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Studying long-term biological processes such as the colonization of aging epithelia by somatic mutant clones has been slowed by the lack of suitable culture systems. Here we describe epithelioids, a facile, cost-effective method of culturing multiple mouse and human epithelia. Esophageal epithelioids self-maintain without passaging for at least a year, recapitulating the 3D structure, cell dynamics, transcriptome, and genomic stability of the esophagus. Live imaging over 5 months showed epithelioids replicatein vivocell dynamics. Epithelioids enable the study of cell competition and mutant selection in 3D epithelia, and how anti-cancer treatments modulate the competition between transformed and wild type cells. Epithelioids are a novel method with a wide range of applications in epithelial tissues, particularly the study of long term processes, that cannot be accessed using other culture models.

During aging, progenitor cells acquire mutations, which may generate clones that colonize the surrounding tissue. By middle age, normal human tissues, including the esophageal epithelium (EE), become a patchwork of mutant clones. Despite their relevance for understanding aging and cancer, the processes that underpin mutational selection in normal tissues remain poorly understood. Here, we investigated this issue in the esophageal epithelium of mutagen-treated mice. Deep sequencing identified numerous mutant clones with multiple genes under positive selection, including Notch1, Notch2 and Trp53, which are also selected in human esophageal epithelium. Transgenic lineage tracing revealed strong clonal competition that evolved over time. Clone dynamics were consistent with a simple model in which the proliferative advantage conferred by positively selected mutations depends on the nature of the neighboring cells. When clones with similar competitive fitness collide, mutant cell fate reverts towards homeostasis, a constraint that explains how selection operates in normal-appearing epithelium.

SummaryHuman epithelial tissues accumulate cancer-driver mutations with age1–7, yet tumor formation remains rare. The positive selection of these mutations argues they alter the behavior and fitness of proliferating cells8–10. Hence, normal adult tissues become a patchwork of mutant clones competing for space and survival, with the fittest clones expanding by eliminating their less-competitive neighbors9–12. However, little is known about how such dynamic competition in normal epithelia impacts early tumorigenesis. Here we show that the majority of newly formed esophageal tumors are eliminated through competition with mutant clones in the surrounding normal epithelium. We followed the fate of microscopic tumors in a mouse model of esophageal carcinogenesis. Most neoplasms are rapidly lost despite no indication of tumor cell death, decreased proliferation, or an anti-tumor immune response. Deep-sequencing of 10-day and 1-year-old tumors shows evidence of genetic selection on the surviving neoplasms. Induction of highly competitive clones in transgenic mice increased tumor removal, while pharmacologically inhibiting clonal competition reduced tumor loss. The results are consistent with a model where survival of early neoplasms depends on their competitive fitness relative to that of mutant clones in the adjacent normal tissue. We have identified an unexpected anti-tumorigenic role for mutant clones in normal epithelium by purging early neoplasms through cell competition, thereby preserving tissue integrity.

SummaryNormal human tissues progressively accumulate cells carrying mutations. Activating mutations inPIK3CAgenerate large clones in the aging human esophagus, but the underlying cellular mechanisms are unclear. Here, we tracked mutantPIK3CAesophageal progenitor cells in transgenic mice by lineage tracing. Expression of an activating heterozygousPik3caH1047Rmutation in single progenitor cells tilts cell fate towards proliferation, generating mutant clones that outcompete their wild type neighbors. The mutation leads to increased aerobic glycolysis through the activation of Hif1α transcriptional targets compared with wild type cells. We found that interventions that level out the difference in activation of the PI3K/HIF1α/aerobic glycolysis axis between wild type and mutant cells attenuate the competitive advantage ofPik3caH1047Rmutant cellsin vitroandin vivo. Our results suggest that clinically feasible interventions that even out signaling imbalances between wild type and mutant cells may limit the expansion of oncogenic mutants in normal epithelia.

The ability of epithelial cells to rewire their cell fate program beyond their physiological repertoire has become a new paradigm in stem cell biology. This plasticity leaves behind the concept of strict stem cell hierarchies, opening up new exciting questions about its limits and underlying regulation. Here we developed a heterotypic 3D culture system to study the mechanisms modulating changes in the identity of adult esophageal epithelial cells. We demonstrate that, when exposed to the foreign stroma of adult skin, esophageal cells transition towards hair follicle identity and architecture. Heterotypic transplantation experiments recapitulated this cell fate conversion processin vivo. Single-cell RNA sequencing and histological analysis, capturing the temporality of this process, reveal that most esophageal cells switching towards skin identity remain in an intermediate state marked by a transient regenerative profile and a particularly strong hypoxic signature. Inhibition of HIF1a establishes the central role of this pathway in regulating epithelial cell plasticity, driving cells away from their transition state in favor of cell fate conversion.

