Chromene-based compounds as drug candidates for renal and bladder cancer therapy - A systematic review.

Renal (RC) and bladder cancers (BC) are common urological malignancies prevalent in the male population. Incidence and mortality rates are expected to increase in the near future. Drug toxicity and development of drug resistance in both diseases are major obstacles to achieve successful treatments. Chromenes are heterocyclic compounds constituted by a benzene ring fused to a pyran nucleus. Natural and synthetic chromene-based compounds have proven to be promising anticancer agents. Additionally, re-sensitization of cancer cells to classical treatments has also been demonstrated. Thus, the aim of this systematic review is to assess the potential of chromene-based compounds in the treatment of RC and BC. Study collection was performed in six different databases, to compile existing information on preclinical (in vitro and in vivo) and clinical studies developed to date. Overall, multiple chromene-based compounds showed potent anticancer effects, affecting several biological features such as reduction in cell viability, proliferation, migration and invasion in vitro, and induction of cell cycle arrest and cell death. Tumor volume and weight were generally decreased in vivo upon chromene-based treatment. Modest results have been obtained in two clinical trials, with reports of a partial response and two objective responses in RC patients. Thus, the chromene family can be considered an attractive chemical scaffold, harboring promising drug candidates for RC and BC therapeutics.

Health and economic burden of wildland fires PM2.5-related pollution in Portugal - A longitudinal study.

Portugal has been affected by wildland fires that destroy thousands of hectares of forest, causing damage to the environment and to the exposed populations. This study aims to assess the influence of wildland fire emissions on air quality, its effect on population health and the related costs, between 2015 and 2018 in Portugal. The cause-specific mortality due to PM2.5 was calculated considering the exposure for five endpoints in adults, twelve age groups for adults and considering children under five years old. The contribution of wildfire emissions to PM2.5 concentrations in Portugal was assessed through EMEP-MSC/W model. Results showed that the average annual fire emissions of PM2.5, CO, CH4, CO2 and NO2 a significant and continuous increase was observed during the first three years (2015, 2016 and 2017) for all pollutants, followed by a decrease in 2018, with values lower than those observed in 2015. Regarding the long-term exposure to PM2.5 emitted by fires a total of 32, 93, 189 and 31 deaths, corresponding to a cost of 59, 174, 360 and 60 million EUR in 2015, 2016, 2017 and 2018, respectively, were estimated. On the other hand, in the first three years an increase in years of life lost (YLL) values of 496, 1608 and 3092 was observed, corresponding to a cost of 16, 54 and 105 million EUR, respectively, followed by a decrease in 2018 with a YLL of 480, corresponding to a cost of 17 M€.

A review of relevant parameters for assessing indoor air quality in educational facilities.

Indoor air quality (IAQ) in educational facilities is crucial due to the extended time students spend in those environments, affecting their health, academic performance, and attendance. This paper aimed to review relevant parameters (building characteristics and factors related with occupancy and activities) for assessing IAQ in educational facilities, and to identify the parameters to consider when performing an IAQ monitoring campaign in schools. It also intended to identify literature gaps and suggest future research directions. A narrative literature review was conducted, focusing on seven key parameters: building location, layout and construction materials, ventilation and air cleaning systems, finishing materials, occupant demographics, occupancy, and activities. The findings revealed that carbon dioxide (CO2) levels were predominantly influenced by classroom occupancy and ventilation rates, while particulate matter (PM) concentrations were significantly influenced by the building's location, design, and occupant activities. Furthermore, this review highlighted the presence of other pollutants, such as trace metals, polycyclic aromatic hydrocarbons (PAHs), carbon monoxide (CO), nitrogen dioxide (NO2), ozone (O3), and radon, linking them to specific factors within the school environment. Different IAQ patterns, and consequently different parameters, were observed in various school areas, including classrooms, canteens, gymnasiums, computer rooms, and laboratories. While substantial literature exists on IAQ in schools, significant gaps still remain. This study highlighted the need for more studies in middle and high schools, as well as in other indoor microenvironments within educational settings beyond classrooms. Additionally, it underscored the need for comprehensive exposure assessments, long-term studies, and the impacts of new materials on IAQ including the effects of secondary reactions on surfaces. Seasonal variations and the implications of emerging technologies were also identified as requiring further investigation. Addressing those gaps through targeted research and considering the most updated standards and guidelines for IAQ, could lead to define more effective strategies for improving IAQ and safeguarding the students' health and performance.

