GEMA-Na and MELD 3.0 severity scores to address sex disparities for accessing liver transplantation: a nationwide retrospective cohort study.

Background: The Gender-Equity Model for liver Allocation corrected by serum sodium (GEMA-Na) and the Model for End-stage Liver Disease 3.0 (MELD 3.0) could amend sex disparities for accessing liver transplantation (LT). We aimed to assess these inequities in Spain and to compare the performance of GEMA-Na and MELD 3.0. Methods: Nationwide cohort study including adult patients listed for a first elective LT (January 2016-December 2021). The primary outcome was mortality or delisting for sickness within the first 90 days. Independent predictors of the primary outcome were evaluated using multivariate Cox's regression with adjusted relative risks (RR) and 95% confidence intervals (95% CI). The discrimination of GEMA-Na and MELD 3.0was assessed using Harrell c-statistics (Hc). Findings: The study included 6071 patients (4697 men and 1374 women). Mortality or delisting for clinical deterioration occurred in 286 patients at 90 days (4.7%). Women had reduced access to LT (83.7% vs. 85.9%; p = 0.037) and increased risk of mortality or delisting for sickness at 90 days (adjusted RR = 1.57 [95% CI 1.09-2.28]; p = 0.017). Female sex remained as an independent risk factor when using MELD or MELD-Na but lost its significance in the presence of GEMA-Na or MELD 3.0. Among patients included for reasons other than tumours (n = 3606; 59.4%), GEMA-Na had Hc = 0.753 (95% CI 0.715-0.792), which was higher than MELD 3.0 (Hc = 0.726 [95% CI 0.686-0.767; p = 0.001), showing both models adequate calibration. Interpretation: GEMA-Na and MELD 3.0 might correct sex disparities for accessing LT, but GEMA-Na provides more accurate predictions of waiting list outcomes and could be considered the standard of care for waiting list prioritization. Funding: Instituto de Salud Carlos III, Agencia Estatal de Investigación (Spain), and European Union.

Labour market participation after sickness absence due to cancer: a dynamic cohort study in Catalonia (Spain).

BACKGROUND: The consequences of cancer on working until retirement age remain unclear. This study aimed to analyse working life considering all possible labour market states in a sample of workers after sickness absence (SA) due to cancer and to compare their working life paths to those of a sample of workers without SA and with an SA due to other diseases. METHODS: This was a retrospective dynamic cohort study among social security affiliates in Catalonia from 2012-2018. Cases consisted of workers with an SA due to cancer between 2012-2015 (N = 516) and were individually age- and sex-matched with those of affiliates with an SA due to other diagnoses and workers without an SA. All workers (N = 1,548, 56% women) were followed up from entry into the cohort until the end of 2018 to characterise nine possible weekly labour states. Sequence analysis, optimal matching, and multinomial logistic regression were used to identify and assess the probability of future labour market participation patterns (LMPPs). All analyses were stratified by sex. RESULTS: Compared with workers with an SA due to cancer, male workers with no SA and SA due to other causes showed a lower probability of being in the LMPP of death (aRRR 0.02, 95% CI: 0.00‒0.16; aRRR 0.17, 95% CI: 0.06‒0.46, respectively) and, among women, a lower probability of permanent disability and death (aRRR 0.24, 95% CI: 0.10‒0.57; aRRR 0.39, 95% CI: 0.19‒0.83, respectively). Compared to workers with SA due to cancer, the risk of early retirement was lower among workers with no SA (women, aRRR 0.60, 95% CI: 0.22‒1.65; men, aRRR 0.64, 95% CI: 0.27‒1.52), although these results were not statistically significant. CONCLUSIONS: Workplaces, many of which have policies common to all diagnoses, should be modified to the needs of cancer survivors to prevent an increasing frequency of early retirement and permanent disability when possible. Future studies should assess the impact of cancer on premature exit from the labour market among survivors, depending on cancer localisation and type of treatment.

Returning to work after a sickness absence due to cancer: a cohort study of salaried workers in Catalonia (Spain).