Human epithelial tissues accumulate cancer-driver mutations with age1–9, yet tumour formation remains rare. The positive selection of these mutations suggests that they alter the behaviour and fitness of proliferating cells10–12. Thus, normal adult tissues become a patchwork of mutant clones competing for space and survival, with the fittest clones expanding by eliminating their less competitive neighbours11–14. However, little is known about how such dynamic competition in normal epithelia influences early tumorigenesis. Here we show that the majority of newly formed oesophageal tumours are eliminated through competition with mutant clones in the adjacent normal epithelium. We followed the fate of nascent, microscopic, pre-malignant tumours in a mouse model of oesophageal carcinogenesis and found that most were rapidly lost with no indication of tumour cell death, decreased proliferation or an anti-tumour immune response. However, deep sequencing of ten-day-old and one-year-old tumours showed evidence of selection on the surviving neoplasms. Induction of highly competitive clones in transgenic mice increased early tumour removal, whereas pharmacological inhibition of clonal competition reduced tumour loss. These results support a model in which survival of early neoplasms depends on their competitive fitness relative to that of mutant clones in the surrounding normal tissue. Mutant clones in normal epithelium have an unexpected anti-tumorigenic role in purging early tumours through cell competition, thereby preserving tissue integrity. The rarity of tumour formation despite the high proportion of cancer-driver mutations in epithelia is explained by the competitive fitness of tumour cells relative to that of surrounding mutant epithelial cells.

Anti-MDA5 antibodies have been strongly associated with rapidly progressive interstitial lung disease (RP-ILD) in dermatomyositis (DM) patients, especially in the clinically amyopathic subset (CADM). We present a case of anti-MDA5 antibody-associated RP-ILD in a patient with arthritis but with no other clinical signs suggestive of DM or CADM successfully treated with a combination of cyclophosphamide, cyclosporine and corticoids. A review of the literature was also done. Despite its rarity, anti-MDA5 antibody-associated ILD should be suspected in cases of RP-ILD even without other signs of DM or CADM as prompt and aggressive treatment could improve prognosis.

During aging, progenitor cells acquire mutations, which may generate clones that colonize the surrounding tissue. By middle age, normal human tissues, including the esophageal epithelium (EE), become a patchwork of mutant clones. Despite their relevance for understanding aging and cancer, the processes that underpin mutational selection in normal tissues remain poorly understood. Here, we investigated this issue in the esophageal epithelium of mutagen-treated mice. Deep sequencing identified numerous mutant clones with multiple genes under positive selection, including Notch1, Notch2 and Trp53, which are also selected in human esophageal epithelium. Transgenic lineage tracing revealed strong clonal competition that evolved over time. Clone dynamics were consistent with a simple model in which the proliferative advantage conferred by positively selected mutations depends on the nature of the neighboring cells. When clones with similar competitive fitness collide, mutant cell fate reverts towards homeostasis, a constraint that explains how selection operates in normal-appearing epithelium.

Adult tissues such as the epidermis of the skin and the epithelium lining the esophagus are continuously turned over throughout life. Cells are shed from the tissue surface and replaced by cell division. Yet, the cellular mechanisms that underpin these tissues homeostasis remain poorly established, having important implications for wound healing and carcinogenesis. Lineage tracing, in which of a cohort of proliferating cells and their descendants are genetically labelled in transgenic mice, has been used to study the fate behavior of the proliferating cells that maintain these tissues. However, based on this technique, distinct mutually irreconcilable models, differing in the implored number and hierarchy of proliferating cell types, have been proposed to explain homeostasis. To elucidate which of these conflicting scenarios should prevail, here we performed cell proliferation assays across multiple body sites in transgenic H2BGFP mouse epidermis and esophagus. Cell-cycle properties were then extracted from the H2BGFP dilution kinetics and adopted in a common analytic approach for a refined analysis of a new lineage-tracing experiment and eight published clonal data sets from esophagus and different skin territories. Our results show H2BGFP dilution profiles remained unimodal over time, indicating the absence of slow-cycling stem cells across all tissues analyzed. We find that despite using diverse genetic labelling approaches, all lineage-tracing data sets are consistent with tissues maintenance by a single population of proliferating cells. The outcome of a given division is unpredictable but, on average the likelihood of producing proliferating and differentiating cells is balanced, ensuring tissue homeostasis. The fate outcomes of sister cells are anticorrelated. We conclude a single cell population maintains squamous epithelial homeostasis.

