Gene expression profiling of head and neck cancer

MDThe purpose of this study was to classify oral squamous cell carcinomas (OSCCs) based on their gene expression profiles, to identify differentially expressed genes in these cancers, and to correlate genetic deregulation with clinical-histopathological data and patient outcome. After conducting proof of principle experiments utilizing six head and neck squamous cell carcinomas (HNSCCs) cell lines, the gene expression profiles of 20 OSCCs and subsequently an additional 8 OSCCs were determined using cDNA microarrays containing 19,200 sequences and the Binary Tree-Structured Vector Quantization (BTSVQ) method of data analysis. Two sample clusters were identified in the group of 20 tumors that correlated with T3-T4 category of disease (P=0.035) and nodal metastasis( p=0.035). Samplec lustering of 28 OSCCsa nd the 6 cell lines revealed a correlation with disease free survival. BTSVQ analysis identified a subset of 23 differentially expressed genes with the lowest quantization error scores in the cluster containing more advanceds taget umors from the 20 OSCC dataset.T he expressiono f six of these differentially expressedg enesw as validated by quantitative real-time RT-PCR. Statistical analysis of quantitative real-time RT-PCR data was performed and, after Bonferroni correction, CLDNI (p = 0.007) over-expressionw as significantly correlated with the cluster containing more advanced stage tumors. Despite the clinical heterogeneity of OSCC, molecular subtyping by cDNA microarray analysis was able to identify distinct patternso f genee xpressiona ssociatedw ith relevant clinical parameters. The application of this methodology represents an advance in the classification of oral cavity tumors, and may ultimately aid in the development of more tailored therapies for oral carcinoma

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds

Report of the Surgeon General

In 1964, the first Surgeon General's report on the effects of smoking on health was released. In the nearly 50 years since, extensive data from thousands of studies have consistently substantiated the devastating effects of smoking on the lives of millions of Americans. Yet today in the United States, tobacco use remains the single largest preventable cause of death and disease for both men and women. Now, this 2010 report of the Surgeon General explains beyond a shadow of a doubt how tobacco smoke causes disease, validates earlier findings, and expands and strengthens the science base. Armed with this irrefutable data, the time has come to mount a full-scale assault on the tobacco epidemic. More than 1,000 people are killed every day by cigarettes, and one-half of all long-term smokers are killed by smoking-related diseases. A large proportion of these deaths are from early heart attacks, chronic lung diseases, and cancers. For every person who dies from tobacco use, another 20 Americans continue to suffer with at least one serious tobacco-related illness. But the harmful effects of smoking do not end with the smoker. Every year, thousands of nonsmokers die from heart disease and lung cancer, and hundreds of thousands of children suffer from respiratory infections because of exposure to secondhand smoke. There is no risk-free level of exposure to tobacco smoke, and there is no safe tobacco product. This new Surgeon General's report describes in detail the ways tobacco smoke damages every organ in the body and causes disease and death. We must build on our successes and more effectively educate people about the health risks of tobacco use, prevent youth from ever using tobacco products, expand access to proven cessation treatments and services, and reduce exposure to secondhand smoke. Putting laws and other restrictions in place, including making tobacco products progressively less affordable, will ultimately lead to our goal of a healthier America by reducing the devastating effects of smoking. This 2010 Surgeon General's report represents another important step in the developing recognition, both in this nation and around the world, that tobacco use is devastating to public health. Past investments in research and in comprehensive tobacco control programs--combined with the findings presented by this new report--provide the foundation, evidence, and impetus to increase the urgency of our actions to end the epidemic of tobacco use.CDC-INFO Pub ID 220456220456U.S. Department of Health and Human Services. How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General. Atlanta, GA: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, 2010.Chapter 1. Introduction, evaluation of evidence on mechanisms of disease production, and summary -- Chapter 2. The changing cigarette -- Chapter 3. Chemistry and toxicology of cigarette smoke and biomarkers of exposure and harm -- Chapter 4. Nicotine addiction: past and present -- Chapter 5. Cancer -- Chapter 6. Cardiovascular diseases -- Chapter 7. Pulmonary diseases -- Chapter 8. Reproductive and developmental effects -- Chapter 9. A vision for the future -- List of abbreviations -- List of tables and figures -- Definitions and alternative nomenclature of genetic symbols used in this report -- Index.2010704

The damaged mucosal barrier in intestinal inflammation: interaction with therapeutic bacteria and roles for metallothioneins as immunomodulators