Development and validation of a photograph-based instrument to assess nutrition literacy: The NUTLY project.

Most instruments measuring nutrition literacy evaluate theoretical knowledge, not necessarily reflecting skills relevant to food choices. We aimed to develop and validate a photograph-based instrument to assess nutrition literacy (NUTLY) among adults in Portugal. NUTLY assesses the ability to distinguish foods with different nutritional profiles; from each of several combinations of three photographs (two foods with similar contents and one with higher content) participants are asked to identify the food with the highest energy/sodium content. The NUTLY version with 79 combinations, obtained after experts/lay people evaluations, was applied to a sample representing different age, gender and education groups (n = 329). Dimensionality was evaluated through latent trait models. Combinations with negative or with positive small factor loadings were excluded after critical assessment. Internal consistency was measured using Cronbach's alpha and construct validity by comparing NUTLY scores with those obtained in the Medical Term Recognition Test and the Newest Vital Sign (NVS), and across education and training in nutrition/health groups. The cut-off to distinguish adequate/inadequate nutrition literacy was defined through ROC analysis using the Youden index criterion, after performing a Latent class analysis which identified a two-class model to have the best goodness of fit. Test-retest reliability was assessed after one month (n = 158). The final NUTLY scale was unidimensional and included 48 combinations (energy: 33; sodium: 15; α = 0.74). Mean scores (±standard deviation) were highest among nutritionists (39.9 ± 4.4), followed by health professionals (38.5 ± 4.1) and declined with decreasing education (p < 0.001). Those with adequate nutrition literacy according to NVS showed higher NUTLY scores (37.9 ± 4.3 vs. 33.9 ± 6.9, p < 0.001). Adequate nutrition literacy was defined as a NUTLY score≥35 (sensitivity: 89.3%; specificity: 93.7%). Test-retest reliability was high (ICC = 0.77). NUTLY is a valid and reliable nutrition literacy measurement tool.

Airborne SARS-CoV-2 is more frequently detected in environments related to children and elderly but likely non-infectious, Norway, 2022.

This study investigates the presence of SARS-CoV-2 in indoor and outdoor environments in two cities in Norway between April and May 2022. With the lifting of COVID-19 restrictions in the country and a focus on vaccination, this research aims to shed light on the potential for virus transmission in various settings. Air sampling was conducted in healthcare and non-healthcare facilities, covering locations frequented by individuals across different age groups. The study found that out of 31 air samples, only four showed the presence of SARS-CoV-2 RNA by RT-qPCR, with no viable virus detected after RNAse pre-treatment. These positive samples were primarily associated with environments involving children and the elderly. Notably, sequencing revealed mutations associated with increased infectivity in one of the samples. The results highlight the importance of considering children as potential sources of virus transmission, especially in settings with prolonged indoor exposure. As vaccination coverage increases globally, and with children still representing a substantial unvaccinated population, the study emphasizes the need to re-implement mask-wearing mandates indoors and in public transport to reduce virus transmission. The findings have implications for public health strategies to control COVID-19, particularly in the face of new variants and the potential for increased transmission during the autumn and winter seasons.

Investigating the role of symptom valorisation in tuberculosis patient delay in urban areas in Portugal.

