Low-density cultured cartilage cells expanded in platelet lysate present distinct features to develop an innovative clinical treatment for diffuse cartilage lesions.

PURPOSE: Chondrocyte-based cell therapies are effective for the treatment of chondral lesions, but remain poorly indicated for diffuse lesions in the context of early osteoarthritis (OA). The aim of this study was to develop a protocol to obtain chondroprogenitor cells suitable for the treatment of diffuse chondral lesions within early OA. METHODS: Cartilage cells were expanded at low density in human platelet lysate (hPL). A test was performed to exclude senescence. The expression of surface cluster of differentiation 146, cluster of differentiation 166, major histocompatibility complex (MHC)-I and MHC-II and of genes of interest were evaluated, as well as the trophic potential of these cells, by the assessment of lubricin and matrix production. The immunomodulatory potential was assessed through their co-culture with macrophages. RESULTS: Cartilage cells expanded at low density in hPL showed higher proliferation rate than standard-density cells, no replicative senescence, low immunogenicity and expression of lubricin. Moreover, they presented an increased expression of chondrogenic and antihypertrophic markers, as well as a superior matrix deposition if compared to cells cultured at standard density. Cartilage cells induced on macrophages an upregulation of CD206, although a higher increase of CD163 expression was observed in the presence of low-density cells. CONCLUSIONS: These findings lay the grounds to explore the clinical usefulness of low-density cultured cartilage cells to treat diffuse lesions in early OA joints for both autologous and allogenic use. LEVEL OF EVIDENCE: Not applicable.

State of the Art and New Trends from the 2022 Gism Annual Meeting.

The 2022 Italian Mesenchymal Stem Cell Group (Gruppo Italiano Staminali Mesenchimali, GISM) Annual Meeting took place on 20-21 October 2022 in Turin (Italy), with the support of the University of Turin and the City of Health and Science of Turin. The novelty of this year's meeting was its articulation, reflecting the new structure of GISM based on six sections: (1) Bringing advanced therapies to the clinic: trends and strategies, (2) GISM Next Generation, (3) New technologies for 3D culture systems, (4) Therapeutic applications of MSC-EVs in veterinary and human medicine, (5) Advancing MSC therapies in veterinary medicine: present challenges and future perspectives, (6) MSCs: a double-edged sword: friend or foe in oncology. National and international speakers presented their scientific works with the aim of promoting an interactive discussion and training for all attendees. The atmosphere was interactive, where ideas and questions between younger researchers and senior mentors were shared in all moments of the congress.

A New Human Platelet Lysate for Mesenchymal Stem Cell Production Compliant with Good Manufacturing Practice Conditions Preserves the Chemical Characteristics and Biological Activity of Lyo-Secretome Isolated by Ultrafiltration.

Recently, we proposed a Good Manufacturing Practice (GMP)-compliant production process for freeze-dried mesenchymal stem cell (MSC)-secretome (lyo-secretome): after serum starvation, the cell supernatant was collected, and the secretome was concentrated by ultrafiltration and freeze-dried, obtaining a standardized ready-to-use and stable powder. In this work, we modified the type of human platelet lysate (HPL) used as an MSC culture supplement during the lyo-secretome production process: the aim was to verify whether this change had an impact on product quality and also whether this new procedure could be validated according to GMP, proving the process robustness. MSCs were cultured with two HPLs: the standard previously validated one (HPL-E) and the new one (HPL-S). From the same pool of platelets, two batches of HPL were obtained: HPL-E (by repeated freezing and thawing cycles) and HPL-S (by adding Ca-gluconate to form a clot and its subsequent mechanical wringing). Bone marrow MSCs from three donors were separately cultured with the two HPLs until the third passage and then employed to produce lyo-secretome. The following indicators were selected to evaluate the process performance: (i) the lyo-secretome quantitative composition (in lipids and proteins), (ii) the EVs size distribution, and (iii) anti-elastase and (iv) immunomodulant activity as potency tests. The new HPL supplementation for MSCs culture induced only a few minimal changes in protein/lipid content and EVs size distribution; despite this, it did not significantly influence biological activity. The donor intrinsic MSCs variability in secretome secretion instead strongly affected the quality of the finished product and could be mitigated by concentrating the final product to reach a determined protein (and lipid) concentration. In conclusion, the modification of the type of HPL in the MSCs culture during lyo-secretome production induces only minimal changes in the composition but not in the potency, and therefore, the new procedure can be validated according to GMP.