Inflammatory bowel diseases (IBD) are a group of chronic, relapsing, immunologically-mediated disorders of the intestine, the main forms being Crohn’s disease and ulcerative colitis. Recent studies underscore the importance of the damaged epithelial barrier and the dysregulated innate immune system in the pathogenesis of IBD. These two components cause defects in the interaction of the host with the bacterial flora. The epithelium must maintain a physical barrier between the lumen and the underlying mucosa. Defects in the mucus layer, integrity, and permeability are found in IBD and are thought to have direct implications on disease initiation and/or progress. Furthermore, it has become clear that the intestinal epithelial cell lining exerts an important role in the identification of bacteria and the appropriate counteraction. The involvement of the innate immune system can be explained by two theories. The first one states that an exaggerated innate response and a loss of tolerance for the commensal intestinal microflora are caused by defective down-regulation of pattern recognition receptor signalling. The second theory encompasses a defective innate immunity originating from failure of recognition of bacterial threats or an ineffective response, which results in persistence of the exogenous stimulus and an exaggerated secondary immune response. The first part of this thesis focuses on the interaction of therapeutic bacteria with the damaged epithelial barrier. Genetically modified Lactococcus lactis bacteria were developed in order to provide a local mucosal delivery of the anti-inflammatory cytokine interleukin (IL)-10. Since local delivery of the cytokine in the tissue seems primordial, we focused on the in vivo interaction of the bacteria with intestinal murine mucosa and observed differences between an intact and an inflamed (damaged) epithelial barrier. In healthy intestinal tissue, we found an occasional transcellular uptake in follicle-associated epithelium. Our data were suggestive of dendritic cell sampling, but not of sampling by M cells. In inflamed mucosal tissue, both confocal and electron microscopic analysis suggested an enhanced uptake of lactococci through the paracellular route, which is likely to be a result of the damaged mucosal barrier. We were able to show the presence of viable and IL-10-producing lactococci in ileal and colonic lamina propria. The absence of lactococci in mesenteric lymph nodes or in the spleen excluded a systemic circulation of the bacteria, being safe for administration in patients. In the second part, the role of metallothioneins (MTs) as immunomodulators in intestinal inflammation is investigated. We found a significantly lower mRNA and protein expression in the epithelium of unaffected tissue of Crohn’s disease patients. In vitro studies with an MT-knockdown intestinal epithelial cell line showed a defective secretion of the neutrophil chemokine IL-8 upon bacterial challenge. A time course study of dextran sodium sulphate-induced colitis revealed that MTnull mice exhibited a significantly more severe histological inflammation in the early phase of colitis compared with wild type mice, a difference which was due to epithelial MTs. Both these in vitro and in vivo studies point to an immunomodulatory role for epithelial MTs in the acute intestinal immune response. In ulcerative colitis, we did not find this impaired basal epithelial MT expression. Here, active inflammation caused a down-regulation of epithelial MT and an up-regulation of non-epithelial MT which was also observed in other forms of intestinal inflammation. The inflammation-dependent down-regulation in the epithelium is in contrast with the known induction of MTs by proinflammatory cytokines and reactive oxygen intermediates, which are produced during inflammation. We found that transforming growth factor (TGF)-β, one of the few inhibitors identified for MT mRNA production, could decrease epithelial MT expression in ex vivo experiments. Contrary to the decrease in the epithelium, an increase in MT-expressing cells occurs in the lamina propria during active inflammation, predominantly caused by strongly MT-expressing fibroblasts of the granulation tissue. Since we did not find a correlation between MT expression and proliferation (a correlation which has been reported in vitro in the literature), we suggest that the role of MTs in these cells is a cytoprotective one, which would be beneficial for host cells in the inflammatory