Endothelial cells line the lumen of all blood vessels and play a critical role in maintaining the barrier function of the vasculature. Sealing of the vessel wall between adjacent endothelial cells is facilitated by interactions involving junctionally expressed transmembrane proteins, including tight junctional molecules, such as members of the junctional adhesion molecule family, components of adherence junctions, such as VE-Cadherin, and other molecules, such as platelet endothelial cell adhesion molecule. Of importance, a growing body of evidence indicates that the expression of these molecules is regulated in a spatiotemporal manner during inflammation: responses that have significant implications for the barrier function of blood vessels against blood-borne macromolecules and transmigrating leukocytes. This review summarizes key aspects of our current understanding of the dynamics and mechanisms that regulate the expression of endothelial cells junctional molecules during inflammation and discusses the associated functional implications of such events in acute and chronic scenarios.

JAM-C is a junctional adhesion molecule, enriched at tight junctions on endothelial and epithelial cells, and also localized to Schwann cells at junctions between adjoining myelin end loops. The role of JAM-C following peripheral nerve injury (PNI) is currently unknown. We examined the localization of JAM-C after sciatic nerve crush injury in adult rats. JAM-C immunoreactivity was present in paranodes and incisures in sham surgery control nerve, but distal to the crush injury significantly decreased at three and 14 days. JAM-C was re-expressed at 28 days and, by 56 days, was significantly increased in the distal nerve compared to controls. In a 7-mm length of sciatic nerve sampled distal to the crush site, the densities of JAM-C immunoreactive paranodes increased in the distal direction. Conversely, the densities of JAM-C immunoreactive incisures were highest immediately distal to the crush site and decreased in the more distal direction. Further analysis revealed a strong correlation between JAM-C localization and remyelination. Fifty-six days after crush injury, greater densities of JAM-C paranodes were seen compared to the nodal marker jacalin, suggesting that paranodal JAM-C precedes node formation. Our data are the first to demonstrate a potential role of JAM-C in remyelination after PNI.

The migration of neutrophils into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarized migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial migration (TEM)) in a luminal-to-abluminal direction. By real-time confocal imaging, we found that neutrophils had disrupted polarized TEM ('hesitant' and 'reverse') in vivo. We noted these events in inflammation after ischemia-reperfusion injury, characterized by lower expression of junctional adhesion molecule C (JAM-C) at EC junctions, and they were enhanced by blockade or genetic deletion of JAM-C in ECs. Our results identify JAM-C as a key regulator of polarized neutrophil TEM in vivo and suggest that reverse TEM of neutrophils can contribute to the dissemination of systemic inflammation.

Objective—Junctional adhesion molecule-C (JAM-C) is an adhesion molecule that has multiple roles in inflammation and vascular biology, but many aspects of its functions under pathological conditions are unknown. Here we investigated the role of JAM-C in leukocyte migration in response to ischemia reperfusion (I/R) injury.Methods and Results—Pretreatment of mice with soluble JAM-C (sJAM-C), used as a pharmacological blocker of JAM-C-mediated reactions, significantly suppressed leukocyte migration in models of kidney and cremaster muscle I/R injury (39 and 51% inhibition, respectively). Furthermore, in the cremaster muscle model (studied by intravital microscopy), both leukocyte adhesion and transmigration were suppressed in JAM-C-deficient mice (JAM-C−/−) and enhanced in mice overexpressing JAM-C in their endothelial cells (ECs). Analysis of JAM-C subcellular expression by immunoelectron microscopy indicated that in I/R-injured tissues, EC JAM-C was redistributed from cytoplasmic vesicles and EC junctional sites to nonjunctional plasma membranes, a response that may account for the role of JAM-C in both leukocyte adhesion and transmigration under conditions of I/R injury.Conclusions—The findings demonstrate a role for EC JAM-C in mediating leukocyte adhesion and transmigration in response to I/R injury and indicate the existence of a novel regulatory mechanism for redistribution and hence function of EC JAM-C in vivo.