A Novel Xeno-Free Method to Isolate Human Endometrial Mesenchymal Stromal Cells (E-MSCs) in Good Manufacturing Practice (GMP) Conditions.

The cyclic regeneration of human endometrium is guaranteed by the proliferative capacity of endometrial mesenchymal stromal cells (E-MSCs). Due to this, the autologous infusion of E-MSCs has been proposed to support endometrial growth in a wide range of gynecological diseases. We aimed to compare two different endometrial sampling methods, surgical curettage and vacuum aspiration biopsy random assay (VABRA), and to validate a novel xeno-free method to culture human E-MSCs. Six E-MSCs cell samples were isolated after mechanical tissue homogenization and cultured using human platelet lysate. E-MSCs were characterized for the colony formation capacity, proliferative potential, and multilineage differentiation. The expression of mesenchymal and stemness markers were tested by FACS analysis and real-time PCR, respectively. Chromosomal alterations were evaluated by karyotype analysis, whereas tumorigenic capacity and invasiveness were tested by soft agar assay. Both endometrial sampling techniques allowed efficient isolation and expansion of E-MSCs using a xeno-free method, preserving their mesenchymal and stemness phenotype, proliferative potential, and limited multi-lineage differentiation ability during the culture. No chromosomal alterations and invasive/tumorigenic capacity were observed. Herein, we report the first evidence of efficient E-MSCs isolation and culture in Good Manufacturing Practice compliance conditions, suggesting VABRA endometrial sampling as alternative to surgical curettage.

A New Human Platelet Lysate for Mesenchymal Stem Cell Production Compliant with Good Manufacturing Practice Conditions.

Mesenchymal stem cells (MSCs) are classified as advanced therapy medicinal products, a new category of GMP (good manufacturing practice)-compliant medicines for clinical use. We isolated MSCs from 5 bone marrow (BM) samples using human platelet lysate (HPL) instead of foetal bovine serum (FBS). We used a new method of HPL production consisting of treating platelet (PLTs) pools with Ca-Gluconate to form a gel clot, then mechanically squeezing to release growth factors. We compared the new HPL (HPL-S) with the standard (HPL-E) obtained by freezing/thawing cycles and by adding heparin. HPL-S had not PLTs and fibrinogen but the quantity of proteins and growth factors was comparable to HPL-E. Therefore, HPL-S needed fewer production steps to be in compliance with GMP conditions. The number of colonies forming unit-fibroblasts (CFU-F) and the maintenance of stem markers showed no significant differences between MSCs with HPL-E and HPL-S. The cumulative population doubling was higher in MSCs with HPL-E in the earlier passages, but we observed an inverted trend of cell growth at the fourth passage. Immunophenotypic analysis showed a significant lower expression of HLA-DR in the MSCs with HPL-S (1.30%) than HPL-E (14.10%). In conclusion, we demonstrated that HPL-S is an effective alternative for MSC production under GMP conditions.

Sleep and new media usage in toddlers.

