Cigarette smoke is well known for its adverse effects on human health, especially on the lungs. Basic research is essential to identify the mechanisms involved in the development of cigarette smoke-related diseases, but translation of new findings from pre-clinical models to the clinic remains difficult. In the present study, we aimed at comparing the gene expression signature between the lungs of human smokers and mice exposed to cigarette smoke to identify the similarities and differences. Using human and mouse whole-genome gene expression arrays, changes in gene expression, signaling pathways and biological functions were assessed. We found that genes significantly modulated by cigarette smoke in humans were enriched for genes modulated by cigarette smoke in mice, suggesting a similar response of both species. Sixteen smoking-induced genes were in common between humans and mice including six newly reported to be modulated by cigarette smoke. In addition, we identified a new conserved pulmonary response to cigarette smoke in the induction of phospholipid metabolism/degradation pathways. Finally, the majority of biological functions modulated by cigarette smoke in humans were also affected in mice. Altogether, the present study provides information on similarities and differences in lung gene expression response to cigarette smoke that exist between human and mouse. Our results foster the idea that animal models should be used to study the involvement of pathways rather than single genes in human diseases. There are more than one billion cigarette smokers on the planet; cigarette smoking being responsible for five million deaths every year worldwide In the past decades, our understanding of the cellular and molecular consequences of cigarette smoke exposure on the lung has expanded tremendously owing to new research tools, and increased availability of clinical samples and animal models. Despite major technological advancements, one important problem remains; observations made in animal models do not always reflect human pathobiology. As a consequence, we are still facing the translational challenge associated with the use of animals to model human diseases, including chronic obstructive pulmonary disease (COPD) There is currently no large-scale study comparing the pulmonary gene expression profile associated to cigarette smoke exposure in humans and mice. Such study has the potential to identify a conserved response to cigarette smoke between the two species, identify new genes and cellular pathways affected by cigarette smoke, validate the use of animal models in investigating the role played by given genes or pathways, and facilitate the translation of knowledge acquired in animal models to clinically relevant findings.Using a human and a mouse whole-genome expression study, we identified the pulmonary genes, pathways and biological functions affected by cigarette smoke in a mouse model of cigarette smoke exposure and validated the findings in a previously described large-scale transcriptomic human data set Institut universitaire de cardiologie et de pneumologie de Québec (IUCPQ) ethics board. All subject provided written informed consent. Animal experiments were performed in respect to the Canadian Council on Animal Care policies and guidelines. McMaster’s Animal Research Ethics Board (AREB) reviewed and approved animal experimentation protocols.Sample collection from human subjects was reviewed and approved by the www.tissuebank.ca). Clinical characteristics of subjects are shown in Human subjects and the lung specimen collection were described previously Female BALB/c mice were obtained from Charles River at 6–8 weeks of age. Using a whole body exposure system , mice (5 per group) were exposed to room air or the mainstream cigarette smoke of twelve 3R4F reference cigarettes  with filters removed 5 days per week, twice daily for 50 minutes/exposure for 8 weeks, as previously described limma package. Wilcoxon tests were used to detect gene differentially expressed between mice exposed and non-exposed to smoking. Genes were considered differentially expressed for Benjamini-Hochberg corrected p-value <0.05. The lists of genes differentially expressed in human and mouse were then compared. A head-to-head comparison was performed considering the interspecies differences in gene names using the Biomart software (www.ensembl.org/biomart/martview).The human and mouse genomic datasets were deposited in the GEO repository with accession number GSE23546 and GSE55127, respectively. All gene expression analyses were carried out with the R statistical software and packages . Human gene expression was analyzed as described previously www.ingenuity.com). Independent core analyses were performed in mice and humans to identify biological functions and canonical pathways enriched for smoking-induced genes. The Gene Set Enrichment Analysis (GSEA) program Biological pathways analyses were performed using the Ingenuity Pathways Analysis software  (34% [human] and 46% [mouse]) were found in both species. Finally, 71 and 75 biological functions were affected by cigarette smoke in the humans and mice, respectively, 68 found in common (96% [human] and 91% [mouse]). These data suggest that the similarities of the effect of cigarette smoke on the lung between humans and mice are mainly found at the pathway and biological function levels rather than the single gene level  . Ingenuine level .Genes are the main unit of comparison in most studies performed on clinical samples and animal models. Moreover, identifying the involvement of a gene in a given disease is a common research approach. Among the 17 genes significantly modulated by cigarette smoke in mice and humans, 10 were previously reported and investigated. These include MMP12, AHRR, SPP1, ALDH3A1, CYP1B1, GDF15, GSTA2, NQO1, PLA2G7, TREM2 and CLEC5A. The genes previously reported to be upregulated in smokers/COPD patients or cigarette smoke-exposed mice provide validation of our gene expression comparison between mice and humans. We also identified 6 new genes namely ACP5, ATP6V0D2, BHLHE41, NEK6, DCSTAMP and LCN2. These new genes associated with cigarette smoke exposure in both humans and mice may be of great interest for future research.While the similarities between the two species are scarce at the gene level, it is possible that both species are initiating a similar response involving different genes. To identify pathways altered by cigarette smoke in humans and mice, canonical pathways from the Ingenuity Pathway Analysis Software were evaluated. The pathways activated by cigarette smoke in both mice and humans are presented in The lung response to cigarette smoke might not activate the same genes or trigger the same cellular and molecular pathways in both humans and mice but can still involve similar biological functions. In an effort to understand the global response of the human and mouse lung to cigarette smoke, genes were grouped under biological functions using the Ingenuity Pathway Analysis Software. Functions related to immune processes, metabolism and cell functions, diseases, tissue injury and repair, and organ development were observed . InteresThe present study aimed at comparing the human and mouse lung gene expression signature to cigarette smoke exposure. To do so, we compared two genome-wide expression experiments, one in human lungs of non-smokers and smokers and the other one in lungs of mice exposed to room air or cigarette smoke. This interspecies gene expression comparison allowed us to identify 6 new genes modulated by cigarette smoke. Moreover, we confirmed the activation of pathways linked to xenobiotics detoxification and oxidative stress defense/generation. We also identified a new response conserved among mice and humans in the activation of pathways involved in phospholipid metabolism and degradation. Finally, we found that biological functions affected by cigarette smoke exposure are largely similar in human and mouse lung.Wu et al. showed that GDF15 expression is increased in the lung of COPD patients as well as in human lung epithelial cells exposed to cigarette smoke Our study confirmed the impact of cigarette smoke on smoking-induced genes previously described in humans and mice. The role of MMP12 + transporting, lysosomal 38kDa, V0 subunit d2 (ATP6V0D2) is a member of the H+-ATPase family and is responsible for the acidification of the extracellular environment by osteoclasts Atp6v0d2 null mice exhibit osteopetrosis also called the “stone bone” disease Acp5 knockout mice have mild osteoporosis The genes ACP5, ATP6V0D2, BHLHE41, NEK6, DCSTAMP and LCN2 were found to be associated to cigarette smoke exposure but to the best of our knowledge, their roles in smoking-related diseases were never studied. Class E basic helix-loop-helix protein 41 (BHLHE41) is a transcription factor that has an impact on a variety of cellular functions. Its expression can also be induced by many stimuli such a growth factors The involvement of MMP12, AHRR, SPP1, GSTA2, ALDH3A1, CYP1B1, NQO1, TREM2 and CLEC5A in the lung response to cigarette smoke can be associated to immune or detoxification responses. However, the reasons for ACP5, ATP6V0D2, BHLHE41, NEK6, TM7SF4, LCN2, GDF15 and PLA2G7 upregulation are less intuitive. Interestingly, the genes ATP6V0D2, ACP5, DCSTAMP and SPP1 have been mainly studied for their role in bone homeostasis and are expressed by bone-resorbing osteoclasts. Further studies on the involvement of those genes in cigarette smoke-associated disease are required.Ahr-deficient mice exhibit a more robust pulmonary inflammation than their wildtype counter-parts when exposed to cigarette smoke We found that the three major clusters of cellular pathways activated in the lung of mice and humans exposed to cigarette smoke were the response to xenobiotics/aryl hydrocarbon receptor activation, oxidative stress defense/generation and lipid/phospholipid degradation. The presence of xenobiotic compounds in cigarette smoke such as poly aromatic hydrocarbons (PAH) and the associated detoxification response involving the aryl hydrocarbon receptor (AHR) and members of the cytochrome p450 family has been well described 15 reactive oxygen species (ROS) per puff as well as reactive aldehydes and heavy metals The role of oxidative stress is one of the oldest research ground in the field of cigarette smoke exposure. Cigarette smoke contains more than 10Pathways involved in phospholipid metabolism and degradation were activated in both the human and mouse lungs. Three genes in humans and four genes in mice were coding for phospholipases. Phospholipases are enzymes that hydrolyze phospholipids into fatty acids and other compounds, releasing modified fatty acids such as arachidonic acid and second messengers that will be used for eicosanoid synthesis or activate intracellular signaling Cigarette smoke exposure in humans and mice were both reflected in the activation of biological functions related to inflammation, metabolism and cell maintenance, injury and repair, diseases and development. The role of inflammation, injury and repair are the focus of most of the research done on cigarette smoke exposure. However, cigarette smoke seems to affect genes involved in a broad range of functions related to cell maintenance and metabolism. More specifically, the metabolism of major cellular constituents such as lipids, carbohydrates, vitamins and nucleic acids is affected. Therefore, it is not surprising that cigarette smoke has so many systemic effects. In fact, many genes and functions related to non-pulmonary diseases and to the development of other organs are affected within the lungs. It is tempting to speculate that genes affected by cigarette smoke within the lungs may also be affected in other tissues/organs. Finally, the near complete overlap of biological functions affected by cigarette smoke between humans and mice validates the use of the animal model to explore the mechanisms leading to their activation.An inherent limitation of this study was the use of a single inbred mouse strain. It is plausible that the regulation of some genes may only be significant in this strain. Further studies are required to compare gene expression profiles across different inbred mouse strains. In addition, the majority of the clinical samples were obtained from subjects undergoing surgery for lung cancer. Although, we collected non-tumor specimens as distant as possible from the tumor, cancer might still interfere with the expression of some genes and pathways.In the present study, we found that similarities in the transcriptomic response of the lungs between humans and mice exposed to cigarette smoke reside more at the pathway and functional levels rather than the single gene level. Beyond the basic research findings, this study provides empirical data to support that limiting analysis to single genes in animal models may, in many cases, compromise the translational potential to the clinic. While mice and humans show great similarities in the response to many different stimuli, it is likely that they activate different genes, or molecular languages, to achieve the same goal. Therefore, rather than studying the involvement of specific genes, efforts should be deployed to understand the role played by specific pathways or functions. This more integrative approach in mouse would likely improve the translational potential of new findings derived from this unsurpassed pre-clinical model to study human biology and disease.File S1Supporting figures and tables. Figure S1. Genes in common (orthologue) between the human and mouse expression microarrays. Human  and mouse  microarrays were compared on the basis of gene names to identify orthologue genes with probes present in both sets. Figure S2. Enrichment plot for genes modulated by cigarette smoke in mice. Genes modulated by cigarette smoke in mice were tested for enrichment against the genes modulated by cigarette smoke in humans pre-ranked according to their fold change. Table S1. Canonical pathways altered by cigarette smoke in the human lung only. Table S2. Canonical pathways altered by cigarette smoke in the mouse lung only.(DOC)Click here for additional data file.


