Corrigendum to "Circulating Cancer Stem Cell-Derived Extracellular Vesicles as a Novel Biomarker for Clinical Outcome Evaluation".

[This corrects the article DOI: 10.1155/2019/5879616.].

Circulating Cancer Stem Cell-Derived Extracellular Vesicles as a Novel Biomarker for Clinical Outcome Evaluation.

The recent introduction of the "precision medicine" concept in oncology pushed cancer research to focus on dynamic measurable biomarkers able to predict responses to novel anticancer therapies in order to improve clinical outcomes. Recently, the involvement of extracellular vesicles (EVs) in cancer pathophysiology has been described, and given their release from all cell types under specific stimuli, EVs have also been proposed as potential biomarkers in cancer. Among the techniques used to study EVs, flow cytometry has a high clinical potential. Here, we have applied a recently developed and simplified flow cytometry method for circulating EV enumeration, subtyping, and isolation from a large cohort of metastatic and locally advanced nonhaematological cancer patients (N = 106); samples from gender- and age-matched healthy volunteers were also analysed. A large spectrum of cancer-related markers was used to analyse differences in terms of peripheral blood circulating EV phenotypes between patients and healthy volunteers, as well as their correlation to clinical outcomes. Finally, EVs from patients and controls were isolated by fluorescence-activated cell sorting, and their protein cargoes were analysed by proteomics. Results demonstrated that EV counts were significantly higher in cancer patients than in healthy volunteers, as previously reported. More interestingly, results also demonstrated that cancer patients presented higher concentrations of circulating CD31+ endothelial-derived and tumour cancer stem cell-derived CD133 + CD326- EVs, when compared to healthy volunteers. Furthermore, higher levels of CD133 + CD326- EVs showed a significant correlation with a poor overall survival. Additionally, proteomics analysis of EV cargoes demonstrated disparities in terms of protein content and function between circulating EVs in cancer patients and healthy controls. Overall, our data strongly suggest that blood circulating cancer stem cell-derived EVs may have a role as a diagnostic and prognostic biomarker in cancer.

Cryopreservation effects on Wharton's Jelly Stem Cells proteome.

Cryopreservation is the only method for long-term storage of viable cells and tissues used for cellular therapy, stem cell transplantation and/or tissue engineering. However, the freeze-thaw process strongly contributes to cell and tissue damage through several mechanisms, including oxidative stress, cell injury from intracellular ice formation and altered physical cellular properties. Our previous proteomics investigation was carried out on Wharton's Jelly Stem Cells (WJSCs) having similar properties to adult mesenchymal stem cells and thus representing a rich source of primitive cells to be potentially used in regenerative medicine. The aim of the present work was to investigate molecular changes that occur in WJSCs proteome in different experimental conditions: fresh primary cell culture and frozen cell. To analyze changes in protein expression of WJSCs undergoing different culturing procedures, we performed a comparative proteomic analysis (2DE followed by MALDI-TOF MS/MS nanoESI-Q-TOF MS coupled with nanoLC) between WJSCs from fresh and frozen cell culturing, respectively. Frozen WJSCs showed qualitative and quantitative changes compared to cells from fresh preparation, expressing proteins involved in replication, cellular defence mechanism and metabolism, that could ensure freeze-thaw survival. The results of this study could play a key role in elucidating possible mechanisms related to maintaining active proliferation and maximal cellular plasticity and thus making the use of WJSCs in cell therapy safe following bio-banking.

Intracranial haemorrhage in patients on antithrombotics: clinical presentation and determinants of outcome in a prospective multicentric study in Italian emergency departments.

BACKGROUND: Intracranial haemorrhage (ICH) is the type of stroke associated with the highest death rate, and about 30% of ICH occurs in patients on antithrombotic treatment. This study relates clinical presentations and outcome of ICH patients on oral anticoagulant (OA) or antiplatelet (AP) therapy admitted to 33 Italian emergency departments (ED). METHODS: Consecutive patients were enrolled after cranial computed tomography (CT). Primary outcome was the Modified Rankin Scale (MRS) score at 3 months of follow-up. Common descriptive statistics were computed after stratification for traumatic or spontaneous ICH and identification of the anatomical location of bleeding. Multivariate logistic regression was used to assess predictors of death. RESULTS: We recruited 434 patients on AP therapy and 232 on OA. There were 432 spontaneous and 234 traumatic ICH patients. The proportions of AP and OA patients undergoing neurosurgery were 21.8 and 19.4%, respectively, while < 30% underwent procoagulant medical treatment. At the 3-month follow-up, the case fatality rate was 42.0%, while disability or death (MRS 3-6) was 68.1%. The odds ratio for death in OA versus AP patients was 2.63 (95% CI 1.73-4.00) in the whole population and 2.80 (95% CI 1.77-4.41) in intraparenchymal event patients. Glasgow Coma Scale, age, spontaneous event and anticoagulant use were found to be predictors of death both in traumatic and spontaneous events. CONCLUSION: This study confirms the high prevalence of death or disability in OA and AP patients with ICH. As far as the determinants of mortality and disability are concerned, the results of this study might be useful in the clinical management and allocation of resources in the ED setting. The observed low use of procoagulant therapy highlights the need for ED educational programmes to heighten the awareness of available and effective haemostatic treatments.