BACKGROUND: Diagnosis delay contributes to increased tuberculosis (TB) transmission and morbimortality. TB incidence has been decreasing in Portugal, but median patient delay (PD) has risen. Symptom valorisation may determine PD by influencing help-seeking behaviour. We aimed to analyse the association between symptom valorisation and PD, while characterising individuals who disregarded their symptoms. METHODS: A cross-sectional study was conducted among TB patients in Lisbon and Oporto in 2019 - 2021. Subjects who delayed seeking care because they did not value their symptoms or thought these would go away on their own were considered to have disregarded their symptoms. PD was categorised using a 21-day cut-off, and a 30-day cut-off for sensitivity analysis. We estimated the effect of symptom valorisation on PD through a directed acyclic graph. Then, a multivariable regression analysis characterised patients that disregarded their symptoms, adjusting for relevant variables. We fitted Poisson regression models to estimate crude and adjusted prevalence ratios (PR). RESULTS: The study included 75 patients. Median PD was 25 days (IQR 11.5-63.5), and 56.0% of participants had PD exceeding 21 days. Symptom disregard was reported by 38.7% of patients. Patients who did not value their symptoms had higher prevalence of PD exceeding 21 days compared to those who valued their symptoms [PR 1.59 (95% CI 1.05-2.42)]. The sensitivity analysis showed consistent point estimates but wider confidence intervals [PR 1.39 (95% CI 0.77-2.55)]. Being a smoker was a risk factor for symptom disregard [PR 2.35 (95% CI 1.14-4.82)], while living in Oporto [PR 0.35 (95% CI 0.16-0.75)] and having higher household incomes [PR 0.39 (95% CI 0.17-0.94)] were protective factors. CONCLUSIONS: These findings emphasise the importance of symptom valorisation in timely TB diagnosis. Patients who did not value their symptoms had longer PD, indicating a need for interventions to improve symptom recognition. Our findings also corroborate the importance of the socioeconomic determinants of health, highlighting tobacco as a risk factor both for TB and for PD.

Ex vivo generation of regulatory T cells from liver transplant recipients using costimulation blockade.

The potential of adoptive cell therapy with regulatory T cells (Tregs) to promote transplant tolerance is under active exploration. However, the impact of specific transplant settings and protocols on Treg manufacturing is not well-delineated. Here, we compared the use of peripheral blood mononuclear cells (PBMCs) from patients before or after liver transplantation to the use of healthy control PBMCs to determine their suitability for Treg manufacture using ex vivo costimulatory blockade with belatacept. Despite liver failure or immunosuppressive therapy, the capacity for Treg expansion during the manufacturing process was preserved. These experiments did not identify performance or quality issues that disqualified the use of posttransplant PBMCs-the currently favored protocol design. However, as Treg input correlated with output, significant CD4-lymphopenia in both pre- and posttransplant patients limited Treg yield. We therefore turned to leukapheresis posttransplant to improve absolute yield. To make deceased donor use feasible, we also developed protocols to substitute splenocytes for PBMCs as allostimulators. In addition to demonstrating that this Treg expansion strategy works in a liver transplant context, this preclinical study illustrates how characterizing cellular input populations and their performance can both inform and respond to clinical trial design and Treg manufacturing requirements.

SARS-CoV-2 air sampling: A systematic review on the methodologies for detection and infectivity.

This systematic review aims to present an overview of the current aerosol sampling methods (and equipment) being used to investigate the presence of SARS-CoV-2 in the air, along with the main parameters reported in the studies that are essential to analyze the advantages and disadvantages of each method and perspectives for future research regarding this mode of transmission. A systematic literature review was performed on PubMed/MEDLINE, Web of Science, and Scopus to assess the current air sampling methodologies being applied to SARS-CoV-2. Most of the studies took place in indoor environments and healthcare settings and included air and environmental sampling. The collection mechanisms used were impinger, cyclone, impactor, filters, water-based condensation, and passive sampling. Most of the reviewed studies used RT-PCR to test the presence of SARS-CoV-2 RNA in the collected samples. SARS-CoV-2 RNA was detected with all collection mechanisms. From the studies detecting the presence of SARS-CoV-2 RNA, fourteen assessed infectivity. Five studies detected viable viruses using impactor, water-based condensation, and cyclone collection mechanisms. There is a need for a standardized protocol for sampling SARS-CoV-2 in air, which should also account for other influencing parameters, including air exchange ratio in the room sampled, relative humidity, temperature, and lighting conditions.