Researchers involved in the delivery of periodontal therapy are currently investigating the possible use of oral fluids in the diagnosis of oral diseases and drug development. Substantial improvements have been made in the understanding of the mediators implicated on the initiation, pathogenesis, and progression of periodontitis. This review will analyze the mechanisms involved in the breakdown of periodontal supporting tissues during chronic periodontitis and highlights the potential array of biomarkers present in gingival crevicular fluid (GCF), which may relate to existing or predicted tissue regions undergoing metabolic change. Periodontitis involves progressive loss of the alveolar bone around the teeth, and if left untreated, can lead to the loosening and subsequent loss of teeth. It is caused by microorganisms that adhere to and grow on the tooth's surfaces, along with an overly aggressive immune response against these microorganisms [2]. Damage to the periodontal tissue is usually detected by means of periodontal probing, which shows loss of attachment of the tooth, or by radiographs that detect alveolar bone loss. These methods also evaluate the damage caused by previous destruction episodes, resulting in a retrospective diagnosis [3]. Of the 3 fluids found within the oral cavity—gingival crevicular fluid (GCF), serum, and total saliva—the first two have been the focus of the most research in recent years. Due to the non-invasive and simple nature of their collection, analysis of saliva and GCF may be especially beneficial in the determination of current periodontal status and a means of monitoring response to treatment [5]. Accurate detection of periodontal sites exhibiting disease progression or those at risk of future deterioration has proven difficult. The development of a test for most mediators associated with the anatomic events of periodontitis may serve as a useful method for identifying and predicting future progression [6]. It is composed of serum and locally generated materials such as tissue breakdown products, inflammatory mediators, and antibodies directed against dental plaque bacteria. The composition of the GCF is the result of the interplay between the bacterial biofilm adherent to the tooth surfaces and the cells of the periodontal tissues. The collection of GCF is a minimally invasive procedure and the analysis of specific constituents in the GCF provides a quantitative biochemical indicator for the evaluation of the local cellular metabolism that reflects a person’s periodontal health status [7]. Since GCF is an inflammatory exudate that reflects ongoing events in the periodontal tissues that produce it, an extensive search has been made for GCF components that might serve as potential diagnostic or prognostic markers for the progression of periodontitis [8]. GCF is an inflammatory exudate that seeps into gingival crevices or periodontal pockets around teeth with inflamed gingival [9] stated that "markers of disease" might encompass three separate categories: Curtis et al. . About 60% of the total acid phosphatase in whole gingival fluid originates from bacteria [14]. The levels of acid phosphatase do not correlate with measurements of disease severity or activity. Acid phosphatase - It has been widely investigated amongst the lysosomal enzymes and has often been used as a lysosomal marker. Quantitative analysis confirmed that gingival fluid contains 10-20 times more acid phosphatase than serum. The host sources are the PMNs and desquamating epithelial cells [13]. The enzyme is liberated from macrophages, fibroblasts and endothelial cells of healthy or chronically inflamed gingival [15]. It is also positively associated with the number of Spirochetes, Porphyromonas gingivalis, Prevotella intermedia and lactose-negative black pigmenting bacteria in the subgingival flora. The level of β-glucuronidase correlates significantly with attachment loss that may subsequently occur in individuals with adult periodontitis [16]. β –Glucuronidase - It is one of the hydrolases found in the azurophilic or primary granules of PMNs [17]. It acts upon elastin, proteoglycans, hemoglobin, fibrinogen and collagen. Leukocyte elastase degrades mature collagen fibers. Amounts of GCF elastase are greater in periodontitis patients than healthy controls [18]. Elastase - Neutrophil elastase is a serine proteinase confined to the azurophil granules of PMNs which are analogous to lysozymes [18]. Both α1-antitrypsin and α2-macroglobulin were found in gingival fluid by Schenkein and Genco [19] in concentrations representing three-fourths of those found in serum. In inflamed gingiva, GCF samples had about twice as much α2- macroglobulin than the samples collected in the same area after therapy.  Elastase inhibitors - The activity of proteases in the tissues is probably modulated by the presence of inhibitors either produced locally or circulating in plasma. The main plasma inhibitors are α2-macroglobulin and α1-antitrypsin, which accounts for more than 90% of the total protease inhibiting capacity of serum. A third physiological inhibitor, α2-antichymotrypsin seems to inactivate only chymotrypsin-like enzymes, for instance Cathepsin G α2-macroglobulin inhibits all three neutral proteinases from PMN’s by a similar mechanism which consists of irreversible trapping of the enzyme molecule by the inhibitor. α1-antitrypsin inactivates mainly serine proteinases, elastase and cathepsin G and partially mammalian collagenase [20]. GCF concentrations of cathepsin B were found to be elevated in patients with periodontal disease, but lower in patients with gingivitis [21]. Thus, it may have a potential use in distinguishing periodontitis from gingivitis and in planning treatment and monitoring treatment outcomes [22]. Cathepsin D, a carboxy endopeptidase, is present at high concentration in inflamed tissues. Its concentration is found to be 10 times higher in GCF during periodontal destruction [23]. Cathepsin G is serine endopeptidase contained in the azurophil granules of PMNs. It is also known as chymotrypsin like, because it attacks a number of synthetic substrates typical for chymotrypsin and is inhibited by the same inhibitors. It hydrolyzes hemoglobin and fibrinogen, casein, collagen and proteoglycans. Measurements of cathepsin and neutral proteases, have also shown a relationship to the severity of inflammation but no association with disease activity has been demonstrated [24].  Cathepsins – It is an enzyme belonging to the class of cysteine proteinases. In GCF, macrophages are the main producers of cathepsin B [25]. Porphyromonas gingivalis, which is frequently isolated from periodontal lesions in adults with advanced periodontitis, possesses a spectrum of proteases including a trypsin-like enzyme [26]. The presence of this trypsin-like enzyme increases the potential of this organism to mediate destruction of periodontal tissues. It has to cleave peptide substrates with arginine terminal groups such as benzoyl-arginine-2 naphthylamide (BANA) or benzoyl-arginine-p-nitroanilide (BAPNA). The trypsin-like enzyme found in P. gingivalis is able to degrade collagen directly and its GCF levels might provide useful information on the periodontal condition [27].Trypsin-like enzymes - Proteolythic activities associated with black-pigmented Bacteroides species have long been considered as virulence factors in the pathogenesis of periodontal disease [28]. Such enzymes have been assumed to facilitate both bacterial colonization on mucous membranes and penetration of bacterial cells and their antigenic products through the mucosal barrier by elimination of immunoglobulins [28]. Consequently, immunoglobulin-degrading enzymes have been demonstrated mainly in pathogenic species and in species closely associated with infectious diseases [28]. GCF IgG antibodies to periodontopathic organisms are present in significantly higher levels in periodontal disease patients than in normal control subjects [29]. Immunoglobulin-degrading proteases – They constitute a group of microbial enzymes that have gained much interest due to their potential significance as virulence factors [30]. They have the capacity to degrade collagen but their main function most likely lies in the activation of pro-forms of other enzymes, cytokines, and other immune mediators. Eley and Cox [31] monitored GCF levels of DPP II and IV and reported higher levels of both enzymes in sites with rapid and gradual attachment loss than in paired sites without attachment loss. Dipeptidyl Peptidases (DPP) - They are derived from lymphocytes, macrophages, and fibroblasts. DPP II has been localised to macrophages and fibroblasts30 in gingival tissue and in cells in GCF. DPP IV has been localised to monocytes, macrophages, fibroblasts and CD4 and CD8 lymphocytes [32]. It has been reported that an elevated level of neutral protease activity suggests an active phase of periodontal disease [32]. Non-specific neutral proteases - Neutral protease is a non-specific metalloprotease. It cleaves fibronectin, collagen IV, and to a lesser extent collagen I, but it does not cleave collagen V or laminin. It hydrolyzes N-terminal peptide bonds of non-polar amino acid residues and may preferentially attack denatured and intercellular proteins with exposed hydrophobic amino acid residues [33]. The neutrophils are the major cells responsible for MMP release at the infected site, specifically MMP-8 (collagenase-2) and MMP-9 (gelatinase-B) [34]. Although MMP-8 is able to potently degrade interstitial collagens, MMP-9 degrades several extracellular matrix proteins [31]. Mammalian collagenases initiate degradation by making a single cut; subsequent degradation of the denatured collagen molecule can be mediated by the gelatinases. GCF collagenase and collagenase activity has been shown to increase with increasing severity of inflammation and increasing pocket depth and alveolar bone loss [35]. Stromelysins (SL) are the major MMPs of fibroblast origin, and can activate fibroblast type collagenase [36]. Birkedal-Hansen et al. [37] have also suggested that SL may act as a marker of stromal cell involvement in the process of tissue degradation. Matrix Metalloproteinases - Host cell-derived enzymes such as matrix metalloproteinases (MMPs) are an important group of neutral proteinases implicated in the destructive process of periodontal disease that can be measured in GCF [38] The mean amounts of SL and TIMP in diseased sites (gingivitis and periodontitis) is significantly higher than the mean amount of these GCF components in healthy sites [39]. TIMPs – They are locally produced and their main role is defending connective tissues in the very local area around the cell from which metalloproteinases are secreted. The tissue degradation is further thought to be induced by an imbalance between MMPs and TIMP [40]. MPO is produced in the phagosome in excess concentrations