Several studies over the years have demonstrated the association between lack of sleep in children and certain physical, psychological, and behavioral disorders. The aim of this study was to disentangle the association between new screen-based electronic devices and sleep problems in toddlers, adjusting for other covariates already known to be associated with sleep quality. We conducted a cross-sectional study with the aid of a national sample of 1117 toddlers. Parents reported children's sleeping habits such as total sleep time and sleep onset latency, recreational activities, bedtime routines, and temperament. An ordered logistic regression was run to assess the associations between new media exposure and two sleep outcomes (total sleep time and sleep onset latency). Everyday use of a tablet or smartphone raised the odds of a shorter total sleep time (OR 1.95 [1.00-3.79], p < 0.05) and a longer sleep onset latency (OR 2.44 [1.26-4.73] p < 0.05) irrespective of other factors, such as temperament (restlessness, sociability), or traditional screen exposure (watching TV or playing videogames).Conclusion: New media usage is a factor associated in toddlers with sleeping fewer hours and taking longer to fall asleep, irrespective of other confounding factors. What is known • Studies have found an association between sleep behavior and the use of computers and video games in early childhood. • The blue light emitted from TV screens suppresses endogenous melatonin. What is new • The study found an association between daily new media (tablet and smartphone) usage and sleep quality in toddlers • New media usage exposes toddlers to the risk of fewer hours of sleep and taking longer to fall asleep, irrespective of other factors.

Problematic social networking site usage and substance use by young adolescents.

BACKGROUND: Substance use and abuse by young adolescents has become a serious issue for public health services, and several socio-environmental factors can influence how vulnerable a young adolescent may be to their appeal. The present study was devised to examine whether substance use in early adolescence is associated with problematic social networking site usage (PSNSU). METHODS: In the academic year 2013-2014, secondary schools in Padua (north-eastern Italy) were involved in a survey called "Pinocchio". A sample of 1325 pupils attending years 6 to 8 (i.e. aged from 11 to 13 years) completed self-administered questionnaires, in which PSNSU was measured by applying the DSM-IV criteria of dependence to identify any social network addiction disorder and its fallout on daily life. Multivariate analysis (ordered logistic regression) was performed to assess an adjusted association between young adolescents' substance use and PSNSU. RESULTS: The percentage of pupils classified as problematic social networking site users rose with age (from 14.6% in year 6 to 24.3% in year 7, and 37.2% in year 8), and it was higher in girls (27.1%) than in boys (23.6%). In a fully-adjusted model, PSNSU conferred a higher likelihood of being substance users (OR 2.93 95% CI 1.77-4.85). CONCLUSION: This study identified an association between PSNSU and the likelihood of substance use (smoking, alcohol and energy drink consumption), providing further evidence of the need to pay more attention to PSNSU in early adolescence.

Value of HCC-MELD Score in Patients With Hepatocellular Carcinoma Undergoing Liver Transplantation.

CONTEXT: Liver transplantation (LT) is considered the ideal therapy for patients with hepatocellular carcinoma (HCC) having cirrhosis but the shortage of liver donors and the risk of dropout from the wait list due to tumor progression severely limit transplantation. A new prognostic score, the HCC-model for end-stage liver disease (HCC-MELD), was developed by combining α-fetoprotein (AFP), MELD, and tumor size, to improve risk stratification of dropout in patients with HCC. OBJECTIVES: In this study, we investigated the ability of the HCC-MELD score in predicting the posttransplant for patients fulfilling Milan criteria (MC). DESIGN: Two hundred patients with stage II tumor were retrospectively reviewed from a total of 1290 transplants performed at our institution from October 1997 through April 2015. Cox regression analysis was performed to identify the prognostic factors impacting the posttransplant survival. RESULTS: Overall survival at 1, 5, and 10 years was 89.3%, 71.1%, and 67.2%, whereas disease-free survival was 86.4%, 66.5%, and 52.4%, respectively. Multivariate analysis showed HCC-MELD score (hazard ratio [HR] 39.6, P < .001) and microvascular invasion (HR 2.41, P = .002) to be independent risk factors for recurrence, whereas HCC diameter (HR 1.15, P = .041), HCC-MELD (HR 15.611, P = .006), and grading (HR 2.17, P = .03) proved to be predictive factors of poor overall survival. CONCLUSION: Our study showed the validity of the HCC-MELD equation in the evaluation of patients undergoing LT for HCC. This score offers a reliable method to assess the risk of waiting list dropout and predict posttransplantation outcomes.

Problematic cell phone use for text messaging and substance abuse in early adolescence (11- to 13-year-olds).