environment. Finally, we reviewed the MT regulatory mechanisms in health and in disease.De ziekte van Crohn en colitis ulcerosa zijn de belangrijkste chronische ontstekingsziekten van de darm. Recente studies benadrukken het belang van schade aan de epitheelbarrière en defecten in het aangeboren immuunsysteem in het ontstaan van beide ziekten. Deze schade zorgt ervoor dat de interactie tussen de microbiële darmflora en de gastheer niet meer optimaal verloopt. Het darmepitheel dient een fysische barrière te vormen tussen het lumen en de onderliggende mucosa. Bij de ziekte van Crohn en colitis ulcerosa worden afwijkingen in de mucuslaag, de integriteit en de permeabiliteit van het epitheel gevonden, waarvan men denkt dat ze een directe invloed hebben op de initiatie en de progressie van de ziekte. Naast zijn rol als fysische barrière heeft het epitheel ook een belangrijke taak in de identificatie van bacteriën en de gepaste reactie hierop. Het aangeboren immuunsysteem kan op twee manieren een rol spelen in the pathogenese. Een eerste theorie stelt dat defecten in het uitschakelen van proinflammatoire signalen een overdreven aangeboren immuunantwoord veroorzaken en een verlies van tolerantie tegenover de commensale microbiota. Volgens de tweede theorie worden pathogenen niet goed herkend, waardoor er een inefficiënte opruiming volgt die ervoor zorgt dat de exogene stimulus blijft bestaan en er een overdreven secundair immuunantwoord op gang komt. Het eerste deel van deze thesis handelt over de interactie van therapeutische bacteriën met de beschadigde epitheelbarrière. Genetisch gemodificeerde Lactococcus lactis bacteriën werden ontwikkeld om te zorgen voor een lokale toelevering van het anti-inflammatoir cytokine interleukine (IL)-10 in de mucosa. Aangezien deze lokale toelevering uitermate belangrijk is voor de therapeutische werking van de bacteriën, hebben we de interactie onderzocht tussen deze bacteriën en de darmmucosa. We vonden dat de bacteriën op transcellulaire wijze kunnen opgenomen worden in gezond follikelgeassocieerd epitheel. Onze data suggereerden eveneens opname door dendritische cellen, maar niet door M cellen. In ontstoken mucosa bemerkten we een verhoogde opname door paracellulair transport. Dit was waarschijnlijk veroorzaakt door de beschadigingen aanwezig in de mucosale barrière. We konden aantonen dat er levende en IL-10-producerende lactococcen in de lamina propria aanwezig waren. Uiteindelijk kon de afwezigheid van lactococcen in de mesenterische lymfeknopen en de milt uitsluiten dat deze bacteriën in de systemische circulatie terechtkomen. In het tweede deel onderzochten we de rol van metallothioneïnes (MTs) in darmontsteking. We vonden een significante daling in de mRNA en eiwitexpressie van MTs in het epitheel van gezond darmweefsel van patiënten met de ziekte van Crohn. Wanneer we bacteriële stimulaties uitvoerden op een darmepitheel cellijn met deficiënte MT expressie zagen we dat deze deficiëntie zorgde voor een daling in de secretie van het chemokine IL-8. Een gedetailleerde studie van dextraan sodiumsulfaat-colitis gaf aan dat MTnull muizen een zwaardere histologische inflammatie vertoonden dan wild type muizen in de vroege fase van colitis, een verschil dat te wijten was aan epitheliaal MT. Beide studies wijzen in de richting van een immunomodulatorische rol voor epitheliaal MT in het acute immuunantwoord van de darm. Deze verminderde basale epitheelexpressie vonden we niet terug bij colitis ulcerosa. In deze en andere ontstekingsziekten van de darm vonden we dat actieve inflammatie zorgde voor een daling in de epitheelexpressie van MT tegenover een stijging in de expressie van MT in de lamina propria. De ontstekingsafhankelijke daling in het epitheel komt niet overeen met de gekende inductie van MTs door proinflammatoire cytokines en reactieve zuurstofradicalen die geproduceerd worden tijdens ontsteking. Wij vonden dat transforming growth factor (TGF)-β de expressie van MT in epitheelcellen kon doen afnemen. In tegenstelling tot deze afname in het epitheel werd tijdens actieve inflammatie een toename in de expressie waargenomen in cellen van de lamina propria, voornamelijk veroorzaakt door fibroblasten van het granulatieweefsel die sterk positief waren. We suggereren dat MTs een cytoprotectieve rol hebben in deze cellen, wat een voordeel betekent in een inflammatoir milieu. Uiteindelijk hebben we een overzicht gemaakt van de mechanismen die de expressie van MTs regelen in ziekte en gezondheid