of those that mediate bacterial killing. It has been suggested that MPO functions primarily to maintain a low concentration of hydrogen peroxide in the phagosome, thereby preserving the function of the granule proteases that otherwise would undergo irreversible oxidant-mediated inactivation if hydrogen peroxide accumulated in the phagosome [41]. High MPO levels in GCF from patients with progressive chronic periodontitis, and their reduction in response to treatment have been reported by Hernandez et al. [42]. Myeloperoxidase - The myeloperoxidase-hydrogen peroxide-chloride system, which is part of the innate host defence mediated by polymorphonuclear leukocytes, possesses potent antimicrobial activity [43]. Although LDH is found in bacteria, most of its GCF concentration originates from the periodontal tissues. No significant correlation is found between the levels of LDH in gingival fluid and disease severity. As such it could reflect metabolic changes, such as the increase in anaerobic glycolysis characteristic of inflamed gingival [44].Lactate dehydrogenase - It catalyses the reversible reduction of pyruvate to lactate. GCF contains 10-20 times more LDH than blood [45] have examined the relationship between β-glucuronidase and arylsulfatase and have shown that levels in the GCF are elevated in inflamed relative to healthy non-inflamed sites, and that these levels decrease following periodontal treatment. Arylsulfatase - It catalyzes the release of esterbound sulfate from a variety of O-sulfate esters and was shown to be higher in activity in GCF in gingivitis and periodontitis patients. Larnster and Co-workers [46]. β-N-acetyl-hexosaminidase (β-NAH) - It is an acid lysosomal hydrolase that emanates into GCF during neutrophilic phagocytosis. Under secretory conditions, the precursor forms of the newly synthesized enzyme will be liberated. During phagocytosis and cellular lysis, the lysosomal beta-N-acetyl-hexosaminidase is present. Untreated periodontitis is associated with elevated levels of myeloperoxidase, β-NAH, β-Glucoronidase, and Cathepsin D that may contribute to promoted loss of periodontal support and periodontal therapy induces a sustained down regulation of leukocyte activity as evidenced by the remission of GCF markers [46] and human [47] studies. Elevated glycosaminoglycan concentrations were also found in aggressive periodontal diseases, and associations have been made with periodontal pathogens such as P. gingivalis [48]. Glycosaminoglycans - Proteoglycans have a core protein on which one or more heteropolysaccharides  are bound covalently. Different glycosaminoglycans can be found, depending on the tissue, although the most common are the nonsulfated hyaluronic acid, and the sulfated heparan sulfate, Dermatan sulphate, chondroitin-4 sulfate and chondroitin-6 sulfate. In general, chondroitin-4-sulfate is the most common glycosaminoglycan in the periodontium. Proteoglycans have the ability to bind most collagens as well as fibronectin. Upon degradation of periodontal tissues, glycosaminoglycans are released, making their way into the GCF. Chondroitin-4-sulfate appears to be the major glycosaminoglycan in untreated chronic periodontitis sites, as shown in both animal [49]. Hydroxyproline - It is a characteristic amino acid is a major component of the collagen. Hydroxyproline and proline play key roles for collagen stability. They permit the sharp twisting of the collagen helix. Thus it is a major breakdown product of collagen present in the GCF [50] its main role is in cell adhesion and proliferation, which explains its potential use in regenerative strategies. Cross-sectional studies have revealed that fibronectin is invariably found in a degraded form in the GCF, [52] and therefore is inactive [53]. Therefore, its presence in GCF would indicate FN fragmentation due to tissue destruction and not simply inflammation [54]. Fibronectin fragments - It is one of the components of the extracellular matrix (ECM) of periodontal tissue [ Connective tissue and Bone proteins55]. In a cross-sectional study by Bowers et al.[56]. GCF samples were analyzed from patients with gingivitis, at moderate or severe periodontal disease states. Osteonectin - Also referred to as secreted protein acidic and rich in cysteine and basement membrane protein (BM-40), osteonectin is a single-chain polypeptide that binds strongly to hydroxyapatite and other extracellular matrix proteins including collagens. Because of its affinity for collagen and hydroxylapatite, osteonectin has been implicated in the early phases of tissue mineralization [57]. Osteocalcin is predominantly synthesized by osteoblasts [58], and it has an important role in both bone resorption and mineralization [59]. Elevated serum osteocalcin levels have been shown in periods of rapid bone turnover [60]. Serum osteocalcin is presently considered a valid marker of bone turnover when resorption and formation are coupled, and a specific marker of bone formation when formation and resorption are uncoupled [59]. Relationship between GCF osteocalcin levels and periodontal disease have been reported [61]. Osteocalcin - It is a small calcium-binding protein of bone, and is the most abundant non collagenous protein of mineralized tissues [52]. Type I collagen peptides - The most common extracellular matrix component is collagen, which is synthesized in a pro-form containing a terminal propeptide. After cleavage, these peptides are eliminated through the gingival pocket where they can be measured, thus they represent collagen biosynthesis and not degradation. Collagen I carboxy-terminal propeptide and collagen III amino-terminal propeptide were detectable in the GCF of patients with periodontitis, but not in healthy subjects, suggesting that turnover is higher in inflamed sites. The GCF levels of these collagens are increased after nonsurgical periodontal treatment, and return to baseline levels after a few days [62]. However, since OPN is produced by both osteoblasts and osteoclasts, it holds a dual function in bone maturation and mineralization as well as bone resorption [63] Sharma et al. [64] published findings from an investigation of GCF OPN that its concentrations increased proportionally with the progression of disease and when nonsurgical periodontal treatment was provided, its levels were significantly reduced. Osteopontin (OPN) - It is a single-chain polypeptide. In bone matrix, OPN is highly concentrated at sites where osteoclasts are attached to the underlying mineral surface, that is, the clear zone attachment areas of the plasma membrane [65] noted that a simple response against chemotactic factors seemed not to lead to basement membrane destruction, while activated neutrophils generated extensive destruction of the basement membrane. Higher amounts of laminin in GCF from patients with periodontitis suggest the presence of hyperactive neutrophils during the transmigration process through the endothelium/epithelium [66]. Laminin - It is a 900-kDa glycoprotein found in all basement membranes. During gingival inflammation, neutrophils leave the blood vessels and migrate through the connective tissue towards the inflammatory lesion, and some of them invade the gingival crevice. Steadman et al. [67] identified calprotectin in GCF and found that GCF concentration levels in patients with periodontal disease were higher than those in GCF from healthy subjects. The expression of calprotectin from inflammatory cells appears to offer protection of the epithelial cells against binding and invasion by P. gingivalis. In periodontal disease, it appears to improve resistance to P. gingivalis by boosting the barrier protection and innate immune functions of the gingival epithelium [68].Calprotectin - It is a 36-kDa protein composed of a dimeric complex of 8- and 14-kDa subunits. Neutrophils are the primary source of calprotectin although other cells, such as activated monocytes and macrophages and specific epithelial cells, are also capable of manufacturing the protein. Calprotectin acts as a calcium- and zinc-binding protein with both antimicrobial and antifungal activities. It also plays a role in immune regulation through its ability to inhibit immunoglobulin production and acts as a proinflammatory protein for neutrophil recruitment and activation. In periodontology, Kido et al.[69]. Both peptides may also act as substrates of proline-specific peptidases studied in treponemes isolated from the human subgingival dental plaque [69]. These two particular Hb β-chain sequences were present in GCF and successful periodontal therapy will reduce the levels of these peptides [70].Hemoglobin β-chain peptides - 2 peptides derived from the hemoglobin (Hb) are β-chain decapeptide and a dodecapeptide. They are pharmacologically and physiologically active, and act as inflammatory mediators [71]. Subsequent to osteoclastic bone resorption and collagen matrix degradation these molecules are released into the circulation. Given their specificity for bone resorption, pyridinoline cross-links represent a potentially valuable diagnostic aid in periodontics, because biochemical markers specific for bone degradation may be useful in differentiating the presence of gingival inflammation from active periodontal and peri-implant bone destruction [72]. Pyridinoline crosslinks (ICTP) – They represent a class of collagen-degrading molecules that include pyridinoline, deoxypyridinoline, N-telopeptides, and C-telopeptides [73]. Several growth factors are concentrated in the organic matrix of bone and released during bone resorption [74], and are therefore suggested to play a role in bone remodelling through regulation of the coupling process of bone resorption and formation [75]. There are several studies in the periodontal literature examining GCF and salivary levels of growth factors for periodontal disease diagnosis including of epidermal growth factor (EGF),[76] transforming growth factor-α (TGF-α) and TGF-β,[74] platelet-derived growth factor (PDGF),[77] and vascular-endothelial growth factor (VEGF) [78]. Polypeptide growth factors are a class of natural biological mediators that regulate key cellular events in tissue repair, including cell proliferation, chemotaxis, differentiation, and matrix synthesis, by binding to specific cell-surface receptors [GCF is a vehicle for monitoring tissue and cell products and allows a degree of non-invasive access to the periodontium, unlike the majority of other tissues in the body. It carries multiple molecular factors derived from the host response and is considered a significant protective mechanism in periodontal infection. Substantial improvements have been made in the understanding of the mediators implicated in the initiation, pathogenesis, and progression of periodontitis. Evaluation of the markers in GCF is considered a good method in the determination of a person’s risk for periodontal disease.