Cancer incidence and survival rates have increased in the last decades and as a result, the number of working age people diagnosed with cancer who return to work. In this study the probability of accumulating days of employment and employment participation trajectories (EPTs) in a sample of salaried workers in Catalonia (Spain) who had a sickness absence (SA) due to cancer were compared to salaried workers with SA due to other diagnoses or without SA. Each individual with SA due to cancer between 2012 and 2015 was matched by age, sex, and onset of time at risk to a worker with SA due to other diagnoses and another worker without SA. Accumulated days of employment were measured, and negative binomial models were applied to assess differences between comparison groups. Latent class models were applied to identify EPTs and multinomial regression models to analyse the probability of belonging to one EPT of each group. Men and women without SA or with SA due to other diagnoses had at least a 9% higher probability of continuing in employment compared to workers who had a SA due to cancer, especially among men without SA (adjusted IRR 1.27, 95% CI 1.06‒1.53). Men without SA had the highest probability of having high stable EPT compared to workers who had a SA due to cancer (adjusted RRR 3.21, 95% CI 1.87‒5.50). Even though workers with SA due to cancer continue working afterwards, they do it less often than matched controls and with a less stable employment trajectory. Health and social protection systems should guaranty cancer survivors the opportunity to continue voluntary participation in the labour market.

Raman micro spectroscopy study of the interaction of vincristine with A549 cells supported by expression analysis of bcl-2 protein.

Understanding the interaction of anticancer drugs with model cell lines is important to elucidate the mode of action of these drugs as well as to develop cost effective and rapid screening methods. Raman spectroscopy has been demonstrated to be a valuable technique for high throughput, noninvasive analysis. The interaction of vincristine with a human lung adenocarcinoma cell line (A549) was investigated using Raman micro spectroscopy. The results were correlated with parallel measurements from the MTT cytotoxicity assay, which yielded an IC50 value of 0.10 ± 0.03 µM. The Raman spectral data acquired from vincristine treated A549 cells was analysed to understand its interaction with the nucleus in the cell and elucidate DNA intercalation. The dose dependent spectral changes in the nucleus are analysed by PLS-Jack knifing for the identification of the more significant changes associated with the mode of action of the drug. Results are correlated with a similar dose dependent expression analysis of the bcl-2 protein, an anti-apoptotic protein associated with DNA damage, in the vincristine treated A549 cells using flow cytometry. The results indicate the co-existence of two modes of action, microtubule binding at low doses and DNA intercalation at high doses.

The importance of serum serotonin levels in the measurement of radiation-induced bystander cell death in HaCaT cells.

PURPOSE: The aim of this study was to investigate the importance of serum serotonin levels in the measurement of bystander cell death. The study was undertaken as part of an intercomparison exercise involving seven European laboratories funded under the European Union Sixth Framework Programme (FP6) Non-Targeted Effects (NOTE) integrated project. MATERIALS AND METHODS: Three batches of foetal bovine serum were tested; serum with high and low serotonin content from the intercomparison exercise as well as serum from the home laboratory. Three sets of human keratinocytes (HaCaT cell line) were cultured in DMEM:F12 medium supplemented with serum with high or low serotonin content or serum from the home laboratory and both donor and recipient HaCaT cells were plated. The donor HaCaT cells were irradiated (0.5 Gy) using a cobalt 60 teletherapy unit, the medium was harvested 1 hour post irradiation and transferred to the recipient HaCaT cells. Bystander induced cell death was measured by the clonogenic survival assay and the Alamar blue viability assay. RESULTS: A significant reduction in cell survival, as measured by the clonogenic assay, and in cell viability, as measured by the Alamar blue assay, was observed in the recipient HaCaT cells treated with medium from irradiated cells compared to the cells treated with medium from unirradiated cells. No significant difference was found between the three batches of serum. CONCLUSIONS: The data suggest that in our cell system and with our endpoints (clonogenic assay and Alamar blue assay), serum serotonin levels do not play a role in bystander-induced cell death.

Identifying and localizing intracellular nanoparticles using Raman spectroscopy.

Raman microscopy is employed to spectroscopically image biological cells previously exposed to fluorescently labelled polystyrene nanoparticles and, in combination with K-means clustering and principal component analysis (PCA), is demonstrated to be capable of localising the nanoparticles and identifying the subcellular environment based on the molecular spectroscopic signatures. The neutral nanoparticles of 50 nm or 100 nm, as characterised by dynamic light scattering, are shown to be non-toxic to a human lung adenocarcinoma cell-line (A549), according to a range of cytotoxicity assays including Neutral Red, Alamar Blue, Coomassie Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Confocal fluorescence microscopy identifies intracellular fluorescence due to the nanoparticle exposure, but the fluorescence distribution is spatially diffuse, potentially due to detachment of the dye from the nanoparticles, and the technique fails to unambiguously identify the distribution of the nanoparticles within the cells. Raman spectroscopic mapping of the cells in combination with K-means cluster analysis is used to clearly identify and localise the polystyrene nanoparticles in exposed cells, based on their characteristic spectroscopic signatures. PCA identifies the local environment as rich in lipidic signatures which are associated with localisation of the nanoparticles in the endoplasmic reticulum. The importance of optimised cell growth conditions and fixation processes is highlighted. The preliminary study demonstrates the potential of the technique to unambiguously identify and locate nonfluorescent nanoparticles in cells and to probe not only the local environment but also changes in the cell metabolism which may be associated with cytotoxic responses.