The transference of the nutritional function from the VYS to the chorioallantoic placenta during middle pregnancy is a key event for the activation of embryo oxidative metabolism. However, the metabolic adaptations occurring in these tissues during this critical period have not been studied to date. Herein, we investigate the VYS and placenta mitochondrial adaptations throughout gestational days 11, 12 and 13. The results reflect that, during the placentation period, mitochondrial proliferation predominates over differentiation in placenta. Besides, VYS development and mitochondriogenesis show a slowdown despite maintaining the mitochondrial OXPHOS capacities, hence becoming a supporting tissue until the placenta functions are completely available.

Mitochondrial biogenesis and function are essential for proper embryo development; however, these processes have not been further studied during the placentation period, when important oxidative metabolism activation is taking place. Thus, the aim of the present study was to investigate the oxidative phosphorylation system (OXPHOS) enzymatic activities as well as the expression of genes involved in the coordinated regulation of both mitochondrial and nuclear genomes (peroxisome proliferator-activated receptor-γ coactivator-1α, nuclear respiratory factors 1 and 2, mitochondrial single-strand DNA-binding protein, mitochondrial transcription factor A), and mitochondrial function (cytochromecoxidase subunit IV, cytochromecoxidase subunit I and β-ATP phosphohydrolase) in rat embryo throughout the placentation period (gestational days 11, 12 and 13). Our results reflect that embryo mitochondria were enhancing their OXPHOS potential capacities, pointing out that embryo mitochondria become more differentiated during the placentation period. Besides, the current findings show that the mRNAs of the nuclear genes involved in mitochondrial biogenesis were downregulated, whereas their protein content together with the mitochondrial DNA expression were upregulated throughout the period studied. These data indicate that the molecular regulation of the mitochondrial differentiation process during placentation involves a post-transcriptional activation of the nuclear-encoded genes that would lead to an increase in both the nuclear- and mitochondrial-encoded proteins responsible for the mitochondrial biogenic process. As a result, embryo mitochondria would reach a more differentiated stage with a more efficient oxidative metabolism that would facilitate the important embryo growth during the second half of the pregnancy.

The effect of gender and caloric restriction on mitochondrial content and oxidative-phosphorylative capacities has been investigated in rat gastrocnemius muscle. Muscle protein, mitochondrial protein and DNA contents, enzymatic activities of mitochondrial oxidative and phosphorylative system, mitochondrial antioxidant enzymes, protein levels of complex IV (subunit I and IV) and ATPase, and the gene and protein expression of mitochondrial transcription factor A (TFAM), involved in mitochondrial replication and transcription, were measured in rats of both genders fed ad libitum and subjected to three months of 40% caloric restriction. Compared to males, gastrocnemius muscle of female rats showed higher mitochondrial DNA and protein contents, TFAM protein level, oxidative and phosphorylative machinery and activities, and glutathione peroxidase activity. In conclusion, the present data show a clear gender dimorphism in rat muscle mitochondrial features, which could explain the higher facility of females to adapt to altered metabolic energy situations.

In the current study, the mitochondrial proliferationdifferentiation process was investigated in rat embryo during the placentation process, straight after organogenesis, when there is an important oxidative metabolism activation. For this purpose, on gestational days 11, 12 and 13 we studied the mitochondrial DNA (mtDNA) content and the relative gene expression of proteins involved in mtDNA replication (mitochondrial single strand DNA binding protein (mtSSB)), mtDNA transcription (mitochondrial transcription factor A (TFAM)), as well as in mitochondrial function (cytochrome c oxidase subunit I (COXI)). The results indicated that during placentation important changes in mitochondrial proliferation-differentiation process take place in rat embryo. There is a great decrease in cellular mtDNA content and a rise in the ratio between TFAM and mtDNA accompanied by an increase in COXI gene expression. Thus, we can conclude that on gestational day 13 mitochondrial differentiation predominates over mitochondrial proliferation in embryo cells. Besides, our work reveals that in a physiological condition such as embryonic development the TFAM levels change in order to regulate the transcriptional activity of mtDNA.