Neurofibromatosis (NF1) represents the most common single gene cause of learning disabilities. NF1 patients have impairments in frontal lobe based cognitive functions such as attention, working memory, and inhibition. Due to its well–characterized genetic etiology, investigations of NF1 may shed light on neural mechanisms underlying such difficulties in the general population or other patient groups. Prior neuroimaging findings indicate global brain volume increases, consistent with neural over-proliferation. However, little is known about alterations in white matter microstructure in NF1. We performed diffusion tensor imaging (DTI) analyses using tract-based spatial statistics (TBSS) in 14 young adult NF1 patients and 12 healthy controls. We also examined brain volumetric measures in the same subjects. Consistent with prior studies, we found significantly increased overall gray and white matter volume in NF1 patients. Relative to healthy controls, NF1 patients showed widespread reductions in white matter integrity across the entire brain as reflected by decreased fractional anisotropy (FA) and significantly increased absolute diffusion (ADC). When radial and axial diffusion were examined we found pronounced differences in radial diffusion in NF1 patients, indicative of either decreased myelination or increased space between axons. Secondary analyses revealed that FA and radial diffusion effects were of greatest magnitude in the frontal lobe. Such alterations of white matter tracts connecting frontal regions could contribute to the observed cognitive deficits. Furthermore, although the cellular basis of these white matter microstructural alterations remains to be determined, our findings of disproportionately increased radial diffusion against a background of increased white matter volume suggest the novel hypothesis that one potential alteration contributing to increased cortical white matter in NF1 may be looser packing of axons, with or without myelination changes. Further, this indicates that axial and radial diffusivity can uniquely contribute as markers of NF1-associated brain pathology in conjunction with the typically investigated measures. Neurofibromatosis Type 1 (NF1) is caused by a mutation in the neurofibromin gene at locus 17q11.2, and is one of the most common single-gene genetic disorders  affecting cognitive function in humans About 65% of children with NF1 have sustained attentional difficulties and 38–50% meet diagnostic criteria for ADHD, with the vast majority fulfilling criteria for the inattentive subtype th percentile) characterizes about half of children with NF1 Neurofibromin, the NF1 gene product, appears early during embryonic development, with high levels of expression in the brain, suggesting that it is important for the orderly differentiation of CNS neurons in vivo. Diffusion tensor imaging (DTI) is the only currently available non-invasive method for investigating white matter microstructure and connectivity in vivo, based on patterns of water diffusion in neural tissue NF1 mutations are known to impact myelin, as the gene encoding the oligodendrocyte-myelin glycoprotein (OMgp) is embedded within an intron of the NF1 gene To date, only a handful of studies have employed DTI measures in NF1 patients, none of which have taken a whole-brain approach. Previous studies have noted increased apparent diffusion coefficient (ADC) values, indicating more diffuse or less organized cerebral tissue, in both children and adults with NF1 relative to control subjects Here, we conducted the first study to date to investigate changes in white matter microstructure across the whole brain using voxel-wise methods in NF1. Secondly, we examined volumetric differences in grey and white matter between groups, to determine whether previously observed global volume alterations were also present in our sample. Based on findings from prior mouse and human studies we predicted that individuals with NF1 would have larger overall grey and white matter volumes, concomitant with increases in overall diffusivity and decreased FA. Moreover, since neurofibromin is expressed throughout the brain and not just in the localized regions that were the focus of prior analyses, we hypothesized that a voxel-wise approach would reveal more widespread differences than had been previously reported.14 individuals with NF1 and 12 unaffected, demographically matched control subjects participated in the study .Subjects were scanned on a 1.5 T Siemens Sonata scanner  at the Ahmanson-Lovelace Brain Mapping Center at UCLA. Head motion was restricted using foam padding. DTI data were acquired using a 6-direction EPI sequence with 75 contiguous 2 mm AC-PC aligned interleaved slices with no gap . Five repetitions of the sequence were acquired, with a total scan time of 6 minutes 20 seconds. In addition, a high resolution T1-weighted structural image was acquired .The five image acquisitions for each direction were merged, aligned with McFlirt  were defined in the anterior thalamic radiation (ATR), cingulate bundle, hippocampal cingulum, inferior longitudinal fasciculus (ILF), superior longitudinal fasciculus (SLF) and uncinate fasciculus (UF), corticospinal tract, and inferior fronto-occipital fasciculus (IFO) based on the John Hopkins University DTI-based probabilistic tractography atlas Statistics on the ROI data were performed in Stata (v8). Independent samples t-tests were performed on the DTI regions, as well as the structural MRI regions. Voxel-wise statistics on the DTI data were performed in FSL’s randomise tool, which performs a permutation analysis. We performed 5000 permutations using the Threshold Free Cluster Environment (TFCE), a rigorous approach to cluster analysis that avoids the need to define and initial cluster-forming threshold or to perform large amounts of data smoothing. Age and sex (demeaned) were included in the model as confound regressors, with groups modeled separately. Family-wise error rate (FWE) was used to correct for multiple comparisons.In order to directly test whether the observed FA effects were secondary to radial or axial changes, we performed an exploratory analysis in which we re-ran two additional versions of the FA randomize analysis (again with demeaned age and sex as covariates), once with demeaned radial diffusion and once with demeaned axial diffusion as covariates. Then, the number of remaining significant voxels were compared to the number of original significant voxels in the FA analysis.d) = 1.17], however, frontal lobe grey matter was significantly larger in controls  and white matter volume  in NF1 patients relative to controls. This finding remained significant when restricted to an ROI of the frontal lobe white matter [t(24) = 3.62, p = .0014; effect size  to look for areas that remained significant at suprathreshold levels, indicating more robust differences. We found that the alterations in FA  and radiOur second approach to probing regional variation was to perform a ROI analysis in a set of white major matter association tracts (ROI definition described above) including those that do and do not connect to the frontal lobe. When corrected for multiple comparisons using Bonferroni correction, the significant p-value threshold was set at.005 (.05/10 = .005) see . At thisThis is the first paper to investigate diffusion tensor imaging measures in adults with NF1 across the entire brain using a rigorous registration approach, tract-based spatial statistics (TBSS). First, our data replicate and support previous sMRI findings indicating white matter volumetric enlargement in NF1 patients By using multiple DTI measures, this study uniquely contributes to our broader understanding of the previously observed white matter volume increases. Our finding of increased ADC reflects a general increase in diffusivity that can arise through a variety of mechanisms see . One posA limitation of DTI methodology is that it is an indirect measure of the distance between axons, and must remain agnostic to which factor  is driving the results. However, given that white matter volume is increased in this sample and in other samples in the literature, a simple finding of decreased myelination  is not sufficient to explain the larger pattern of results. Therefore, our data support the novel hypothesis that while there may be myelination differences in NF1, there are also likely to be differences in the degree of axonal packing, with the larger distances between axons contributing to the white matter volume increase. Given our findings of increased ADC and radial diffusion in the context of decreased FA, this hypothesis is consistent with histological findings that axonal packing density (amount of space between axons) is positively correlated with ADC and radial diffusion and negatively correlated with FA The possibility that the alterations in axial diffusion may be the result of increased space between axons (less dense tract packing) is supported by some of the known functions of neurofibromin and the molecules it interacts with. Developmentally, when white matter tracts are formed, cellular adhesion molecules play an important role in axon guidance along tracts, and in holding tracts together. There is some evidence that OMgp has the potential to function as an adhesion molecule Certain limitations of the current study must be noted. First of all, the sample size is small, due to limitations in recruiting individuals with this rare disorder and future studies in larger populations, as well as prospective longitudinal studies in developmental samples, will be useful for further probing the etiology and developmental trajectory of structural brain changes in NF1. Secondly, our DTI acquisition sequence had a limited number of directions and thus, we were not able to do tractography analyses. Further, while an increasing number of studies are beginning to include measures of radial and axial diffusion, as they provide more specific information about the nature of white matter microstructural changes than do FA and ADC, there are limitations to these measures. One particular issue is related to the reliability of these measures in areas where there are crossing fibers. This issue may be intensified in low directional data, as we have here. While our results indicate widespread changes across large areas of white matter, regional interpretation of these measures should be considered with caution. This may be better addressed in future studies with higher directional data. Finally, particularly interesting for future studies will be explorations of the relationship of these changes to clinical symptoms, such as those associated with ADHD. Although no studies, to our knowledge, have previously examined the impact of ADHD diagnosis on DTI indices in NF1, Cutting et al. Overall, the current study demonstrates that the finding of white matter overgrowth, as measured by sMRI, persists into adulthood in NF1 patients and that white matter microstructural changes, as measured by DTI, also persist and are more pronounced in the frontal lobe. Notably, we show a novel pattern of DTI findings that may indicate that one contributing factor to this increase in volume may be related to looser fiber packing. Further, our findings indicate that indices of axial and radial diffusivity have added utility as markers of NF1-associated brain pathology beyond the typically investigated measures of diffusion .