Street food in Eastern Europe: a perspective from an urban environment in Moldova.

Street food is popular in Eastern Europe, but its diversity and nutritional value are unknown. This study aimed to characterise the street food environment in Chisinau, Moldova, including the vending sites and vendors, food availability and nutritional composition of foods and beverages. All street food vending sites (single point of sale) located in a 1-km buffer centred on the main public market were systematically selected (n 439; n 328 participants). Data on vending sites' characteristics (mobility, type of physical set-up and access to electricity), operating periods and food availability were collected. Samples of the most commonly available foods of unknown composition were collected (twenty-eight home-made and twenty-four industrial). Macronutrients, Na and K were quantified through chemical analysis. Fruits, beverages and food other than fruits were available in 2·5, 74·3 and 80·8 % of the vending sites, respectively. Among the latter, 66·4 % sold only industrial foods (e.g. pretzels, biscuits, wafers, chocolate and ice cream), 21·5 % only home-made (e.g. savoury and sweet pastries) and 12·1 % both. Home-made foods presented larger serving sizes and energy/serving (median kJ/serving: 1312·5 v. 670·3, P = 0·022); industrial foods were more energy-dense (median kJ/100 g: 1797·0 v. 1269·8, P = 0·002). High SFA, trans-fat and Na contents were found, reaching 10·9 g/serving, 1·4 g/serving and 773·7 mg/serving, respectively. Soft drinks and alcoholic beverages were available in 80·7 and 42·0 % of the vending sites selling beverages, respectively. Concluding, industrial snacks and home-made pastries high in Na and unhealthy fat were frequent in Chisinau. Prevention of diet-related diseases in Moldova may benefit from the improvement of the nutritional profile of street food.

Street food in Maputo, Mozambique: Availability and nutritional value of homemade foods.

BACKGROUND:: A nutrition transition is occurring in the urban areas of developing countries, where street food makes an important contribution to daily food intake. AIM:: We aimed to characterise street food offer in Maputo, Mozambique, and to evaluate the nutritional composition of the most common homemade foods. METHODS:: A cross-sectional study was conducted in 2014. Streets in the surroundings (500 m buffer) of randomly selected public transport stops in KaMpfumu district, Maputo, were canvassed to identify all street food vending sites ( n = 968). Information regarding vending site characteristics and the food offered was gathered through interview and observation. Samples ( n = 80) of the most common homemade foods were collected for laboratorial analysis. RESULTS:: Most street food vending sites identified were stationary (77.4%) and sold exclusively industrial food (51.9%). Frequency of fruit, beverages and food other than fruit was 24.5%, 32.5% and 73.9%, respectively. Fried cakes were the most energy-dense (430 kcal/100 g), and richest in fats (21.0g/100 g) and carbohydrates (53.4 g/100 g). The richest sources of protein were the stewed meat/fish/liver dishes (10.7-11.6 g/100 g). Fried cakes showed the lowest sodium and potassium content (90 mg/100 g and 81 mg/100 g, respectively) whereas hamburgers exhibited the highest content of those micronutrients (455 mg/100 g and 183 mg/100 g, respectively). Stewed liver dishes presented the highest sodium/potassium ratio (11.95). Fried snacks presented the highest trans-fatty acid content (0.20 g/100 g). CONCLUSIONS:: Street food in Maputo is abundant and scattered throughout the urban district, exhibiting high variability in the nutritional composition of homemade foods. Public health policies should be targeted to improve the street food offer, promoting nutrient-dense foods and the reduction of added salt.

Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease.