UNLABELLED: The aim of our study was to examine the association between problematic cell phone use (PCPU) for text messaging and substance abuse in young adolescents. This cross-sectional study was conducted on the basis of an ad hoc questionnaire, during the 2014-2015 school year in a province of the Veneto Region (Italy); it involved a sample of 1156 students in grades 6 to 8 (11 to 13 years old). A self-report scale based on the Short Message Service (SMS) Problem Use Diagnostic Questionnaire (SMS-PUDQ) was administered to assess the sample's PCPU. A multivariate logistic regression model was applied to seek associations between PCPU (as the dependent variable) and independent variables. The proportion of students who reported a PCPU increased with age in girls (13.5% in 6th grade, 16.4% in 7th grade, and 19.5% in 8th grade), but not in boys (14.3% in 6th grade, 18.0% in 7th grade, and 14.8% in 8th grade). Logistic regression showed that drunkenness at least once and energy drink consumption raised the odds of PCPU, whereas reading books, higher average school marks, and longer hours of sleep were associated with lower odds of PCPU in early adolescence. CONCLUSION: our findings confirm a widespread PCPU for text messaging among early adolescents. The odds of PCPU is greater in young people at risk of other substance abuse behavior.

Exploring patient reported quality of life in lung cancer patients: A qualitative study of patient-reported outcome measures.

OBJECTIVE: Lung cancer is the leading cause of cancer-related death globally and provides a major disease burden likely to substantially impact quality of life (QoL). Patient-reported outcome measures (PROMs) have been identified as effective methods of evaluating patient QoL. Existing lung cancer-specific PROMs however have uncertain utility and minimal patient involvement in their design and development. This qualitative study aimed to evaluate the patient perspective of existing PROMs and to explore their appropriateness for population-based descriptions of lung cancer-related QoL. METHODS: A descriptive qualitative study was conducted consisting of semi-structured interviews with 14 patients recruited from the Victorian Lung Cancer Registry and Alfred Hospital using purposive sampling. Interviews first explored the factors most important to lung cancer patients QoL, and second, patient's perspectives on the appropriateness of existing PROMs. Thematic analysis was used to develop themes, and content analysis was conducted to determine PROM acceptability. RESULTS: Five novel themes were identified by patients as being important impacts on QoL: Personal attitude toward the disease is important for coping; independence is valued; relationships with family and friends are important; relationships with treating team are meaningful; personal and public awareness of lung cancer is limited. These patient-identified impacts are poorly covered in existing lung cancer-specific PROMs. Patients welcomed and appreciated the opportunity to complete PROMs; however, they identified problems with existing PROMs relevance, tone, and formatting. CONCLUSION: Existing lung cancer PROMs poorly reflect the five themes identified in this study as most important to lung cancer patients QoL. This study reaffirms the need to review existing PROMs to ensure utility and construct validity. Future PROM development must engage key patient-generated themes and evolve to reflect the changing management and therapeutic landscape.

A New Paclitaxel Formulation Based on Secretome Isolated from Mesenchymal Stem Cells Shows a Significant Cytotoxic Effect on Osteosarcoma Cell Lines.

BACKGROUND: Osteosarcoma (OS) represents a rare cancer with an unfavorable prognosis that needs innovative treatment. The aim was to isolate a secretome from mesenchymal stem cells (MSCs) that are treated with paclitaxel (PTX)-containing microvesicles as a drug delivery system and analyze its cytotoxic effects on OS cell lines (SJSA, MG63, and HOS). METHODS: Three batches of secretome (SECR-1, SECR-2, and SECR-3) were produced from three bone marrow (BM) MSCs samples treated for 24 h with 15 µg/mL of PTX or with a standard medium. The viability of the OS cell lines after 5 days of exposure to SECR-1-2-3 (pure and diluted to 1:2 and 1:4) was analyzed with an MTT assay. The same SECR batches were analyzed with high-performance liquid chromatography (HPLC) and with a nanoparticle tracking assay (NTA). RESULTS: A statistically significant decrease in the viability of all OS cell lines was observed after treatment with SECR-PTX 1-2-3 in a dose-response manner. The NTA analyses showed the presence of nanoparticles (NPs) with a mean size comparable to that of extracellular vesicles (EVs). The HPLC analyses detected the presence of PTX in minimal doses in all SECR batches. CONCLUSIONS: This proof-of-concept study showed that the conditioned medium isolated from MSCs loaded with PTX had a strong cytotoxic effect on OS cell lines, due to the presence of EV and PTX.