Vegfa120/120 or Tie2-Cre;Nrp1fl/− mice or using an in vitro Wnt1-Cre;Rosa26Yfp/+ mouse model of ENS development, ENCC still colonised the entire length of the gut, including the terminal hindgut. These results demonstrate that blood vessel networks are not necessary to guide migrating ENCC during ENS development. Conversely, in miRet51 mice, which lack ENS in the hindgut, the vascular network in this region appeared to be normal suggesting that in early development both networks form independently of each other.The vasculature and nervous system share striking similarities in their networked, tree-like architecture and in the way they are super-imposed in mature organs. It has previously been suggested that the intestinal microvasculature network directs the migration of enteric neural crest cells (ENCC) along the gut to promote the formation of the enteric nervous system (ENS). To investigate the inter-relationship of migrating ENCC, ENS formation and gut vascular development we combined fate-mapping of ENCC with immunolabelling and intravascular dye injection to visualise nascent blood vessel networks. We found that the enteric and vascular networks initially had very distinct patterns of development. In the foregut, ENCC migrated through areas devoid of established vascular networks. In vessel-rich areas, such as the midgut and hindgut, the distribution of migrating ENCC did not support the idea that these cells followed a pre-established vascular network. Moreover, when gut vascular development was impaired, either genetically in  •The gut vasculature is thought to direct migrating enteric neural crest cells (ENCC).•We visualised ENCC and the vasculature in the developing gastrointestinal tract.•Enteric and vascular networks initially had little correlation.•in vivo or in vitro.ENCC colonised the gut when vascular development was impaired •Blood vessels are thus not necessary to guide migrating ENCC during ENS formation. During vertebrate development, organs and tissues must connect to the blood vascular system to receive fluids, nutrients and oxygen, and to the nervous system to receive and send sensory, autonomic or functional information. Consequently, both mature networks share obvious similarities at the anatomical level, and they also use similar cellular and molecular mechanisms to orchestrate their parallel developmental programs . Recent Although the nervous system of the gut contains extrinsic components, its main functional unit is the intrinsic enteric nervous system (ENS). The ENS consists of networks of interconnected ganglia embedded in the wall of the digestive tract and is the largest division of the peripheral nervous system . The ENSGFP–chick intraspecies grafting to permanently label and fate-map vagal ENCC with GFP, combined with intravascular injection of the lipophilic dye DiI to stain the nascent blood vessel network. Because DiI highlighted only patent blood vessels, we also labelled quail embryos with QH1 and HNK1 antibodies to mark endothelial and neural crest-derived networks, respectively. The analysis of wholemount preparations by high magnification confocal imaging showed little correlation between migrating NCC and blood vessel development. Consistent with these observations we showed, using mouse models defective in vascular development, and an in vitro assay, that ENCC can colonise the entire length of the gut independently of the vascular network.We used chickGFP–chick grafting, the neural tube and associated neural crest, adjacent to somites 2–6 inclusive, was microsurgically removed from normal chick embryos at embryonic day E1.5 and replaced with equivalent stage-matched tissue obtained from chickGFP embryos (TM CM-DiI (Molecular Probes®) was injected into the peripheral vascular network using a fine glass needle connected to a mouth pipette  (Nrp1 (Nrp1fl/f)) (MGI:3512103) (Tie2 promoter (MGI:2450311)  (miRet51 mutation (MGI:2387446) have been described . Mice carrying the 1931047)  have bee1931047) . Conditi3512103)  were mat2450311) . Mice ca2449038)  were mat2449038) . Mice caescribed . Genotypin vitro as previously described  supplemented with 1 mM l-Glutamine  and 1 mM Penicillin/Streptomycin antibiotic mixture . Gastrointestinal tracts were cultured up to 4 days then fixed in 4% PFA for 1 h and processed for immunofluorescence labelling as described below.Foetal mouse intestines were cultured escribed . BrieflyFor wholemount immunofluorescence labelling of quail  and mouse tissues, dissected gastrointestinal tracts were fixed for 1–2 h in 4% PFA in PBS then rinsed 3 times in PBS at room temperature and processed as previously described . BrieflyX axis for ZX orthogonal projections and the Y axis for YZ projections.Confocal Z-stacks of chicken tissues from GFP-WT chimera injected with DiI, or from quail and mouse intestine double immunostained for endomucin and TuJ1, acquired with the Zeiss LSM 710, were reconstructed with the 3D viewer in Fiji ImageJ . Images Following intraspecies grafting and DiI intra-vascular injection, wholemount preparations of the gastrointestinal (GI) tract and respiratory system at E4.5 (HH24) showed the developing ENS labelled in green with GFP, because vagal ENCC are derived from the GFP graft B and C, doi:10.1016/j.ydbio.2013.11.007.Supplementary materials related to this article can be found online at 3-dimensional rotation of the image in The following is the Supplementary material related to this article ⁎) and migrating ENCC  reduction in TuJ1+ ENS cells in the mutants .To investigate whether ENCC were present in gastrointestinal tracts with disrupted vasculature, we studied  stomach . Whilst culature . Despite120 mice A–D. At Econtrols I and J.  the gut I and J. al cells  The vasc120 mice E–H. Desp120 mice E and F a120 mice G and H, in vitro, the vascular system collapses and degenerates due to the lack of blood flow, to determine whether ENCC could migrate along the hindgut in the absence of a vascular system. We used Wnt1-Cre;Rosa26Yfp/+ mice, which express YFP in NCC , as shown in FSerial Z stack of confocal images showing reconstruction of the hindgut ENS (TuJ1), vascular system (endomucin) and tissue histology (DAPI) at E19.5 in control littermates (upper panel) and The following is the Supplementary material related to this article in vitro gut culture system and found, once again, that ENCC were able to complete their migration and form an ENS along the entire hindgut. Finally, using a mouse model of Hirschsprung disease that lacks hindgut ENS, we showed that early vascular patterning was unaffected by the absence of an ENS.Here we investigated the interrelationship between migrating ENCC and the developing vasculature of the gut. In avian models we found that both networks displayed very different early developmental patterns: ENCC migrated in areas devoid of vasculature, and in areas where the vasculature was present, they frequently migrated without closely associating with existing blood vessels. These results suggest that the presence of a vascular network is not necessary for ENCC to colonise the digestive tract. To corroborate these findings, we looked at two different strains of mutant mice where the gut vasculature is defective and found that ENCC migrated along the entire length of the gut and formed an ENS despite the presence of severe vascular defects. We also tested the migratory ability of ENCC in the complete absence of vasculature, using an In this study we used the well established technique of neural tube transplantation to label ENCC . We sele165, VEGF121, Ang-1 or Flt-1/Fc resulted in normal peripheral nerve patterning. Bates and colleagues concluded that neurovascular congruency was determined by shared molecular pathways, including Sema3A and NRP1, guiding both developing networks in the vicinity of each other. Our results are consistent with a similar model in the digestive tract where shared mechanisms between neural and vascular development have also been demonstrated. In particular, β-1 integrins have been shown to be required cell autonomously for the migration of both EC and ENCC , regulating migration of both NCC and EC, are present only in discrete gut territories, potentially restricting the colonisation path of both networks and enforcing their proximity in some areas and not others  suggesting that its development is based on an ancient developmental programme, predating and independent of vascular development . Our resand ENCC . This mot others .Our results, however, are in contradiction with a previous study from Nagy and colleagues, looking at the migration of ENCC in the hindgut of quail embryos, which suggested an essential role for the microvasculature in ENS formation by guiding ENCC migration via β-1 integrin signalling . A possiDespite the differences between our findings and those of Nagy et al., both studies clearly document examples of close associations between the developing vascular network and migrating ENCC. This suggests that blood vessels, although not necessary for ENCC migration, may provide a suitable substrate for their migration. Indeed, the basement membrane of EC has been shown to express all the required extracellular matrix components to support NCC migration  and can in situ hybridisation that tmENCC do not express cxcr4, one of the chemokine receptors for SDF1 , migrate through the gut mesentery as a shortcut to reach the terminal hindgut, and that these cells represent the principal source for ENS formation in the colon of mice . Strikinfor SDF1 .VEGF120/120 and Tie2-Cre;Nrp1fl/− mice demonstrated the ability of ENCC to colonise the entire length of the hindgut and form the ENS, despite severe blood vessels defects. Although the ENCC were able to migrate all the way to the terminal hindgut, the resulting ENS was disorganised and sparse compared with wild type controls. In the case of the Tie2-Cre;Nrp1fl/− mice, we observed a statistically significant reduction in the number of ENS cells. Likewise, our results in chick suggest a connection between the density of the ENS and that of the vascular bed, with the oesophageal ENS being sparser than that of the stomach or hindgut, which are both richly vascularised. The interpretation of such results is, however, complicated by the fact that vascular insufficiency not only reduces the ability of the ENCC to interact with EC, but also has indirect effects due to impaired oxygen and nutrient supply. For that reason, in vitro experiments, where nutrients are provided artificially, are a valuable experimental tool. Here we used gut explant culture, which interrupts blood flow to the explant from the moment it is cultured. It has been well documented that interruption of the blood flow leads to the collapse of the capillaries and a catastrophic induction of apoptosis in EC, both in vivo and in vitro (Our results using in vitro . We tookin vitro . NeverthIn conclusion, our results show that blood vessels are not necessary for guiding migrating ENCC during ENS development. However, there may be interactions between ENCC and endothelial cells prior to the establishment of patent blood vessels, and/or the establishment of functional neuro-vascular units in the gut later in development, as the ENS is subtly altered when vascularisation is reduced. Recent advances in imaging techniques and “optical clearing” of gut tissues now make it possible to investigate later developmental stages at which the gut neuro-vascular units likely become established. These, and further studies in mice deficient in ENS or in the gut vasculature, will shed light on the shared developmental mechanisms that underlie normal gut neurovascularisation.