Transcriptional deregulation and changes in mitochondrial bioenergetics, including pyruvate dehydrogenase (PDH) dysfunction, have been described in Huntington's disease (HD). We showed previously that the histone deacetylase inhibitors (HDACIs) trichostatin A and sodium butyrate (SB) ameliorate mitochondrial function in cells expressing mutant huntingtin. In this work, we investigated the effect of HDACIs on the regulation of PDH activity in striatal cells derived from HD knock-in mice and YAC128 mice. Mutant cells exhibited decreased PDH activity and increased PDH E1alpha phosphorylation/inactivation, accompanied by enhanced protein levels of PDH kinases 1 and 3 (PDK1 and PDK3). Exposure to dichloroacetate, an inhibitor of PDKs, increased mitochondrial respiration and decreased production of reactive oxygen species in mutant cells, emphasizing PDH as an interesting therapeutic target in HD. Treatment with SB and sodium phenylbutyrate, another HDACI, recovered cell viability and overall mitochondrial metabolism in mutant cells. Exposure to SB also suppressed hypoxia-inducible factor-1 (HIF-1α) stabilization and decreased the transcription of the two most abundant PDK isoforms, PDK2 and PDK3, culminating in increased PDH activation in mutant cells. Concordantly, PDK3 knockdown improved mitochondrial function, emphasizing the role of PDK3 inactivation on the positive effects achieved by SB treatment. YAC128 mouse brain presented higher mRNA levels of PDK1-3 and PDH phosphorylation and decreased energy levels that were significantly ameliorated after SB treatment. Furthermore, enhanced motor learning and coordination were observed in SB-treated YAC128 mice. These results suggest that HDACIs, particularly SB, promote the activity of PDH in the HD brain, helping to counteract HD-related deficits in mitochondrial bioenergetics and motor function.SIGNIFICANCE STATEMENT The present work provides a better understanding of mitochondrial dysfunction in Huntington's disease (HD) by showing that the pyruvate dehydrogenase (PDH) complex is a promising therapeutic target. In particular, the histone deacetylase inhibitor sodium butyrate (SB) may indirectly (through reduced hypoxia-inducible factor 1 alpha stabilization) decrease the expression of the most abundant PDH kinase isoforms (e.g., PDK3), ameliorating PDH activity and mitochondrial metabolism and further affecting motor behavior in HD mice, thus constituting a promising agent for HD neuroprotective treatment.

Bone-Targeted Therapies in Cancer-Induced Bone Disease.

Cancer-induced bone disease is a major source of morbidity and mortality in cancer patients. Thus, effective bone-targeted therapies are essential to improve disease-free, overall survival and quality of life of cancer patients with bone metastases. Depending of the cancer-type, bone metastases mainly involve the modulation of osteoclast and/or osteoblast activity by tumour cells. To inhibit metastatic bone disease effectively, it is imperative to understand its underlying mechanisms and identify the target cells for therapy. If the aim is to prevent bone metastasis, it is essential to target not only bone metastatic features in the tumour cells, but also tumour-nurturing bone microenvironment properties. The currently available bone-targeted agents mainly affect osteoclasts, inhibiting bone resorption (e.g. bisphosphonates, denosumab). Some agents targeting osteoblasts begin to emerge which target osteoblasts (e.g. romosozumab), activating bone formation. Moreover, certain drugs initially thought to target only osteoclasts are now known to have a dual action (activating osteoblasts and inhibiting osteoclasts, e.g. proteasome inhibitors). This review will focus on the evolution of bone-targeted therapies for the treatment of cancer-induced bone disease, summarizing preclinical and clinical findings obtained with anti-resorptive and bone anabolic therapies.

Butyrophilin 3A/CD277-Dependent Activation of Human γδ T Cells: Accessory Cell Capacity of Distinct Leukocyte Populations.