[The clinical pathway of treatment of hepatocellular carcinoma]. / Vía clínica. Tratamiento del hepatocarcinoma.

INTRODUCTION: The regular clinical practise slows a wide variability present in medical care in patients with the same pathology. The clinical pathways are integrated and systematized care plans for certain processes. The development of the hepatocarcinoma's (HCC) health care treatment is within the care frame of Tumoral Area's departments, Hepatology and General Surgery in the Clinic University of Navarra (CUN). OBJECTIVES: To conduct a clinical pathway able to organize, homogenize and standardize the care of patients diagnosed with hepatocarcinoma and that could be applied from the beginning to the end of the course of this disease. METHODOLOGY: The identification of the set of care procedures wich would be necessary to achieve the best possible result in a patient who comes to the clinic to be treated for an hepatocarcinoma. The study scope has taken place at the CUN, a private clinic that is a part of the University of Navarra. RESULT: The clinical pathway of the hepatocarcinoma's treatment has been developed centered on the surgical treatment and the hepatic transplant. CONCLUSION: The clinical pathways represents a decrease in variability and quality control in clinical practice, and the ability to analyze the information that indicators provide us, the satisfaction surveys and the computerized registration of electronic data.

Mitophagy and mitochondrial morphology in human melanoma-derived cells post exposure to simulated sunlight.

PURPOSE: To assess changes in mitochondrial morphology and mitophagy induced by simulated sunlight irradiation (SSI) and how these changes are modulated by mitochondrial activity and energy source. MATERIALS AND METHODS: Human malignant amelanotic melanoma A375 cells were pre-treated with either a mitochondrial activity enhancer, uncoupler or were either melanin or glutamine supplemented/starved for 4 hours pre-exposure to sunlight. A Q-Sun Solar Simulator (Q-Lab, Homestead, FL, USA) was employed to expose cells to simulated sunlight. Confocal microscopy imaging of A375 cells co-loaded with mitochondria and lysosome-specific fluorescent dyes was used to identify these organelles and predict mitophagic events. RESULTS: SSI induces pronounced changes in mitochondrial dynamics and mitophagy in exposed skin cells compared to control and these effects were modified by both glutamine and melanin. CONCLUSIONS: Mitochondrial dynamics and rate of mitophagy in melanoma cells are sensitive to even short bursts of environmentally relevant SSI. Mitochondrial dynamics, and its modulation, may also play a role in mitophagy regulation, cell survival and proliferation post SSI.

Correlation of p16(INK4A) expression and HPV copy number with cellular FTIR spectroscopic signatures of cervical cancer cells.

Cervical cancer, a potentially preventable disease, has its main aetiology in infection by high risk human papillomavirus (HR-HPV). Approaches to improving cervical cancer screening and diagnostic methodologies include molecular biological analysis, targeting of biomarker proteins, but also exploration and implementation of new techniques such as vibrational spectroscopy. This study correlates the biomarker protein p16(INK4A) expression levels dependent on HPV copy number with the infrared absorption spectral signatures of the cervical cancer cell lines, HPV negative C33A, HPV-16 positive SiHa and CaSki and HPV-18 positive HeLa. Confocal fluorescence microscopy demonstrated that p16(INK4A) is expressed in all investigated cell lines in both nuclear and cytoplasmic regions, although predominantly in the cytoplasm. Flow cytometry was used to quantify the p16(INK4A) expression levels and demonstrated a correlation, albeit nonlinear, between the reported number of integrated HPV copies and p16(INK4A) expression levels. CaSki cells were found to have the highest level of expression, HeLa intermediate levels, and SiHa and C33A the lowest levels. FTIR spectra revealed differences in nucleic acid, lipid and protein signatures between the cell lines with varying HPV copy number. Peak intensities exhibited increasing tendency in nucleic acid levels and decreasing tendency in lipid levels with increasing HPV copy number, and although they were found to be nonlinearly correlated with the HPV copy number, their dependence on p16(INK4A) levels was found to be close to linear. Principal Component Analysis (PCA) of the infrared absorption spectra revealed differences between nuclear and cytoplasmic spectroscopic signatures for all cell lines, and furthermore clearly differentiated the groups of spectra representing each cell line. Finally, Partial Least Squares (PLS) analysis was employed to construct a model which can predict the p16(INK4A) expression level based on a spectral fingerprint of a cell line, demonstrating the diagnostic potential of spectroscopic techniques.