To establish the role of mitochondrial subpopulations in the mitochondrial maturation process, we studied morphological and functional changes in the mitochondria of different mammalian conceptus tissues during the organogenic and the placentation processes. Mitochondrial subpopulations of three different conceptus tissues, embryo and visceral yolk sac placenta on gestational days 11, 12 and 13 and placenta on days 12 and 13, were examined morphologically by transmission electron microscopy. Cytochrome oxidase activity and protein levels were also measured in each mitochondrial subpopulation. The results indicate two different mitochondrial subpopulation profiles: a homogeneous one, which corresponds to immature mitochondria, and a heterogeneous one, which represents the mature mitochondria. The three tissues studied show different morphologic and metabolic patterns of mitochondrial maturation during the placentation process, rendering them suitable as experimental models to establish the possible relationship between mitochondrial maturation and the mitochondrial subpopulations.

The incidence of cardiac disease is age and sex dependent, but the mechanisms governing these associations remain poorly understood. Mitochondria are the organelles in charge of producing energy for the cells, and their malfunction has been linked to cardiovascular disease and heart failure. Interestingly, heart mitochondrial content and functionality are also age and sex dependent. Here we investigated the combinatory effects of age and sex in mitochondrial bioenergetics that could help to understand their role on cardiac disease. Cardiac mitochondria from 6- and 24-month-old male and female Wistar rats were isolated, and the enzymatic activities of the oxidative-phosphorylative complexes I, III, and IV and ATPase, as well as the protein levels of complex IV, β-ATPase, and mitochondrial transcription factor A (TFAM), were measured. Furthermore, heart DNA content, citrate synthase activity, mitochondrial protein content, oxygen consumption, and H2O2 generation were also determined. Results showed a reduction in heart mitochondrial mass and functionality with age that correlated with increased H2O2 generation. Moreover, sex-dependent differences were found in several of these parameters. In particular, old females exhibited a significant loss of mitochondrial function and increased relative H2O2 production compared with their male counterparts. The results demonstrate a sex dimorphism in the age-associated defects on cardiac mitochondrial function.

Endogenous protective pathways mitigate the overshooting of inflammation after sterile or infectious injury. Here we report that formyl peptide receptor 2 (Fpr2/3) null mice display a major phenotype with exacerbated vascular inflammation observed postischemia reperfusion (IR) injury of the mesenteric artery, characterized by marked neutrophil adhesion and extravasation as visualized by intravital microscopy. Analysis of endogenous agonists for Fpr2/3 revealed that lipoxin A4 (LXA4) was generated by platelet/neutrophil aggregates during ischemia: this cellular response was attenuated in Fpr2/3(-/-) mice; hence, LXA4 levels were lower after 30 minutes' ischemia, and associated with augmented vascular inflammation in the reperfusion (45-180 minutes) phase. Exogenous delivery of LXA4 attenuated IR-mediated inflammation in Fpr2/3(+/+) but not Fpr2/3(-/-) mice; conversely, an Fpr2/3 antagonist skewed the vascular phenotype of Fpr2/3(+/+) mice to that of Fpr2/3(-/-) animals. Such LXA4-based circuit could be activated by aspirin (30-100 mg/kg), which triggered formation of 15-epi-LXA4 in wild-type mice, yet it was effective in Fpr2/3(-/-) mice. In summary, we propose that during ischemia, neutrophil Fpr2/3 controls platelet/neutrophil aggregates with the rapid generation of circulating LXA4, which in turn modulates downstream vascular inflammatory responses evident during the reperfusion phase.

Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed at junctions between adjacent endothelial and epithelial cells and implicated in multiple inflammatory and vascular responses. In addition, we recently reported on the expression of JAM-C in Schwann cells (SCs) and its importance for the integrity and function of peripheral nerves. To investigate the role of JAM-C in neuronal functions further, mice with a specific deletion of JAM-C in SCs (JAM-C SC KO) were generated. Compared to wild-type (WT) controls, JAM-C SC KO mice showed electrophysiological defects, muscular weakness, and hypersensitivity to mechanical stimuli. In addressing the underlying cause of these defects, nerves from JAM-C SC KO mice were found to have morphological defects in the paranodal region, exhibiting increased nodal length as compared to WTs. The study also reports on previously undetected expressions of JAM-C, namely on perineural cells, and in line with nociception defects of the JAM-C SC KO animals, on finely myelinated sensory nerve fibers. Collectively, the generation and characterization of JAM-C SC KO mice has provided unequivocal evidence for the involvement of SC JAM-C in the fine organization of peripheral nerves and in modulating multiple neuronal responses.—Colom, B., Poitelon, Y., Huang, W., Woodfin, A., Averill, S., Del Carro, U., Zambroni, D., Brain, S. D., Perretti, M., Ahluwalia, A., Priestley, J. V., Chavakis, T., Imhof, B. A., Feltri, M. L., Nourshargh, S. Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves.