Contrast adaptation of flankers reduces collinear facilitation and inhibition.

Increase (facilitation) or decrease (inhibition) of contrast sensitivity for a Gabor patch presented between two collinear flankers is a well-studied contextual modulation phenomenon. It has been suggested that this effect has its neural bases in the primary visual cortex, specifically the horizontal connections between hypercolumns with similar orientation and spatial frequency selectivity. Another typical phenomenon dependent on early visual areas is contrast adaptation, in which the neural response to a contrast stimulus is decreased after exposure. Here, we investigated the effect of contrast adaptation of the flankers on the magnitude of collinear modulation by testing whether contrast adaptation reduced collinear facilitation and collinear inhibition. Results showed dissociation in the effect of collinear flanker adaptation, which increased contrast thresholds for the target in the facilitatory configuration and reduced them in the inhibitory configuration. Moreover, the effect was specific for the collinear configuration, since contrast adaptation of orthogonal flankers did not affect the contrast of the target, pointing towards the involvement of early visual units specific for orientation. Surprisingly, the same pattern of results was also confirmed when the inhibitory configuration was tested with low-contrast flankers, indicating that the effect of adaptation does not depend on a decrease in perceived contrast of the flankers. Taken together, these results suggest that contrast adaptation disrupts collinear modulation and that contrast thresholds can be affected by adapting portions of the visual field outside the receptive field of the units processing the contrast of the target (i.e., the flankers).

Investigating the role of the foveal cortex in peripheral object discrimination.

Peripheral object discrimination is hindered by a central dynamic mask presented between 150 and 300 ms after stimulus onset. The mask is thought to interfere with task-relevant feedback coming from higher visual areas to the foveal cortex in V1. Fan et al. (2016) supported this hypothesis by showing that the effect of mask can be further delayed if the task requires mental manipulation of the peripheral target. The main purpose of this study was to better characterize the temporal dynamics of foveal feedback. Specifically, in two experiments we have shown that (1) the effect of foveal noise mask is sufficiently robust to be replicated in an online data collection (2) in addition to a change in sensitivity the mask affects also the criterion, which becomes more conservative; (3) the expected dipper function for sensitivity approximates a quartic with a global minimum at 94 ms, while the best fit for criterion is a quintic with a global maximum at 174 ms; (4) the power spectrum analysis of perceptual oscillations in sensitivity data shows a cyclic effect of mask at 3 and 12 Hz. Overall, our results show that foveal noise affects sensitivity in a cyclic manner, with a global dip emerging earlier than previously found. The noise also affects the response bias, even though with a different temporal profile. We, therefore, suggest that foveal noise acts on two distinct feedback mechanisms, a faster perceptual feedback followed by a slower cognitive feedback.

Inactivated Platelet Lysate Supports the Proliferation and Immunomodulant Characteristics of Mesenchymal Stromal Cells in GMP Culture Conditions.

Mesenchymal stromal cells (MSCs) isolated from bone marrow (BM-MSCs) are considered advanced therapy medicinal products (ATMPs) and need to be produced according to good manufacturing practice (GMP) in their clinical use. Human platelet lysate (HPL) is a good GMP-compliant alternative to animal serum, and we have demonstrated that after pathogen inactivation with psoralen, it was safer and more efficient to use psoralen in the production of MSCs following GMP guidelines. In this study, the MSCs cultivated in fetal bovine serum (FBS-MSC) or inactivated HPL (iHPL-MSC) were compared for their immunomodulatory properties. We studied the effects of MSCs on (1) the proliferation of total lymphocytes (Ly) and on naïve T Ly subsets induced to differentiate in Th1 versus Th2 Ly; (2) the immunophenotype of different T-cell subsets; (3) and the cytokine release to verify Th1, Th2, and Th17 polarization. These were analyzed by using an in vitro co-culture system. We observed that iHPL-MSCs showed the same immunomodulatory properties observed in the FBS-MSC co-cultures. Furthermore, a more efficient effect on the increase of naïve T- cells and in the Th1 cytokine release from iHPL was observed. This study confirms that iHPL, used as a medium supplement, may be considered a good alternative to FBS for a GMP-compliant MSC expansion, and also to preserve their immunomodulatory proprieties.