A model for single and multiple knowledge based networks.

The inherent black-box nature of neural networks is an important drawback with respect to the problem of explanation of neural network responses. Although several articles have tackled the problem of rule extraction from a single neural network, just a few papers have investigated rule extraction from several combined neural networks. In this article we describe how to translate symbolic rules into the Discretized Interpretable Multi-Layer Perceptron (DIMLP) and how to extract rules from one or several combined neural networks. Our approach consists of characterizing discriminant hyperplane frontiers. Unordered rules are extracted in polynomial time with respect to the size of the problem and the size of the network. Moreover, the degree of matching between extracted rules and neural network responses is 100% on training examples. We applied single DIMLP networks to 17 data sets related to medical diagnosis and medical prognosis problems. Results based on 10-fold cross-validation showed that the DIMLP model was on average as accurate as standard multi-layer perceptrons (MLP). Furthermore, DIMLP networks were significantly more accurate than CN2 on eight problems, whereas only on one problem CN2 was better than DIMLP. Finally, a non-Hodgkin lymphoma diagnosis problem based on classification of electrophoresis gels was defined. It turned out that ensembles of DIMLP networks were significantly more accurate than CN2 (96.1% +/- 1.4 versus 82.7% +/- 4.0). Finally, symbolic rules revealed the presence of five important spots for the discrimination of the class of Lymphocyte Leukemia/Chronic Lymphoid Leukemia (Lc/LLc), and the class of Centrocytic Lymphoma (Cc).

A study on rule extraction from several combined neural networks.

The problem of rule extraction from neural networks is NP-hard. This work presents a new technique to extract "if-then-else" rules from ensembles of DIMLP neural networks. Rules are extracted in polynomial time with respect to the dimensionality of the problem, the number of examples, and the size of the resulting network. Further, the degree of matching between extracted rules and neural network responses is 100%. Ensembles of DIMLP networks were trained on four data sets in the public domain. Extracted rules were on average significantly more accurate than those extracted from C4.5 decision trees.

Shortage of circulating naive CD8(+) T cells provides new insights on immunodeficiency in aging.

Clinical observations indicate that elderly people are prone to severe, often lethal infectious diseases induced by novel pathogens. Since the ability to mount primary immune responses relies on the availability of naive T cells, the circulating naive T-cell reservoir was evaluated throughout the human life span. Naive T cells were identified as CD95(-) T lymphocytes for their phenotypic and functional features. Indeed, the lack of CD95 marker is sufficient to identify a population of naive T cells, as defined by coincidence with previously characterized CD45RA(+) CD62L(+) T cells. Naive CD95(-) T cells, as expected, require a costimulatory signal, such as CD28, to optimally proliferate after anti-CD3 stimulation. Cytofluorimetric analysis of circulating T lymphocytes from 120 healthy subjects ranging in age from 18 to 105 years revealed that naive T cells decreased sharply with age. The younger subjects had a naive T-lymphocyte count of 825 +/- 48 cells/microL, and the centenarians had a naive T-lymphocyte count of 177 +/- 28 cells/microL. Surprisingly, the naive T-cell count was lower in CD8(+) than in CD4(+) subsets at any age, and the oldest individuals were almost completely depleted of circulating naive CD8(+) T cells (13 +/- 4 cells/microL). Concomitantly, a progressive expansion of CD28(-) T cells occurs with age, which can be interpreted as a compensatory mechanism. These data provide new insights into age-related T-cell-mediated immunodeficiency and reveal some analogies of T-cell dynamics between advanced aging and human immunodeficiency virus (HIV) infection. In conclusion, the exhaustion of the naive CD8(+) T-cell reservoir, which has never been reported before, suggests that this T-cell pool is a major target of the aging process and may define a parameter possibly related to the life span of humans. (Blood. 2000;95:2860-2868)

[Multiple symmetrical lipomatosis (MSL): a clinical case and a review of the literature]. / La lipomatosi multipla simmetrica (LMS): contributo clinico e revisione della letteratura.

