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The feeling of hunger or satiety results from integration of the sensory nervous system with other physiological and metabolic cues. This regulates food intake, maintains homeostasis and prevents disease. InC. elegans, chemosensory neurons sense food and relay information to the rest of the animal via hormones to control food-related behaviour and physiology. Here we identify a new component of this system, SKN-1B which acts as a central food-responsive node, ultimately controlling satiety and metabolic homeostasis. SKN-1B, an ortholog of mammalian NF-E2 related transcription factors (Nrfs), has previously been implicated with metabolism and respiration, because can mediate the increased lifespan incurred by dietary restriction. We show that actually SKN-1B is not essential for dietary restriction longevity and instead, controls a variety of food-related behaviours. It acts in two hypothalamus-like ASI neurons to sense food, communicate nutritional status to the organism, and control satiety and exploratory behaviours. This is achieved by SKN-1B modulating endocrine signalling pathways (IIS and TGF-β), and by promoting a robust mitochondrial network. Our data suggest a food-sensing and satiety role for mammalian Nrf proteins.

The feeling of hunger or satiety results from integration of the sensory nervous system with other physiological and metabolic cues. This regulates food intake, maintains homeostasis and prevents disease. In C. elegans, chemosensory neurons sense food and relay information to the rest of the animal via hormones to control food-related behaviour and physiology. Here we identify a new component of this system, SKN-1B which acts as a central food-responsive node, ultimately controlling satiety and metabolic homeostasis. SKN-1B, an ortholog of mammalian NF-E2 related transcription factors (Nrfs), has previously been implicated with metabolism, respiration and the increased lifespan incurred by dietary restriction. Here we show that SKN-1B acts in two hypothalamus-like ASI neurons to sense food, communicate nutritional status to the organism, and control satiety and exploratory behaviours. This is achieved by SKN-1B modulating endocrine signalling pathways (IIS and TGF-β), and by promoting a robust mitochondrial network. Our data suggest a food-sensing and satiety role for mammalian Nrf proteins., Author summary Deciding when and how much to eat is important for maintaining health and preventing disease. It requires an intricate molecular level of communication between our nervous, physiological, and metabolic systems. These signals stimulate food intake, and afterwards the feeling of satiety which makes us stop eating. We have studied these phenomena using the simple nematode worm C. elegans which has a fully mapped nervous system and quantifiable food-related behaviours. In C. elegans, chemosensory neurons sense food and communicate this to the rest of the animal via hormones to control food-related behaviour and associated physiological changes. Here we identify a new central node of this system, the C. elegans gene SKN-1B, which acts in two sensory neurons to sense food, communicate food-status to the rest of the worm, and control satiety and exploratory behaviours. It does this by altering hormonal signalling (Insulin and Transforming Growth Factor-β), and by promoting a strong mitochondrial network. The mammalian equivalents of SKN-1B are the NF-E2 related transcription factors (Nrfs), which have previously been implicated with metabolism and respiration. Our data suggest a new food-sensing and satiety role for mammalian Nrf proteins.

High mobility group box 1 (HMGB1) is a non-histone protein localised in the cell nucleus, where it interacts with DNA and promotes nuclear transcription events. HMGB1 levels are elevated during acute myeloid leukaemia (AML) progression followed by participation of this protein in triggering signalling events in target cells as a pro-inflammatory stimulus. This mechanism was hypothesised to be employed as a survival pathway by malignant blood cells and our aims were therefore to test this hypothesis experimentally. Here we report that HMGB1 triggers the release of tumour necrosis factor alpha (TNF-α) by primary human AML cells. TNF-α induces interleukin 1 beta (IL-1β) production by healthy leukocytes, leading to IL-1β-induced secretion of stem cell factor (SCF) by competent cells (for example endothelial cells). These results were verified in mouse bone marrow and primary human AML blood plasma samples. In addition, HMGB1 was found to induce secretion of angiogenic vascular endothelial growth factor (VEGF) and this process was dependent on the immune receptor Tim-3. We therefore conclude that HMGB1 is critical for AML progression as a ligand of Tim-3 and other immune receptors thus supporting survival/proliferation of AML cells and possibly the process of angiogenesis.