Human Vγ9Vδ2 T cells recognize in a butyrophilin 3A/CD277-dependent way microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) or endogenous pyrophosphates (isopentenyl pyrophosphate [IPP]). Nitrogen-bisphosphonates such as zoledronic acid (ZOL) trigger selective γδ T cell activation because they stimulate IPP production in monocytes by inhibiting the mevalonate pathway downstream of IPP synthesis. We performed a comparative analysis of the capacity of purified monocytes, neutrophils, and CD4 T cells to serve as accessory cells for Vγ9Vδ2 T cell activation in response to three selective but mechanistically distinct stimuli (ZOL, HMBPP, agonistic anti-CD277 mAb). Only monocytes supported γδ T cell expansion in response to all three stimuli, whereas both neutrophils and CD4 T cells presented HMBPP but failed to induce γδ T cell expansion in the presence of ZOL or anti-CD277 mAb. Preincubation of accessory cells with the respective stimuli revealed potent γδ T cell-stimulating activity of ZOL- or anti-CD277 mAb-pretreated monocytes, but not neutrophils. In comparison with monocytes, ZOL-pretreated neutrophils produced little, if any, IPP and expressed much lower levels of farnesyl pyrophosphate synthase. Exogenous IL-18 enhanced the γδ T cell expansion with all three stimuli, remarkably also in response to CD4 T cells and neutrophils preincubated with anti-CD277 mAb or HMBPP. Our study uncovers unexpected differences between monocytes and neutrophils in their accessory function for human γδ T cells and underscores the important role of IL-18 in driving γδ T cell expansion. These results may have implications for the design of γδ T cell-based immunotherapeutic strategies.

A new interministerial strategy for the promotion of healthy eating in Portugal: implementation and initial results.

OBJECTIVE: To describe the implementation, main intervention areas and initial results of the Integrated Strategy for the Promotion of Healthy Eating (EIPAS) in Portugal. METHODS: EIPAS was published as a Law, in December of 2017, as a result of a collaboration between several ministries, including the Finance, Internal Affairs, Education, Health, Economy, Agriculture, and Sea Ministries, aiming at improving the dietary habits of the Portuguese population. The working group, led by the Ministry of Health, developed this strategy for over a year. The framework produced was based on WHO and European Commission recommendations as well as on relevant data from the last Portuguese dietary intake survey (2015/2016). EIPAS also reflects the results of a public hearing, including the food industry, among others, and the experience gathered, since 2012, through the National Programme for the Promotion of Healthy Eating. It considers the 'health in all policies' challenge set by WHO and has four different strategic areas, namely (1) creation of healthier food environments, (2) improvement of the quality and accessibility of healthy food choices for consumers, (3) promotion and development of literacy, in order to encourage healthy food choices, and (4) promotion of innovation and entrepreneurship. In order to achieve these goals, a set of 51 actions was established and assigned to the seven ministries involved. RESULTS: Under the scope of this strategy, Portugal has already implemented several actions, including (1) definition of standards for food availability at all public healthcare institutions; (2) implementation of a sugar tax on sweetened beverages; (3) implementation of a voluntary agreement with the food industry sector for food reformulation (work in progress); (4) design of a proposal for an interpretative model of front-of-pack food labelling; (5) improvement of the nutritional quality of food aid programmes for low-income groups; and (6) regulation of marketing of unhealthy foods to children. CONCLUSIONS: For the first time, Portugal has a nutrition policy based on the WHO concept of 'health in all policies' and on the national data on food intake. The implementing process of all 51 actions and the inherent complexities and difficulties found so far have made this process be an authentic political and social laboratory that deserves to be followed.

Dynamic metabolic flux analysis using a convex analysis approach: Application to hybridoma cell cultures in perfusion.