Lynch syndrome is an autosomal dominantly inherited disorder of cancer susceptibility caused by germline mutations in the DNA mismatch repair (MMR) genes. Mutation carriers have a substantial burden of increased risks of cancers of the colon, rectum, endometrium and several other organs which generally occur at younger ages than for the general population. The issue of whether breast cancer risk is increased for MMR gene mutation carriers has been debated with evidence for and against this association.Using the PUBMED, we identified all relevant studies of breast cancer associated with Lynch syndrome that were published by 15 December 2012. In the review, we included: (i) molecular studies that reported microsatellite instability and/or immunohistochemistry in breast cancer tumors of MMR gene mutation carriers; and (ii) risk studies that investigated risk of breast cancer for confirmed MMR gene mutation carriers or families or clinically and/or pathologically defined Lynch syndrome families.We identified 15 molecular studies and, when combined, observed 62 of 122  breast cancers in MMR gene mutation carriers were MMR-deficient. Of the 21 risk studies identified, 13 did not observe statistical evidence for an association of breast cancer risk with Lynch syndrome while 8 studies found an increased risk of breast cancer ranging from 2- to 18-fold compared with the general population (or non-carriers). There is only one prospective study demonstrating an elevated risk of breast cancer for MMR gene mutation carriers compared with the general population .Since breast cancer is a relatively common disease in the general population, more precise estimates of risk and gene-specific risks will need to utilize large prospective cohort studies with a long follow-up. While current data are inconclusive at a population level, individual tumor testing results suggest that MMR deficiency is involved with breast cancers in some individuals with Lynch syndrome. MLH1, MSH2, MSH6 and PMS2. The prevalence of individuals who carry a pathogenic germline mutation in one of these genes in the population is estimated to be, depending on various assumptions, from 1 in 370 to 1 in 3,000 [Lynch syndrome, formerly known as hereditary nonpolyposis colorectal cancer (HNPCC) , is an ain 3,000 . Though in 3,000 .Lynch syndrome-associated cancers typically exhibit DNA microsatellite instability (MSI) and loss of MMR protein expression. Instability of microsatellite DNA sequences characterized by the presence of random contractions or expansions in the length of simple sequence repeats, can be detected by polymerase chain reaction-based methods ,7. The lThese cancers include adrenocortical adenocarcinoma -11, thyrInitially raised by Henry Lynch and colleagues several decades ago , the issWe used PUBMED  to searcFor molecular studies, studies were eligible if they reported mismatch repair deficiency status (either by MSI and/or IHC) of breast cancer tumors of MMR gene mutation carriers. For risk studies, studies were eligible (i) if they were case-control or cohort studies, and (ii) if they investigated the risk of breast cancer for confirmed MMR gene mutation carriers or families or clinically and/or pathologically defined Lynch syndrome families compared with non-carriers or the general population. Studies were excluded if they were editorials or literature reviews.For molecular studies, we extracted the following data: first author's name, year of publication, country in which the study was performed, number of breast cancers observed in MMR gene mutation carriers, specific mutated MMR gene, age at diagnosis of breast cancer, and the MSI and/or IHC status of the breast tumor.For risk studies, we extracted the following data: first author's name, year of publication, country in which the study was performed, recruitment and ascertainment method of families or mutation carriers, definition of Lynch syndrome, numbers of MMR gene mutation carriers, specific mutated MMR gene, observed and expected numbers of breast cancers, estimates of breast cancer risk for mutation carriers compared with non-carriers or the general population  and absolute or cumulative risk of breast cancer for mutation carriers (expressed as lifetime risk or 10-year risk).For molecular studies, we defined a tumor with high level of microsatellite instability (≥ 30% of markers tested) or those with loss of normal expression of any of the four DNA MMR genes by IHC to be a MMR-deficient tumor. We divided the total number of MMR-deficient breast cancers by the total number of breast cancers in MMR gene mutation carriers to estimate the proportion of breast cancers in MMR gene mutation carriers that were MMR-deficient.For risk studies that did not provide the relative risk (or standardized incidence ratio), we calculated the estimated the standardized incidence ratio (SIR) by dividing the observed numbers of breast cancers by the reported expected numbers. When not provided by the study, we calculated the 95% CI assuming that the numbers of observed cases followed a Poisson distribution. All statistical analyses were conducted using Stata 11.2 .We identified 638 studies identified through the literature search, and retrieved 129 of these studies for full assessment for eligibility. A total of thirty-four studies were included in the systematic review including fifteen molecular studies and twenty-one risk studies (two studies were both molecular and risk studies)  or accelerate carcinogenesis in breast cancers that occur in MMR gene mutation carriers (as an accelerator). However, these observations are also consistent with MMR deficiency being a phenotype arising within breast cancer that was caused by another factor (as a passenger), rather than the phenotype driving or accelerating carcinogenesis.One method to overcome this problem of cause or consequence is to determine whether breast cancer is more likely to develop in carriers of MMR gene mutations compared with non-carriers , that is, empirical evidence of risk. Eight studies ,47-53 esOne retrospective study  comparedSix studies -60 estimTwo studies ,62 from Prospective studies avoid the ascertainment issue described above because the carriers used to estimate cancer risk are unaffected at the time of recruitment or first observation. To date, there is only one prospective study of MMR gene mutation carriers without prior diagnosis of cancer . An incrThe two other studies of the Colon Cancer Family Registry observed an approximately 2-fold increased risk of breast cancer for MMR gene mutation carrying women. These included those who had a previous diagnosis of colorectal cancer  or endomet al. [MLH1 c.C1528T mutation. In this study design, unaffected siblings of familial cases have the same familial risk profile  and therefore, share potential confounders that are correlated within siblings, in other words, it is an informative comparison within the same study base [Blokhius et al.  comparedudy base . The lacet al. [et al. [in situ were predominantly of higher nuclear grade, suggesting that aberrations in MMR function may lead to a more aggressive phenotype in breast cancer.The estimates of an increased risk of breast cancer in Lynch syndrome described in the previous studies are relatively small compared with the increased risks observed for colorectal and endometrial cancers . Howeveret al.  hypothes [et al.  who obseThe suggested small increased risk of breast cancer in Lynch syndrome is important to confirm, given that breast cancer is a common disease in women. The average lifetime risk of breast cancer in women is estimated to be 12.2% . The thret al. [MLH1 mutation-carrying families but not in MSH2 families  for the risk studies included in the review [e review -65,78-80Click here for file


Despite several decades of intensive effort to improve the imaging techniques for lung cancer diagnosis and treatment, primary lung cancer is still the number one cause of cancer death in the United States and worldwide. The major causes of this high mortality rate are distant metastasis evident at diagnosis and ineffective treatment for locally advanced disease. Indeed, approximately forty percent of newly diagnosed lung cancer patients have distant metastasis. Currently, the only potential curative therapy is surgical resection of early stage lung cancer. Therefore, early detection of lung cancer could potentially increase the chance of cure by surgery and underlines the importance of screening and detection of lung cancer. In the past fifty years, screening of lung cancer by chest X-Ray (CXR), sputum cytology, computed tomography (CT), fluorescence endoscopy and low-dose spiral CT (LDCT) has not improved survival except for the recent report in 2010 by the National Lung Screening Trial (NLST), which showed a 20 percent mortality reduction in high risk participants screened with LDCT compared to those screened with CXRs. Furthermore, serum biomarkers for detection of lung cancer using free circulating DNA and RNA, exosomal microRNA, circulating tumor cells and various lung cancer specific antigens have been studied extensively and novel screening methods are being developed with encouraging results. The history of lung cancer screening trials using CXR, sputum cytology and LDCT, as well as results of trials involving various serum biomarkers, are reviewed herein. Cancer is the leading cause of death worldwide and accounted for 7.6 million deaths in 2008 with primary lung cancer responsible for 1.37 million deaths alone . With thThe results of primary lung cancer screening trials using imaging modalities such as chest X-RAYs combined with sputum sampling, CT scans and LDCT will be reviewed. In addition, the role of serum biomarkers and biological modalities, circulating DNA and RNA, exosomal microRNA and circulating tumor cells (CTCs) and thermogram and nanopore sensor technologies will be discussed.The first large prospective trial using chest X-Ray as a screening tool for the early detection of lung cancer was performed in the UK and published in 1968. In that study there were 29723 men aged 40 years or older who had six-monthly chest radiograph screening for three years compared with a control group of 25311 men who received chest radiograph at the beginning and the end of the study. Lung cancer annual mortality rate did not differ statistically in the two groups, with 62 deaths in the screened group and 59 in the control group, but the 5-year survival rate for participants with lung cancer diagnosed by screening chest radiographs was 23% versus 6% in the control group 46] The LThe National Comprehensive Cancer Network (NCCN) published Lung Cancer Screening Guidelines , recommeCurrently, there are no validated biomarkers for early lung cancer detection. Extensive efforts have been made for the development of useful biomarkers and novel biomarkers from recent studies will be discussed here.CTCs are circulating and free floating malignant cells that have migrated from the primary cancer or metastatic sites in the peripheral blood. Advanced techniques have been developed to identify, isolate and characterize these CTCs . Differeet al., published a case–control study demonstrating that the quantity of free circulating DNA can be measured in lung cancer patients by real-time quantitative polymerase chain reaction (RT-PCR) of the human telomerase reverse transcriptase gene (hTERT) [et al., designed a 5-year prospective trial that enrolled 1,035 heavy smokers aged 50 years or older who were monitored by annual LDCT for 5 years and plasma circulating DNA levels were measured by RT-PCR. The authors reported no improvement in the accuracy of lung cancer screening by LDCT in heavy smokers by measurement of the baseline assessment of plasma circulating DNA level, but higher levels of plasma circulating DNA at surgery was a risk factor for aggressiveness of lung cancer with a decreased 5-year survival [hTERT gene is not specific for lung cancer and that the level of free circulating DNA may be influenced by other benign diseases [In 2003, Sozzi  (hTERT) . Subsequsurvival . They hydiseases .et al., studied the thermal properties of plasma by highly sensitive differential scanning calorimetry (DSC) which can generate unique and statistically different DSC thermograms in several diseases, such as SLE, Lyme disease, and rheumatoid arthritis or cancers including cervical, lung, melanoma, ovarian, and uterine [et al., a case–control study [Abundant proteins in the plasma are an underutilized source with valuable information for clinical usage . Chairs  uterine -60. In Xol study  involvinol study  of 30 paet al.,[et al.,[et al.,[The discovery of tumor-derived exosomes was first reported in the peripheral blood of ovarian cancer patients in 1979 ,64. Exoset al.,. showed ,[et al.,,70. repo,[et al.,. reporteet al.,[Based on evidence that specific microRNA profiles are unique to different types of cancers , Rabinowet al.,. designeet al.,[let-7 microRNAs. This strategy may be used in concert with unique lung cancer microRNA profiles for lung cancer screening and/or monitoring treatment responses in lung cancer patients.In the last twenty years, various sensitive quantitative techniques have been developed to detect and analyze macromolecules at a single molecular level in biomedical research . With inet al.,. describTelomerase, a