Mechanistic studies of in vitro cytotoxicity of poly(amidoamine) dendrimers in mammalian cells.

Poly(amidoamine) (PAMAM) dendrimer nanoparticles have been demonstrated to elicit a well defined cytotoxicological response from mammalian cell lines, the response increasing systematically with dendrimer generation and number of surface amino groups. In this work, using generation G4, G5, and G6 dendrimers, this systematic response is furthermore demonstrated for the generation of reactive oxygen species, lysosomal activity, and the onset of apoptosis and levels of DNA damage. The results are consistent with a pathway of localisation of PAMAM dendrimers in the mitochondria leading to ROS production causing oxidative stress, apoptosis and DNA damage. ROS production is co-located in the mitochondria, and both generated levels and timescales are systematically generation dependent (G4

TRIL, a functional component of the TLR4 signaling complex, highly expressed in brain.

TLR4 is the primary sensor of LPS. In this study, we describe for the first time TLR4 interactor with leucine-rich repeats (TRIL), which is a novel component of the TLR4 complex. TRIL is expressed in a number of tissues, most prominently in the brain but also in the spinal cord, lung, kidney, and ovary. TRIL is composed of a signal sequence, 13 leucine-rich repeats, a fibronectin domain, and a single transmembrane spanning region. TRIL is induced by LPS in the human astrocytoma cell line U373, in murine brain following i.p. injection, and in human PBMC. Endogenous TRIL interacts with TLR4 and this interaction is greatly enhanced following LPS stimulation. TRIL also interacts with the TLR4 ligand LPS. Furthermore, U373 cells stably overexpressing TRIL display enhanced cytokine production in response to LPS. Finally, knockdown of TRIL using small interfering RNA attenuates LPS signaling and cytokine production in cell lines, human PBMC, and primary murine mixed glial cells. These results demonstrate that TRIL is a novel component of the TLR4 complex which may have particular relevance for the functional role of TLR4 in the brain.

Interleukin-6, endothelial activation and thrombogenesis in chronic atrial fibrillation.

BACKGROUND: A prothrombotic or hypercoagulable state has been described in AF, which could increase the risk of thromboembolism. As inflammation has been related to thrombogenesis and endothelial activation, we hypothesised that the prothrombotic state in AF (as assessed by an index of thrombogenesis, prothrombin fragment 1+2 [F1+2]) and endothelial activation (soluble E-selectin (sEsel)) could be related to an index of inflammation (interleukin-6 (IL-6)). PATIENTS AND METHODS: We studied 191 consecutive patients (98 male; mean age 72.3+/-9.2 years) with chronic non-rheumatic AF who were not on anticoagulant therapy. Plasma IL-6, sEsel and F1+2 were measured by ELISA. Research indices were compared to 74 controls in sinus rhythm matched for age and sex. In 43 patients with AF, the effects of introducing anticoagulation (INR 2.0-3.0) were also studied. RESULTS: Patients with AF had elevated levels of F1+2 (p<0.001) and IL-6 (p=0.045), but not sEsel. There was no significant correlation between F1+2 and IL-6. In multivariate analysis, only F1+2 levels were independently associated with the presence of AF (p=0.001). After oral anticoagulation, plasma levels of F1+2 and sEsel were significantly decreased (both p<0.01). CONCLUSION: High levels of IL-6 in AF suggest an inflammatory state, which appears to be more related to clinical variables of the patients, rather than to the presence of AF per se. There was no association of inflammation with endothelial activation (sEsel) or the presence of abnormal thrombogenesis (high F1+2 levels) in AF. Moreover, no changes in IL-6 levels were found despite the reduction of the other markers by oral anticoagulant therapy.