Objective: Gender and diet have an important effect in cardiovascular disease and other aging-associated disorders, whose initiation and/or worsening seem to be delayed in females from different species and in animals subjected to caloric restriction (CR). The aim of the present study was to investigate whether cardiac muscle bioenergetic mitochondrial features could be responsible for these beneficial effects. Methods: Fifteen-month-old male and female Wistar rats were fed ad libitum or subjected to 40% CR for 3 months. Cardiac mitochondrial function (citrate synthase activity, oxygen consumption), activity of complexes I, III, IV and ATPase of the OXPHOS system, antioxidant activities (MnSOD, GPx), mitochondrial DNA and protein content, mitochondrial H2O2 production, heart oxidative damage, complex IVand ATPase content and efficiency, as well as protein levels of mitochondrial transcription factor A (TFAM) and peroxisome-proliferatoractivated receptor-gamma co-activator 1 alpha (PGC1α) were measured. Results: Female and CR rats exhibited lower cardiac mitochondria content, which were more efficient and generated less H2O2 than in males and ad libitum fed animals, with their consequent lower heart oxidative damage. Conclusion: Higher mitochondrial differentiation becomes a metabolic adaptation to increase energy efficiency, as what happens in female and CR rats. This adaptation is associated with their lower mitochondrial free radical production and oxidative damage, which could help to understand the mechanism by which these animals exhibit a lower incidence of aging-related disorders, including cardiovascular disease.

Sex-related differences in energy balance were studied in young Wistar rats fed standard chow pellets either ad libitum or in restricted amounts (60% of ad libitum intake) for 100 days. Caloric intake, indirect calorimetry, organ and adipose tissue weights, energy efficiency, liver mitochondrial respiration rate, and brown adipose tissue (BAT) uncoupling protein-1 (UCP1) content were measured. Ad libitum-fed females showed greater oxygen consumption (V̇o2) and carbon dioxide production (V̇co2) and lower energy efficiency than males. Caloric restriction induced a chronic drop of V̇o2 and V̇co2 in females but not in males over the period studied. Restricted females showed a better conservation of metabolic active organ mass and a greater decrease in adipose depots than restricted males. Moreover, changes of BAT size and UCP1 content suggest that BAT may be the main cause responsible for sex differences in the response of energy balance to caloric restriction. In conclusion, our results indicate that females under caloric restriction conditions deactivate facultative thermogenesis to a greater degree than males. This ability may have obvious advantages for female survival and therefore the survival of the species when food is limiting.

The goal of this paper is to establish the nature of the scholastic-linguistic model in non-university education in the Balearics: the free choice model (which allows pupils to choose the language of instruction freely) or the total bilingualism model (in which pupils receive instruction in both official languages). The Statute of Autonomy of the Balearic Islands (1983) does not define the scholastic-linguistic model, unlike the Language Normalisation Act (1986). The author draws attention to articles such as 1.2.b, which mentions the goal of ensuring that Catalan is used progressively as the language of instruction in education, or 18.1, which restricts the right to free choice of the language of instruction to "early education". This analysis of the Language Normalisation Act enables the author to conclude that the scholastic-linguistic model in the Balearics is that of total bilingualism: beyond early instruction, pupils are not allowed to choose the language of instruction, a role that, following an administrative decision, is to be played by both Catalan and Castilian. The paper ends with an analysis of Decree 92/1997, which sets the necessary measures to implement the total bilingualism model, e.g., by stipulating the subjects that must be taught in Catalan in primary and compulsory secondary education. In this connection, the author notes that the implementation of this Decree may be delayed if the Language Normalisation Act is not observed as far as the language training of teachers is concerned.

e11036 Background: It remains controversial if young age at diagnosis is an independent prognostic factor for recurrence in breast cancer (BC) patients. Data regarding recurrence with long-term follow-up in premenopausal women are sparse. The aim of the study was to compare the outcome of young patients (39 years of age at diagnosis. Five and 10-year disease-free survival rate was 61 % and 48% respectively for the younger group and 77% and 74% for the older group (p2 cm), N (negative versus positive nodes), Grade (grade 1-2 versus grade 3) and Hormonal Receptor status (positive versus negative), age less than 40 remains and independent prognostic factor for recurrence with an adjusted HR of 2.42 (95%CI: 1.42-4.10) (p