ribonucleoprotein complex with reverse transcriptase activity to synthesis DNA termini at the end of eukaryotic chromosomes during each cell division, plays a vital role in the replication of chromosomal termini or telomeres. It is inactivated in most normal tissues at an undetectable level and reactivated or upregulated in all cancer tissues investigated, such as breast, prostate, lung, liver, and pancreatic cancer -79. Compet al.,[Miyazu et al.,. evaluatet al.,[Aberrant mRNA expression from normal genes has also been investigated in association with tumorigenesis. Using reverse transcription polymerase chain reaction (RT-PCR), studies have demonstrated the detection of tyrosinase mRNA in whole blood samples of melanoma patients  and teloet al.,. evaluatet al., reported that LunX mRNA was overexpressed in 14 of 24 (58%) blood samples from NSCLC patients [There are several other molecular candidates that have been used as biomarkers. The LunX (lung-specific X protein) is a human lung-specific gene identified in 2001 . Mitas epatients . Cytokerpatients .et al., evaluated DNA microsatellite in 34 patients with lung cancer, including 11 patients with small cell lung cancer (SCLC) and 23 patients with NSCLC and 20 healthy controls. A panel of 12 microsatellites was selected to perform allelotyping. Plasma DNA allelic imbalance (AI) was noted in 88% of cancer patients (30/34) compared to no plasma or bronchial DNA alteration in 20 healthy controls [Using fluorescent microsatellite analysis, Beau-Faller Decades of research have evaluated various approaches for lung cancer screening, including routine chest radiograph, sputum cytology, CT scanning, LDCT and, most recently, molecular biomarkers. Early screening tests of high risk individuals based on sputum cytology or chest X-Ray have not shown improvement in disease-specific survival. Chest LDCT scan has been proven as an effective tool for the detection of early stage resectable disease. Indeed, results from the NLSCT showed a 20 percent mortality reduction in high-risk participants screened with LDCT compared to those with chest X-Rays. Recent NCCN guidelines endorse LDCT as a screening tool for lung cancer. However, questions remain regarding the definition of high risk patients; how long and how frequent to screen; and how to address the false positive results and high cost and potential toxicity from radiation exposure. Additional randomized trials using LDCT are ongoing and will address these unanswered questions and may eventually demonstrate the real impact of early detection on mortality at a population level.Several serum tumor protein markers have been studied extensively, and are currently available for blood testing, such as carcinoembryonic antigen, serum cytokeratin 19 fragment, and progastrin-releasing peptide, but none of them is satisfactory for diagnosis of early stage lung cancer because of their relatively low sensitivity and specificity in detecting the presence of cancer cells -91. DiffWith the advances of our understanding of key cellular changes of lung cancer and the rapid development of new technologies, recent diagnostic approaches have focused on genomics and proteomics. The Human Genome Project (HGP) announced the first working draft in 2000 and opened the genomic era, which has provided opportunities for gene expression analysis and several studies have identified differences in gene expression between normal and lung cancer cells using techniques such as serial analysis of gene expression  and cDNAResearchers have been studying tumorigenesis, targeting subtle changes at the molecular level to facilitate early diagnosis, identify clinically relevant biological triggers related to cancer development, and identify useful biomarkers. In this regard, CTCs, exosomal microRNA, free circulating DNA, telomerase, LunX and CYFRA21-1 have demonstrated encouraging results. Investigators have taken advantage of technical advances that allow rapid high-throughput assays to interrogate the genome, proteome, and epigenome. Results of such Phase I studies are expected to be reported in 2012.Based on screening guidelines suggested by the NCCN, significant strides have been made in effective screening methods to diagnosis lung cancer at early stages for high risk patients with a 30 pack-year smoking history. But challenges in early detection of lung cancer remain. As one can anticipate, in the near future, effective biomarkers or evidence-based image scans used alone or in combination may provide the clinician with effective tools for the early detection of lung cancer in the era of molecular and digital oncology, and hopefully alter the natural course of lung cancer and improve the survival of patients with lung cancer.The authors declare that they have no competing interests.All authors participated equally in draft writing and revising the manuscript. All authors read and approve the final manuscript.


Ageing is a multifactorial process often characterized by a progressive decline in physiological function(s). Ageing can and is often associated with an increased incidence of cardiovascular and renal disease. Klotho is a novel antiageing gene that encodes a protein with multiple pleiotropic functions including an emerging role in cardiorenal disease. Mice deficient for this gene display a phenotype of premature human ageing characterized by diffuse vascular calcification, altered calcium/phosphate metabolism, and shortened lifespan. Klotho is mainly expressed in the renal tubules but it also exists as circulating soluble form detectable in the blood, with systemic effects. Reduction in soluble Klotho has been associated with renal disease, hyperphosphataemia, increased oxidative stress, endothelial dysfunction, and diffuse vascular calcification. Conversely, overexpression of Klotho promotes cardiovascular-renal protection. The majority of the research on Klotho has been conducted in vitro and in animal studies but there is emerging data from human studies which suggest that Klotho may be a modifiable factor involved in the pathogenesis of cardiovascular and renal disease in at-risk populations. Further data is required to confirm if this novel protein can emerge as therapeutic tool that may be used to prevent or slow progression of cardiorenal disease. Ageing is a complex phenomenon resulting from the interaction between genetic and environmental factors . AdvanceKlotho is a gene that encodes a novel protein regulating multiple functions, fortuitously discovered in 1997 by Kuro-o and colleagues and named after the goddess who spins the thread of life in Greek mythology . In mice, the deletion of Klotho gene causes a phenotype of premature human aging including vascular calcification, altered calcium/phosphate metabolism with hyperphosphataemia, and shortened lifespan. Klotho protein exists in two forms: a type I transmembrane protein (1014 amino acids) with a large extracellular domain and a short intracellular portion (10 amino acids), predominantly expressed in the renal tubules, and a circulating soluble factor detectable in blood and in lesser extent in other biological fluids .Soluble Klotho is produced either by proteolytic cleavage of the extracellular domain of the transmembrane form (130 kDa isoform) operated by the membrane-anchored proteases ADAM10 and ADAM17 or by alternative mRNA splicing (isoform 70 kDa) . The sysThe fact that FGF23 requires Klotho for binding to its receptor explains why Klotho- and FGF23-deficient mice display identical phenotype , 13. TheKlotho/FGF23 signalling induces phosphaturia by suppressing the sodium-dependent phosphate cotransporters type IIa (NPT2a) expressed on the brush border membrane of renal tubular cells. Soluble Klotho has also been found to regulate directly the phosphate transport, in the proximal tubule of the kidney by deglycosylation of NaPi-2a cotransporters . The resHigh FGF23 levels in patients with chronic kidney disease are due to the declining renal clearance and also may represent a compensatory response to hyperphosphataemia .Recent observational data suggest that FGF23 is associated with and may represent an independent risk factor for cardiovascular and all-cause mortality in patients with chronic kidney diseases stage of 4 and 5 (eGFR < 30 mL/min) , 18.The reduction in Klotho expression observed in chronic kidney disease may be an important event contributing to the above with accumulation of FGF23 being a compensatory mechanism to the increase of phosphate levels driven by the primary reduction in Klotho. Recent data support this hypothesis as changes in Klotho levels appear to precede changes in phosphate levels, the key driver of FGF23 balance in renal disease .Klotho expression is affected by physiological and pathological factors. Renal expression of Klotho in rat is minimal in prenatal life but increases after birth . A reducConversely, there is evidence in animals that the overexpression of soluble Klotho can reverse the ageing process and provides cardiovascular-renal protection possibly by inducing resistance to oxidative stress and protecting tissues from oxidative damage , 26. In this paper, we will summarize the key areas of research on the putative role of Klotho in prevention or delay of cardiorenal progression. We performed a Pub Med/Medline search for the terms Klotho, cardiovascular disease, and renal disease from 2000 to 2010 with a focus on recent mechanistic and proof-of-concept studies evaluating the role of Klotho in the prevention and treatment of cardiorenal disease.Klotho is predominantly expressed in the renal distal tubular cells . Animal Klotho expression is reduced in renal distal tubules, urine, and blood of rats subjected to bilateral renal ischemia . InteresThe nephroprotective effects of Klotho have also been tested in an animal model of glomerulonephritis . The traβ1 signalling [βR2) suppressing the activation of the type I receptor (TGFβR1) that phosphorylates Smad2/3 proteins (transcription factors regulating the expression of TGFβ1 target genes) [In vivo, the intraperitoneal administration of soluble Klotho recombinant protein, immediately after the induction of unilateral ureteral obstruction, prevents the acute renal fibrosis through the inhibition of TGFgnalling . Specifit genes) .Studies conducted in humans have reported a reduction of Klotho, both tissue (transmembrane) and soluble forms, in acute and chronic kidney disease. Koh et al. examined the kidneys of 10 patients with clinical or histological diagnosis of chronic kidney disease and demonstrated that the expression of Klotho protein was significantly reduced when compared to healthy control . There are limited studies evaluating changes in serum and urine levels of Klotho in humans; Yamazaki and his colleagues were the first to establish a novel assay to detect circulating serum Klotho . In 181 A reduction of Klotho levels in urine has been reported in patients with acute kidney injury and in subjects with chronic kidney disease , 36. Hu Klotho may be an early clinical biomarker of acute and chronic renal injury CKD as its diminution precedes changes of other well-established markers/factors involved in the progression of renal failure. However, further long-term prospective studies are required to establish the utility/value of Klotho as an early marker of acute and chronic renal disease.Soluble Klotho has an important role in maintaining endothelial wall homeostasis and promoting the health of the vasculature –39. In eEndothelial dysfunction results from the imbalance between the release of vasodilator and vasoconstrictor factors and is an early step in the development and progression of cardiovascular disease. This disrupted equilibrium is predominantly due to the reduced bioavailability of nitric oxide (NO) because of its inactivation by ROS . NO not Saito et al. demonstrated that in Klotho heterozygous mutant mice endothelium-dependent vasodilatation in the aorta and arterioles in response to acetylcholine is attenuated and the excretion of urinary nitric oxide metabolites is reduced . In KlotKlotho gene delivery, mediated by adenoviral vector, in a rat model of atherosclerosis, increased endothelium-dependent NO synthesis and prevented adverse vascular remodelling . Klotho is also involved in the modulation of endothelial inflammation as demonstrated in vitro by Maekawa et al. In human umbilical vein endothelial cells (HUVECs), soluble recombinant protein might suppress the expression adhesion molecules involved in the pathogenesis of vascular disease such as intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) . Hu et al. have documented that Klotho is a direct inhibitor of vascular smooth muscle cell (VSMC) calcification. The authors showed in an elegant mouse model that Klotho deficiency is associated with more severe calcification, undetectable levels of soluble Klotho, and higher serum levels of phosphorus, whereas Klotho overexpression was accompanied by less calcification, preserved levels of Klotho and normal renal function . In vitrSoluble Klotho is a novel humoral factor that confers resistance to oxidative stress associated with ageing and several pathological conditions predisposing to cardiovascular-renal damage. There is emerging evidence highlighting the essential involvement of Klotho in calcium/phosphate metabolism and the maintenance of vascular integrity.