Unravelling Lung Cancer Heterogeneity and Associated Therapeutic Responses using in vivo and ex vivo Model Systems

Lung cancer is the major cause of cancer-related deaths worldwide (Ferlay et al., 2015; Siegel et al., 2018). Tobacco smoking is the primary risk factor, contributing to nearly 85% of the lung cancer deaths (Furrukh, 2013). Lung cancer is histologically and genetically a heterogeneous disease (Gridelli et al., 2015). Comprehensive genomic profiling has led to the classification of lung cancer into distinct molecular subtypes, and the development of targeted therapies against specific genetic alterations (Bronte et al., 2010). However, as of yet, no clinically approved therapies available for the treatment of lung cancer patients carrying common driver genes, namely mutations in oncogenic KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog), and the tumor suppressor STK11 (Serine/Threonine Kinase 11, also known as LKB1). This thesis investigates the role of niche-specific lung progenitor cells in defining NSCLC histotype spectra and immune microenvironment heterogeneity, using a murine model driven by oncogenic Kras and loss of the tumor suppressor Lkb1. In addition, using Kras-driven NSCLC mouse models, this thesis also demonstrates histotype-associated spatial oncogenic signaling heterogeneity and its importance in defining therapeutic responses. Furthermore, it establishes Kras-driven NSCLC tumor tissue slice cultures and shows their utility as ex vivo models in predicting spatial responses to drug combinations. Finally, by performing drug screening on murine NSCLC primary cell cultures coupled with in vivo acute drug response analysis, this thesis identifies tumor subtype-selective therapeutic sensitivity and resistance mechanisms, as well as effective drug combinations.Keuhkosyöpä on syöpäperäisten kuolemien suurin syy maailmanlaajuisesti (Ferlay et al., 2015; Siegel et ai., 2018). Tupakan tupakointi on ensisijainen riskitekijä, joka myötävaikuttaa lähes 85 prosenttiin keuhkosyöpäkuolemista (Furrukh, 2013). Keuhkosyöpä on histologisesti ja geneettisesti heterogeeninen sairaus (Gridelli ym., 2015). Kattava genominen profilointi on johtanut keuhkosyövän luokitteluun erillisiin molekyylialatyyppeihin ja kohdennettujen terapioiden kehittämiseen tiettyjä geneettisiä muutoksia vastaan ​​(Bronte et al., 2010). Kuitenkin vieläkään kliinisesti hyväksyttyjä terapioita ei ole saatavilla keuhkosyöpäpotilaille, joilla on tavallisia kuljettajageenejä, nimittäin onkogeenisen KRAS: n (Kirsten Rat Sarcoma Viral Oncogene Homolog) ja kasvaimen suppressori STK11 (Serine / Threonine Kinase 11, myös tunnetaan nimellä LKB1). Tämä opinnäytetyö tutkii niche-spesifisten keuhkojen progenitorisolujen roolia määriteltäessä NSCLC-histotyyppisiä spektrejä ja immuuni-mikroympäristön heterogeenisyyttä käyttämällä hiirimallia, jota onkologinen Kras ja peräsuolen vaimennin Lkb1. Lisäksi tässä tutkimuksessa käytetään Kras-ohjattujen NSCLC GEM -mallien avulla loogisesti tyypillistä spatiaalisen onkogeenisen signaloinnin heterogeenisyyttä ja sen merkitystä terapeuttisten vasteiden määrittämisessä. Lisäksi se luo Kras-ohjatut NSCLC-tuumorikudosviipaleet ja osoittaa niiden käyttökelpoisuuden ex vivo -malleina spatiaalisten vasteiden ennustamiseksi lääkekombinaatioille. Lopuksi, tekemällä huumeiden seulonta hiiren NSCLC-primaarisoluviljelmillä, joihin on kytketty in vivo akuutti lääkeanalyysianalyysi, tässä työssä tunnistetaan kasvain-alatyyppiselektiivinen terapeuttinen herkkyys ja vastustusmekanismit sekä tehokkaat lääkeyhdistelmät

Translational Studies on Inflammation

Inflammation is known worldwide, from the bench to the bedside, but it is a hard theme to approach with one single point of view.In this sense, a selection of translational studies would support the medical-scientific community to better understand the complex network of the inflammatory process, its maintenance, and potential treatment targets. The eleven chapters that compose this book present interesting insights into inflammation and its mechanisms, merging classic background with innovative approaches. From the molecular basis to experimental models, the chapters selected for this book bring to readers at different academic levels updated and practical data on inflammation. Find out what drives interdisciplinary medical research on inflammation and enjoy this informative collection

TRP Channels in Health and Disease

Almost 25 years ago, the first mammalian transient receptor potential (TRP) channel was cloned and published. TRP channels now represent an extended family of 28 members fulfilling multiple roles in the living organism. Identified functions include control of body temperature, transmitter release, mineral homeostasis, chemical sensing, and survival mechanisms in a challenging environment. The TRP channel superfamily covers six families: TRPC with C for “canonical”, TRPA with A for “ankyrin”, TRPM with M for “melastatin”, TRPML with ML for “mucolipidin”, TRPP with P for “polycystin”, and TRPV with V for “vanilloid”. Over the last few years, new findings on TRP channels have confirmed their exceptional function as cellular sensors and effectors. This Special Book features a collection of 8 reviews and 7 original articles published in “Cells” summarizing the current state-of-the-art on TRP channel research, with a main focus on TRP channel activation, their physiological and pathophysiological function, and their roles as pharmacological targets for future therapeutic options

Cellular Oxidative Stress

This book collects 17 original research papers and 9 reviews that are part of the Special Issue “Cellular Oxidative Stress”, published in the journal Antioxidants. Oxidative stress on a cellular level affects the function of tissues and organs and may eventually lead to disease. Therefore, a precise understanding of how oxidative stress develops and can be counteracted is of utmost importance. The scope of the book is to emphasize the latest findings on the cellular targets of oxidative stress and the potential beneficial effect of antioxidants on human health