In recent years, dynamic metabolic flux analysis (DMFA) has been developed in order to evaluate the dynamic evolution of the metabolic fluxes. Most of the proposed approaches are dedicated to exactly determined or overdetermined systems. When an underdetermined system is considered, the literature suggests the use of dynamic flux balance analysis (DFBA). However the main challenge of this approach is to determine an appropriate objective function, which remains valid over the whole culture. In this work, we propose an alternative dynamic metabolic flux analysis based on convex analysis, DMFCA, which allows the determination of bounded intervals for the fluxes using the available knowledge of the metabolic network and information provided by the time evolution of extracellular component concentrations. Smoothing splines and mass balance differential equations are used to estimate the time evolution of the uptake and excretion rates from this experimental data. The main advantage of the proposed procedure is that it does not require additional constraints or objective functions, and provides relatively narrow intervals for the intracellular metabolic fluxes. DMFCA is applied to experimental data from hybridoma HB58 cell perfusion cultures, in order to investigate the influence of the operating mode (batch and perfusion) on the metabolic flux distribution.

Human breast cancer cells educate macrophages toward the M2 activation status.

INTRODUCTION: The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. METHODS: Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). RESULTS: Clinically, we found that high numbers of CD163(+) M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type (p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. CONCLUSIONS: This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.

Liposome encapsulated zoledronate favours M1-like behaviour in murine macrophages cultured with soluble factors from breast cancer cells.

BACKGROUND: Tumour stromal macrophages differentiate to tumour-associated macrophages (TAMs) with characteristics of immunosuppressive M2-type macrophages, having a central role in promoting tumour vascularisation, cancer cell dissemination and in suppressing anti-cancer immune responses. Bisphosphonates (BPs) are a group of drugs commonly used as anti-resorptive agents. Further, nitrogen containing BPs like Zoledronate (ZOL), are known to cause unspecific inflammatory reactions hence the hypothesis that its use could modulate TAMs polarization toward a more inflammatory phenotype. METHODS: We studied the in vitro polarization of J774 murine macrophages upon culture in 4T1 breast cancer cell-conditioned medium (4T1CM) and stimulation with LPS and free and liposome-encapsulated bisphosphonates. RESULTS: In this system, breast cancer soluble factors reduced the pro-inflammatory activation of macrophages but increased the secretion of matrix metalloproteinases (MMPs). In the presence of 4T1CM, a non-cytotoxic dose of liposome-encapsulated ZOL (ZOL-LIP) enhanced the expression of iNOS and TNF-α, markers of M1 activation, but did not diminish the expression of M2-type markers. In contrast, clodronate treatment either as a free drug (CLO) or liposome-encapsulated (CLO-LIP) decreased the expression of the M1-type markers and was highly cytotoxic to the macrophages. CONCLUSIONS: Breast cancer cells soluble factors modulate macrophages toward M2 activation state. Bisphosphonates may be applied to counteract this modulation. We propose that ZOL-LIP may be suitable for favouring cytotoxic immune responses by TAMs in breast cancer, whereas CLO-LIP may be appropriate for TAM depletion.

CDX2 regulation by the RNA-binding protein MEX3A: impact on intestinal differentiation and stemness.

The homeobox transcription factor CDX2 plays a crucial role in intestinal cell fate specification, both during normal development and in tumorigenic processes involving intestinal reprogramming. The CDX2 regulatory network is intricate, but it has not yet been fully uncovered. Through genome-wide screening of a 3D culture system, the RNA-binding protein MEX3A was identified as putatively involved in CDX2 regulation; therefore, its biological relevance was addressed by setting up cell-based assays together with expression studies in murine intestine. We demonstrate here that MEX3A has a repressive function by controlling CDX2 levels in gastric and colorectal cellular models. This is dependent on the interaction with a specific binding determinant present in CDX2 mRNA 3'untranslated region. We have further determined that MEX3A impairs intestinal differentiation and cellular polarization, affects cell cycle progression and promotes increased expression of intestinal stem cell markers, namely LGR5, BMI1 and MSI1. Finally, we show that MEX3A is expressed in mouse intestine, supporting an in vivo context for interaction with CDX2 and modulation of stem cell properties. Therefore, we describe a novel CDX2 post-transcriptional regulatory mechanism, through the RNA-binding protein MEX3A, with a major impact in intestinal differentiation, polarity and stemness, likely contributing to intestinal homeostasis and carcinogenesis.