In the present review, we will discuss the role of Ang II and oxidative stress in hypertension, the relationship between the BRS and the genesis of hypertension and how the oxidative stress triggers baroreflex dysfunction in several models of hypertension. Finally, we will describe some novel therapeutic drugs for improving the BRS during hypertension.Hypertension is a multifactorial disorder, which has been associated with the reduction in baroreflex sensitivity (BRS) and autonomic dysfunction. Several studies have revealed that increased reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase, following activation of type 1 receptor (AT This system consists mainly of an enzymatic cascade catalyzed by renin and angiotensin converting enzyme (ACE), generating Ang II Peach, . The eff1R induces potent vasoconstriction   release    Braga, .1R axis and may be beneficial for the cardiovascular system  cleaves Ang I and Ang II into Ang-(1–9) and Ang-(1–7), respectively  , including superoxide anion formation precedes the development of hypertension in SHR and in Ang II-infused mice H oxidase is a multi-subunit enzyme and is one of the enzymatic sources of superoxide production. The NAD(P)H oxidase has five subunits: p47phox (“phox” stands for phagocyte oxidase), p67phox, p40phox, p22 phox, and the catalytic subunit gp91phox   in the short term. Previous reports have suggested that baroreflex sensitivity (BRS) is reduced during hypertension and the mechanisms underlying its reduction involves ROS Braga, .In recent decades, several research groups have worked extensively to improve the treatment of hypertension and its complication, including reduction in BRS, focusing on the discovery of new therapy strategies and drugs  and HR in SHRSP but not in WKY  in SFO or RVLM demonstrated that the pressor effects caused by Ang II infusion were attenuated. Girouard et al.  found thin situ abolished the Ang II-mediated depression of cardiac and sympathetic baroreceptor reflex gain in the WKY. In contrast, PKC inhibition in the NTS of SHR only partially reduced the effect of Ang II on the baroreceptor reflex gain (Sun et al., Studies aiming to characterize the signal transduction mechanism of PI3-kinase involvement in Ang II-induced stimulation of central neuronal activity in cultured neurons and Ang II-induced inhibition of baroreflex in SHR vs. WKY rats showed that application of Ang II to neurons produced a 42% greater increase in neuronal firing in cells from the SHR compared to WKY. Interestingly, the Ang II-mediated increase in firing rate was abolished entirely by the PKC inhibitor GF109230 in the WKY while it was necessary to block both PKC and PI3K activity to produce the same effect in the SHR (Sun et al., Furthermore, aiming to determine whether or not chronic reduction of ROS in the RVLM improves impaired BRS in hypertensive rats, Ogawa et al.  transfecStudies from our laboratory revealed that natural products were capable of improving BRS through ROS scavenging mechanisms. The flavonoid quercetin improves both sympathetic and parasympathetic components of baroreflex and reduces MAP in SHR due to reduction of systemic oxidative stress. The mechanism is unknown, but it was suggested that this antioxidant can interfere with three or more different free radical–producing systems (Monteiro et al., In conclusion, natural products with antioxidant properties have emerged as new potential therapeutic tools for improving BRS during hypertension. Although promising, there is still a long way until their use in the clinics becomes reality.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DNA methylation is an epigenetic alteration, involved in human cancers. “Field cancerization”, also known as “field defect” in cancerogenesis has been explained by the presence of cells with genetic and epigenetic alterations, which are predisposed to cancer development. DNA methylation has been proposed as a candidate mediator of this field cancerization.The aim of the study was to assess the methylation status of 6 genes promoter  in the tumor and in the adjacent normal tissue in patients suffering from head and neck squamous cell carcinoma (HNSCC).The samples of tumor and adjacent normal tissue were obtained during surgery from 55 patients hospitalized at Maria Skłodowska-Curie Cancer Center and Institute of Oncology, Gliwice. After DNA isolation the methylation-specific polymerase chain reaction (MSP) was used to analyze the methylation status of the gene promoter mentioned above. Conventional histopathological parameters were assessed to prove the presence of squamous cell carcinoma while no neoplastic changes were detected histopathologically in the adjacent normal tissue. In order to establish the clinical relevance of methylation in HNSCC, correlation between methylation frequency of these genes promoter as well as the clinical stage of tumor, the patient’s age and gender were also considered. Ethics approval for this study was received from the Maria Skłodowska-Curie Cancer Center and Institute of Oncology Ethics Committee (No. KB/493-31/08).Methylation of MGMT, APC, TIMP3, RAR, ECAD and p16 were detected in 38%, 33%, 61%, 67%, 29%, 49% of tumors, respectively. Some of the patients who delivered methylation–positive tumor samples, had also methylation–positive adjacent normal tissues. The genes promoter methylation were present in 20%, 31%, 39%, 49%, 11%, 42% of the tumor-adjacent normal tissues, respectively.To sum up, the epigenetic modifications represented by DNA methylation in the adjacent normal tissue in the course of HNSCC point to the usefulness of DNA methylation as a marker of epigenetic cancerization field and of the future risk of cancer development.


HIV hijacks the body's immune system to have itself delivered to T cells in which it can explosively replicate. It does this partly by subverting the function of various types of dendritic cells (DCs) that are present in many of the tissues exposed to the external environment. To date, however, it has been unclear how HIV is transmitted in the gut. A recent paper in EMBO Molecular Medicine goes much of the way to elucidating this mechanism, implicating DCs in the lamina propria that sit beneath the DC-free columnar epithelia (CE) of the gut and extend and then return processes between the CE cells to capture HIV , and promote the migration of other immune cells to the site of infection through the release of cytokines and chemokines. DCs are derived originally from bone marrow and divided into two major types: the myeloid and plasmacytoid DCs (which are major producers of type I interferons). Myeloid DCs consist of sub-populations found in diverse anatomical locations throughout the body, and especially in tissues in contact with the external environment. These include the blood, secondary lymphoid tissue and especially the anogenital and intestinal epithelium. DC sub-populations have been best defined in mice, but the human homologues are now being described , immature Langerhans cells (LC) – a type of DC – express the C-type lectin receptor (CLR) langerin and patrol this epithelium which covers the lower female genital tract, anus and male foreskin in humans. Myeloid DCs thus act as sentinels, specialized for pathogen detection. The underlying dermis contains at least three subsets of DCs that are characterized by their surface receptors: CD1a, CD14 or, as recently discovered, blood dendritic cell antigen 3 (BDCA3). Most express CLRs, a mannose receptor and/or DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), as well as different complements of Toll-like receptors (TLRs). Unlike the SSE, the CE coating the respiratory tract, gut and endocervix, is devoid of resident DCs. However, DC populations found in the underlying lamina propria closely resemble dermal DCs ; adhesion molecules are up-regulated (ICAM1) and lysosomal enzyme expression is down-regulated. The viral and cellular stimuli modulating such changes are still being elucidated. However, we do know that the HIV gene and protein product Vpr inhibits type I IFN production, and that signalling through CCR5 and components of the HIV inoculum might induce maturation and migration. HIV nucleic acids might interact with cytoplasmic RNA or DNA sensors to induce these changes  is transferred to CD4/CCR5 receptors, leading to neutral fusion with the plasma membrane. Once the infected DC contacts CD4 T cells, the large portion of HIV residing in the vesicular caves is transferred to the T cells by expulsion into the virologic synapse at the contact region. HIV also enters the endosomal system where it is lysed by acid-proteolytic digestion prior to antigen presentation to T cells. HIV entering via the CD4/CCR5 receptor undergoes HIV exposure to the intestinal mucosa allows viral penetration through transiently open tight junctions of the CE, thus creating a gradient that attracts the emigration of lamina propria DCs and extension of their processes through this route.This response was dependent upon the interaction of the HIV envelope protein with CCR5, but not CXCR4. This viral protein is known to signal through CCR5 and to induce DC migration (Anand et al, de novo replication or vesicular caves, will be interesting to elucidate.The paper by Cavarelli et al. extends our knowledge of such mechanisms. They have shown that HIV exposure to the intestinal mucosa allows viral penetration through transiently open tight junctions of the CE, thus creating a gradient that attracts the emigration of lamina propria DCs and extension of their processes through this route.These studies provide a further rationale for developing inhibitors of HIV-CCR5 interactions as microbicides, especially in the anal mucosa and perhaps in combination with suppressive therapy in late pregnancy and delivery (Lederman & Este, The authors declare that they have no conflict of interest.