HIV-1 matrix protein p17 binds to monocytes and selectively stimulates MCP-1 secretion: role of transcriptional factor AP-1.

HIV-1 matrix protein p17 activates a variety of cell responses which play a critical role in viral replication and infection. Its activity depends on the expression of p17 receptors (p17R) on the surface of target cells. Whether p17 also plays a role in stimulating human monocytes, a major HIV-1 reservoir, is not known. Here we show that human monocytes constitutively express p17Rs and that p17 selectively triggers these cells to produce MCP-1. The effect of p17 on MCP-1 expression was observed at the transcriptional level and was primarily dependent on the activation of the transcription factor AP-1. p17 increased the binding activity of AP-1 complexes in a time- and dose-dependent manner. Deletion of the AP-1 binding sites in the MCP-1 promoter resulted in the lack of p17-induced MCP-1 transcription. In particular, the P3 binding site located between -69 and -63 position seems to be essential to MCP-1 mRNA induction in p17-treated monocytes. An ever increasing amount of evidences shows a tight link between biologically dysregulated monocytes, AP-1 activation, MCP-1 release and HIV-1 pathogenesis. Overall our results suggest that p17 may play a critical role in the monocyte-mediated inflammatory processes, which are suspected to be major precipitating events in AIDS-defining diseases.

Replication-deficient mutant Herpes Simplex Virus-1 targets professional antigen presenting cells and induces efficient CD4+ T helper responses.

Both neutralizing antibodies and cytotoxic T-cells are necessary to control a viral infection. However, vigorous T helper responses are essential for their elicitation and maintenance. Here we show that a recombinant replication-deficient Herpes Simplex Virus (HSV)-1 vector encoding the Human Immunodeficiency Virus (HIV)-1 matrix protein p17 (T0-p17) was capable of infecting professional antigen presenting cells (APCs) in vitro and in vivo. The injection of T0-p17 in the mouse dermis generated a strong p17-specific CD4+ T helper response preceding both p17-specific humoral and effector T cell responses. Moreover, we show that T0-p17 infection did not interfere with the endogenous processing of the transgene encoded antigen, since infected APCs were able to evoke a strong recall response in vitro. Our results demonstrate that replication-deficient HSV vectors can be appealing candidates for the development of vaccines able to trigger T helper responses.

HIV-1 matrix protein p17 modulates in vivo preactivated murine T-cell response and enhances the induction of systemic and mucosal immunity against intranasally co-administered antigens.

HIV-1 p17 is a viral cytokine that acts on preactivated, but not on resting, human T cells promoting proliferation, proinflammatory cytokines release and HIV-1 replication, after binding to a cellular receptor (p17R). Here, we demonstrate that p17Rs are expressed on activated murine T cells, which respond to p17 stimulation similarly to their human counterpart. We developed a mouse model of abortive HSV-1 infection to induce T cell activation in vivo. Preactivated cells expressed p17Rs and were highly susceptible to p17 stimulation, which triggered proinflammatory cytokines release and promoted CD4+ T cell survival and expansion. Coculture of in vivo activated splenocytes with macrophages in the presence of p17 further increased their ability to produce IFN-gamma. The presence of macrophages and activated T cells at mucosal sites prompted us to investigate the immunomodulatory activities of p17 in vivo. Intranasal coadministration of p17 with beta-galactosidase (beta-gal) resulted in improved beta-gal specific cellular and humoral immune responses at systemic and mucosal levels. It is well established that HIV-1 replication is driven in an autocrine/paracrine manner by endogenously produced proinflammatory cytokines. Our results highlight the role of p17 in sustaining cellular activation and inflammation, thereby promoting a permissive microenvironment for HIV-1 replication. In addition, p17 is a promising candidate antigen, exhibiting immunomodulatory/adjuvant properties, that need to be exploited in the development of HIV/AIDS vaccines.