Multiple symmetric lipomatosis (MSL) is a rare condition of the fatty tissue affecting mostly white men between 20 and 65 years old especially in the mediterranean region. The disease is characterized by a massive development of large unencapsulated lipomas mainly located on the subcutaneous tissue of the cervical, deltoid, thoracic, abdominal and lumbar areas and it is often accompanied by hyperuricemia, dyslipemia, macrocytic anaemia, peripheral neuropathy, impaired glucose tolerance and alcohol consumption. Alcohol could both promote the development of lipomas through changes in the number and function of beta-adrenergic receptors and because of its lipogenic and antilipolytic action. Other authors have hypothesized that the defective lipolysis is due to a disorder in the mitochondria of brown fat whose distribution is similar to the peculiar position of the lipomas in the MSL. In this report the authors describe an atypical clinical picture of MSL in a 65-years-old white man.

T lymphocyte proliferative capability to defined stimuli and costimulatory CD28 pathway is not impaired in healthy centenarians.

It is generally assumed that T cell proliferation is impaired in aged individuals. We report data on the proliferative capability of peripheral blood mononuclear cells (PBMC) and T lymphocytes from 40 healthy people of different ages, (19-107 years), including 14 centenarians, to defined mitogenic stimuli. We observed no age-related proliferative impairment both in PBMC and in purified T cells stimulated by anti-CD3 mAb or phorbol myristate acetate (PMA). Furthermore, T cells stimulated by anti-CD3 mAb or PMA and costimulated by CD28 mAb did not proliferate differently among young, middle aged subjects and centenarians. Thus, short term T cell proliferation is not affected even at extreme age when well defined stimuli are used on cells deriving from carefully selected healthy subjects.

A double-blind, placebo-controlled study of preventive immunotherapy with E.P.D., in the treatment of seasonal allergic disease.

Control of seasonal symptoms by means of a preventive and easy to use (only one intradermal injection eight weeks before the pollen peak) immunotherapy, is recommended nowadays. We verified the clinical efficacy of E.P.D. (Enzyme Potentiated Desensibilization) in a double-blind, placebo-controlled study. This particular immunotherapy consists of an intradermal injection mix, made up of allergenic extracts at extremely low doses and an enzyme called beta-glucuronidase. The vaccine is administered once a year, eight weeks before pollen peaks. We studied a group of 40 patients allergic to grass pollen. The results, analysed statistically on the basis of a symptoms score, showed good clinical efficacy and a significant reduction of drug consumption during the high pollen period. Due to the clinical effectiveness, easy administration (only on injection) and excellent tolerance of the immunotherapy, E.P.D. is particularly suited for the prevention of seasonal symptoms in patients allergic to grass pollen.

Expansion of cytotoxic CD8+ CD28- T cells in healthy ageing people, including centenarians.

Ageing is associated with complex remodelling in the phenotypic and functional profiles of T lymphocytes. We investigated whether expression of CD28 antigen on T cells is conserved throughout adulthood and ageing in humans. For this purpose we analysed T cells obtained from peripheral blood of 102 healthy people of ages ranging from 20 to 105 years. We found an age-related increase of CD28- T cells in percentage and absolute number, predominantly among CD8+ T cells. CD28- T cells from aged donors analysed by flow cytometry appeared as resting cells (not expressing CD25, CD38, CD69, CD71, DR), bearing markers of cytotoxic activity (CD 11b and CD 57) and with a phenotype compatible with 'memory' cells (up-regulated CD2 and CD11a; CD62L absent). At the functional level, freshly isolated purified CD28- CD8+ T cells showed high anti-CD3 redirected cytotoxic activity against Fc-bearing P815 cells. The same activity tested on freshly isolated bulk T lymphocytes was significantly augmented with age. We found a positive correlation between age, number of CD8+ CD28- T cells and anti-CD3 redirected cytotoxicity by freshly isolated T cells. These data suggest that an activation of unknown nature within the cytotoxic arm of the immune system occurs with age. We speculate that these cytotoxic T lymphocytes (CTL) in vivo may constitute armed effector cells for immediate killing of targets bearing peptides from pathogens of intracellular origin.

Morphometric evaluation of CD16-positive cells with respect to CD2 antigen coexpression.

We examined morphometric as well as functional characteristics of CD16-positive human peripheral blood lymphocytes on the basis of the coexpression of the CD2 antigen. For morphometric analyses, nuclear area and cellular area were determined by counting line cross-points of a superimposed quadratic lattice test system overlying nuclei and the whole cell, respectively. Moreover, to evaluate the cellular villousity degree, the maximum inscrible circle and an irregular polygon were inscribed within cell profiles. The cytoplasm fraction included between the plasmalemma and the traced irregular polygon was considered as the villous portion of the cell. Finally, the NK capability was measured in a 6-hr 51Cr-release assay with human K-562 myeloid cells as targets. Within the CD16-positive cell population, the CD16-positive/CD2-negative cells seem to represent the most efficient NK cell subset. To the higher NK capability correspond a higher villousity degree and a lower nuclear area/cellular area ratio of the CD2-negative/CD16-positive subset, when compared with CD2-positive/CD16-positive cells.