Acute myeloid leukaemia (AML) is a blood/bone marrow cancer originating from myeloid cell precusors capable of self-renewing. AML cells implement biochemical mechanisms which allow them not only to survive, but also to successfully escape immune surveillance. ln this work, we discuss crucial molecular mechanisms used by human AML cells in order to evade immune attack.

The T cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated receptor which is involved in a variety of biological responses in human immune cells. It is highly expressed in most acute myeloid leukaemia (AML) cells and therefore may serve as a possible target for AML therapy. However, its biochemical activities in primary human AML cells remain unclear. We therefore analysed the total expression and surface presence of the Tim-3 receptor in primary human AML blasts and healthy primary human leukocytes isolated from human blood. We found that Tim-3 expression was significantly higher in primary AML cells compared to primary healthy leukocytes. Tim-3 receptor molecules were distributed largely on the surface of primary AML cells, whereas in healthy leukocytes Tim-3 protein was mainly expressed intracellularly. In primary human AML blasts, both Tim-3 agonistic antibody and galectin-9 (a Tim- 3 natural ligand) significantly upregulated mTOR pathway activity. This was in line with increased accumulation of hypoxia-inducible factor 1 alpha (HIF-1α) and secretion of VEGF and TNF-α. Similar results were obtained in primary human healthy leukocytes. Importantly, in both types of primary cells, Tim-3- mediated effects were compared with those induced by lipopolysaccharide (LPS) and stem cell factor (SCF). Tim-3 induced comparatively moderate responses in both AML cells and healthy leukocytes. However, Tim-3, like LPS, mediated the release of both TNF-α and VEGF, while SCF induced mostly VEGF secretion and did not upregulate TNF-α release.

Acute myeloid leukemia (AML) is a severe and often fatal systemic malignancy. Malignant cells are capable of escaping host immune surveillance by inactivating cytotoxic lymphoid cells. In this work we discovered a fundamental molecular pathway, which includes ligand-dependent activation of ectopically expressed latrophilin 1 and possibly other G-protein coupled receptors leading to increased translation and exocytosis of the immune receptor Tim-3 and its ligand galectin-9. This occurs in a protein kinase C and mTOR (mammalian target of rapamycin)-dependent manner. Tim-3 participates in galectin-9 secretion and is also released in a free soluble form. Galectin-9 impairs the anti-cancer activity of cytotoxic lymphoid cells including natural killer (NK) cells. Soluble Tim-3 prevents secretion of interleukin-2 (IL-2) required for the activation of cytotoxic lymphoid cells. These results were validated in ex vivo experiments using primary samples from AML patients. This pathway provides reliable targets for both highly specific diagnosis and immune therapy of AML.

// Vadim V. Sumbayev 1 , Isabel Goncalves Silva 1 , Jennifer Blackburn 1 , Bernhard F. Gibbs 1 , Inna M. Yasinska 1 , Michelle D. Garrett 2 , Alexander G. Tonevitsky 3 , Yuri A. Ushkaryov 1 1 School of Pharmacy, University of Kent, Chatham, Kent, ME4 4TB, United Kingdom 2 School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, United Kingdom 3 Hertsen Moscow Oncology Research Institute, Branch of The National Medical Research Radiological Center, Ministry of Health of The Russian Federation, 125284, Moscow, Russian Federation Correspondence to: Vadim V. Sumbayev, email: V.Sumbayev@kent.ac.uk Yuri A. Ushkaryov, email: Y.Ushkaryov@kent.ac.uk Keywords: latrophilin, myeloid leukaemia, mammalian target of rapamycin Received: March 17, 2016 Accepted: May 29, 2016 Published: June 14, 2016 ABSTRACT Acute myeloid leukaemia (AML) is a blood cancer affecting cells of myeloid lineage. It is characterised by rapid growth of malignant leukocytes that accumulate in the bone marrow and suppress normal haematopoiesis. This systemic disease remains a serious medical burden worldwide. Characterisation of protein antigens specifically expressed by malignant cells, but not by healthy leukocytes, is vital for the diagnostics and targeted treatment of AML. Here we report, for the first time, that the neuronal receptor latrophilin-1 is expressed in human monocytic leukaemia cell lines and in primary human AML cells. However, it is absent in healthy leukocytes. Latrophilin-1 is functional in leukaemia cells tested, and its biosynthesis is controlled through the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways. Our findings demonstrate that latrophilin-1 could be considered as a novel biomarker of human AML, which offers potential new avenues for AML diagnosis and treatment.

Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells.

The immune receptor Tim-3 is often highly expressed in human acute myeloid leukemia (AML) cells where it acts as a growth factor and inflammatory receptor. Recently, it has been demonstrated that Tim-3 forms an autocrine loop with its natural ligand galectin-9 in human AML cells. However, the pathophysiological functions of Tim-3 in human AML cells remain unclear. Here, we report for the first time that Tim-3 is required for galectin-9 secretion in human AML cells. However, this effect is cell-type specific and was found so far to be applicable only to myeloid (and not, for example, lymphoid) leukemia cells. We concluded that AML cells might use Tim-3 as a trafficker for the secretion of galectin-9 which can then be possibly used to impair the anticancer activities of cytotoxic T cells and natural killer (NK) cells.

Monografia apresentada à Universidade Fernando Pessoa para obtenção do grau Licenciada em Ciências Farmacêuticas Considerados durante muito tempo microrganismos de baixa patogenicidade Enterococcus spp são, actualmente, agentes nosocomiais importantes, principalmente em imunocomprometidos. Uma vez que estes cocos de Gram positivo acumulam facilmente diferentes mecanismos de resistência a antibióticos, são frequentemente seleccionados em ambientes com elevada pressão selectiva, como os hospitais e a indústria agropecuária. Nos últimos anos tem sido descrito o aparecimento de bactérias com resistência múltipla em animais para consumo humano de vários países. Uma vez que grandes quantidades de antibióticos têm sido consumidas no ambiente de produção animal em Portugal (INFARMED, 2007) e se desconhece qual a incidência de bactérias resistentes aos antibióticos em suiniculturas no nosso país, pretende-se com este trabalho avaliar a susceptibilidade, a diferentes famílias de antibióticos, de Enterococcus spp provenientes de diferentes tipos de amostras de suiniculturas com dois tipos de produção: intensiva e extensiva. Foram identificados 185 Enterococcus spp resultantes de 53 amostras colhidas em 2 suiniculturas de produção intensiva do Norte e 1 suinicultura de produção extensiva do Sul de Portugal. O estudo da susceptibilidade aos antibióticos foi realizado em todos os isolados de Enterococcus spp pelo método de difusão com discos segundo as normas de CLSI. A identificação das espécies E. faecalis, E. faecium, E. gallinarum, E. casseliflavus, E. durans e E. hirae e a pesquisa dos genes que mais frequentemente conferem resistência aos glicopéptidos, foi realizada através de dois PCR multiplex. Foram realizados ensaios de conjugação em 76 isolados representativos das três suiniculturas de modo a estudar a transferência de resistências a quatro classes de antibióticos: tetraciclina, aminoglicosídeos, macrólidos e glicopéptidos. Nas três suiniculturas estudadas, mais de 75% dos isolados apresentaram diminuição de susceptibilidade às tetraciclinas ou macrólidos. Os antibióticos que apresentaram maior actividade sobre as bactérias estudadas foram o cloranfenicol, a ampicilina e os glicopéptidos. Para a maioria dos antibióticos, foram registadas maiores percentagens de resistências nas suiniculturas intensivas do que na suinicultura extensiva. Na pesquisa dos genes que conferem resistência aos glicopéptidos apenas foram encontrados os genes vanA (2%) e vanC1 (4%). Nos ensaios de conjugação obtiveram-se transconjugantes para todas as classes de antibióticos estudadas. Após o estudo de susceptibilidade aos antibióticos nas bactérias transconjugantes observou-se que ocorreram fenómenos de co-transferência, sugerindo que diferentes genes de resistência se encontram nas mesmas plataformas genéticas ou que se disseminam em eventos genéticos comuns. Os dados obtidos permitiram concluir que as suiniculturas portuguesas são reservatório de Enterococcus spp com resistência múltipla aos antibióticos. Estas bactérias, através da cadeia alimentar ou do contacto directo do homem com os animais ou ambiente de produção, podem colonizar pele e intestino e contribuírem para a dinâmica da disseminação da resistência aos antibióticos entre bactérias comensais e patogénicas.