Functions of the HIV-1 matrix protein p17.

HIV-1 replication is a dynamic process influenced by a combination of viral and host factors. The HIV-1 matrix protein p17 is a structural protein critically involved in most stages of the life cycle of the retrovirus. It participates in the early stages of virus replication as well as in RNA targeting to the plasma membrane, incorporation of the envelope into virions and particle assembly. Besides its well established functions, p17 acts as a viral cytokine that works on preactivated--but not on resting--human T cells promoting proliferation, proinflammatory cytokines release and HIV-1 replication after binding to a cellular receptor (p17R). Thus, p17 might play a key role in the complex network of host- and virus-derived stimulatory factors contributing to create a favourable environment for HIV-1 infection and replication. Here, we present a brief overview of the functions played by the matrix protein p17 in the HIV-1 life cycle and summarize the current understanding of how p17 could contribute to the pathogenesis of HIV-1 disease.

Experience with Gambling in Late Childhood and Early Adolescence: Implications for Substance Experimentation Behavior.

OBJECTIVE: Previous research among late adolescents suggests an additive association between levels of engagement in gambling and vulnerability to substance use disorder. The aim of our research was to investigate the frequency of gambling experience in childhood and early adolescence and to examine the association between alcohol/cigarette/energy drink consumption and gambling in this young population. METHODS: A survey called "Pinocchio" was conducted during the 2013 to 2014 school year at primary and secondary schools in Padua (north-eastern Italy) on a sample of 1325 students in sixth to eighth grade (11-13 year olds). Multilevel analysis, taking the school-level variance into account, established an adjusted association between gambling and attitude to risk-taking among early adolescents. RESULTS: Among eighth graders, 45.8% of the boys and 35.4% of the girls reported at least 1 type of gambling. In a fully-adjusted model, having experience of gambling confers a higher likelihood of being consumers (at least once a month) of other substances (alcohol, cigarettes, energy drinks, or marijuana). CONCLUSION: Gambling behavior is widespread among adolescents. An association with other risk-taking behavior was found in this study, and this provides further evidence of the need for a greater awareness of gambling behavior in early adolescence.

Predictive value of nodule size and differentiation in HCC recurrence after liver transplantation.

BACKGROUND: Liver transplantation (LT) is considered the best treatment option for HCC patients with cirrhosis. However, the scarce availability of liver donors and the risk of dropout from the waiting list due to the tumor progression severely limit LT for HCC. In this study, we evaluate the survival and recurrence in a cohort of patients undergoing LT for HCC fulfilling "Milan Criteria" (MC) pre-LT. In this study, we propose the development of a new prognostic score which could improve the accuracy in predicting recurrence post-LT. METHODS: Between 1997 and 2011, out of 1010 LT performed in our unit, 131 patients had T2 staged HCC (inside MC). The prognostic model predicting HCC recurrence post-LT was derived from Cox regression analysis. The performance of this model was validated in an external cohort of 198 HCC patients transplanted at another center. RESULTS: Overall survival at 1-3-5 years was 87%, 74.4%, 68.2%, whereas recurrence-free survival was 94.1%, 81.4%, 77.6%, respectively. Predictive factors for recurrence-free survival included high tumor grading (HR 5.01; p = 0.006) and tumor diameter (HR 1.46; p = 0.045). According to this model, the estimated relative risk of HCC recurrence after LT is given by this formula: 0.382 × (Tumor size [cm]) + 1.613 × (if Grading 3-4). The ROC curve was 0.878 (p < 0.001) in predicting HCC recurrence. CONCLUSION: In conclusion, our study showed that the use of this new prognostic score, taking into account maximal tumor diameter and tumor differentiation, improves the accuracy of Milan criteria in predicting HCC recurrence.