 protein was expressed by tumor cells. In a cohort of 120 patients, the amount of ARG2-positive tumor cells was significantly higher in small cell lung cancers (SCLC) than in non-small cell lung cancers (NSCLC). Large cell undifferentiated carcinomas had twice ARG2 than the other NSCLC subtypes. ARG2 expression was increased in Grade 3 tumors, as compared to Grades 1 and 2. However, no relationship was found with tumor size and stage, and with patient survival. Indeed, the enzyme was active, since the Arginine catabolite Ornithine was produced, but Arginine depletion was not attained. In addition, nitrotyrosine was not found in tumor tissue. Accordingly, when tumor cells isolated from lung cancer were incubated with activated autologous T cells, no inhibition of proliferation was detected. Our results indicate that ARG2 is expressed in lung cancer, but it does not induce tumor immune escape and does not affect disease progression, most probably due to the lack of concomitant NOS expression.

Objective: Several requirements need to be fulfilled for clinical use of expanded hematopoietic stem cells (HSCs). Because most cord blood (CB) samples are frozen in single bags and only an aliquot ( approximately 25%) of the blood can be expanded, the thawing and refreezing of samples must be validated in the current European and Italian Good Manufacturing Practice (eIGMP) conditions. Here, we describe in vitro and in vivo validation of the phase I/II protocol for CD34+ expansion of thawed CB units according to the current Cell Therapy Products (CTPs) Guidelines., Materials and Methods: CB units were thawed and 25% of the total volume was processed for CD34+ selection by CliniMACS. The 75% of the unit was immediately refrozen. CD34+ cells were expanded for 3 weeks with stem cell factor, Flt-3/Flk-2 ligand, thrombopoietin, and interleukin-6., Results: In vitro results demonstrated that this culture system induces expansion of thawed CD34+ (median value = 8.3). In vivo data demonstrated that after culture, the final CTPs maintain their repopulating ability in nonobese diabetic severe combined immunodeficient (SCID) mice. Limiting dilution assays performed by injecting decreasing doses of expanded CD34+ cells revealed that the frequency of SCID repopulating cells after ex vivo expansion is 1:8,034. Analyses for sterility, viability, cell senescence, and cytogenetic assessment demonstrated that expansion procedures in eIGMP conditions are safe for clinical protocols., Conclusions: This offers promising new options for expansion of allogenic HSCs and also for autologous usage in transplantation and other cell therapy protocols.

An immunological comparison of three different third companions (abacavir [ABC], efavirenz [EFV], ritonavir-boosted indinavir [IDVr]) on a backbone of either zidovudine plus didanosine (AZT/ddI) or zidovudine plus lamivudine (AZT/3TC) was performed in 76 HIV-infected, advanced-naive patients. Baseline median CD4 count and viremia were 217/microL and 238,301 copies/mL, respectively. Immunologic parameters were measured at baseline and after months of therapy. By the end of the study, 36 patients (mostly in the protease inhibitor [PI]-containing arms) had dropped out of the study; 22/36 cases of drop out were due to tolerability issues. All regimens resulted in increases in CD4 counts, with the most solid changes seen in patients using ABC as a third companion. Median HIV plasma viremia at month 12 was <50 copies/mL, and viremia was undetectable in 26/38 patients (68%). At the end of the study period, HIV antigen- and mitogen-stimulated proliferation overall was better in patients using either of the PI-boosted third companions. In these patients, the strongest down-modulation of activation marker-bearing cells was also observed. Finally, CD8+/28-/CD45RA+ lymphocytes (effector cells) were increased in all groups of patients with the exception of individuals receiving PI-boosted therapies. Results of this pilot study, although very preliminary, suggest that different combinations of antivirals result in a range of effects on immune cell functions. The clinical implications of these results need to be further analyzed in follow-up studies and in larger cohorts of patients.

Purpose: To evaluate the clinical features of breast cancer patients with genetic susceptibility to this disease and to investigate the contribution of BRCA1 germline mutations to the phenotype of these tumors., Patients and Methods: We reviewed the clinical and pathological records of 102 women with suspected inherited susceptibility to breast cancer consecutively seen at the Genetic Oncology Service of Parma, Italy. Sixty-two patients with a high probability of harboring a germline, cancer-predisposing mutation were tested for BRCA1 mutations. Exon 11 was screened using the protein truncation test and detected mutations were confirmed by direct sequencing (DS). All other exons were analyzed by DS., Results: Among the 62 patients with a completed mutation analysis, 48 (77.4%) had wild-type BRCA1, six (9.6%) had variants of unclear significance, eight (13%) had deleterious mutations. BRCA1-associated breast cancers (BABC) were significantly less likely to be diagnosed at stage I than breast cancers in women without mutations (12.5% vs 51%; P = 0.045), more likely to have a high proliferation rate (100% vs 24%, P < 0.001), and more likely to be histological grade 3 (100% vs 14%, P < 0.001), estrogen and progesterone receptor negative (87.5% vs 13%, P < 0.001; 75% vs 23%, P = 0.004), and p53 positive (87.5% vs 30%, P = 0.023). All tumors with BRCA1 mutations were HER-2/neu negative compared with 57% of the non-BRCA1 tumors (P = 0.04). There were no significant differences between BABC and non-BABC in 20-year relapse-free survival, 20-year event-free survival, and 20-year overall survival., Conclusion: In this population-based study, BABC seems to present with adverse molecular features when compared with non-BABC, although the prognosis appears to be similar.