Background. This study aimed to establish the expression profile of circulating microRNAs (miRNAs) during nerve allotransplantation in the presence and absence of FK506 immunosuppression. Methods. A 1 cm BALB/c donor sciatic nerve graft was transplanted into the sciatic nerve gaps created in recipient C57BL/6 mice with or without daily FK506 immunosuppression [1 mg/(kg·d)]. At 3, 7, and 14 d after nerve allotransplantation, serum samples were collected for miRNA expression analysis by Illumina small RNA deep sequencing. Results. Sequence analysis showed that the dominant size of circulating small RNAs after nerve allotransplantation was 22 nucleotides, followed by 23-nucleotide sequences. Nine upregulated circulating miRNAs  were identified at 3 d, but none was identified at 7 or 14 d. Among them, miR-9-5p had the highest fold-change of >50-fold, followed by miR-340-5p with 38.8-fold. The presence of these nine miRNAs was not significant at 7 and 14 d after nerve allotransplantation with or without immunosuppression, showing that these miRNAs are not ideal biomarkers for monitoring rejection of deep-buried nerve allografts, a response usually observed later. Conclusions. We identified nine upregulated circulating miRNAs, which may have a biological function, particularly during the early stages after nerve allotransplantation under FK506 immunosuppression. Streptomyces tsukubaensis, is used to suppress immunological responses and demonstrates relatively good results without significant rejection when applied to nerve allografts [At present, nerve allograft transplantation is a clinical reality for select patients who sustain considerable nerve injury with nerve defect . In the lografts . Followilografts . Importalografts –6. Howevlografts –9. Lack Micro-RNAs (miRNAs) are endogenous, small (comprising ~ 22  nucleotides  (nt)), single-stranded, and noncoding RNAs that regulate gene expression and are detectable in whole blood, serum, plasma, urine, and other body fluids in a highly stable form –12. Incrn = 3 animals/group at each time point), whole blood samples were collected at the indicated times in tubes containing anticoagulant. The labels (F)3d, (F)7d, and (F)14d were used to indicate the serum samples of mice undergoing allotransplantation with FK506 immunosuppression for 3, 7, and 14 d, respectively, while the labels (N)3d, (N)7d, and (N)14d were used to indicate the serum samples of mice undergoing allotransplantation without FK506 immunosuppression and sacrificed at postoperative days 3, 7, and 14, respectively. After the whole blood samples were incubated at room temperature for 15 min, they were centrifuged at 3,000 ×g for 10 min, white blood cells were slowly removed from the corresponding layers, and the serum was extracted and stored at –80°C before processing for RNA analyses. All housing conditions and surgical procedures, analgesia, and assessments were in accordance with national and institutional guidelines; an Association for Assessment and Accreditation of Laboratory Animal Care- (AAALAC-) accredited SPF facility was used. Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Chang Gung Memorial Hospital.Male BALB/c and C57BL/6 mice  were purchased from BioLasco . The BALB/c mice served as sciatic nerve allograft donors. The C57BL/6 mice were randomized into two groups of nerve allograft transplantation: one with FK506 treatment and another without FK506 treatment. These species of mice were selected on the basis of disparity at the MHC locus and prior experience with reciprocal rejection of grafts between these murine strains . The micTotal RNA was extracted from the harvested nerve graft, whole blood, and serum by using the mirVana miRNA Isolation Kit . For miRNA array, purified RNA yield was determined on the basis of the absorbance at 260 nm by using an SSP-3000 NanoDrop spectrophotometer , and RNA quality was evaluated with a Bioanalyzer 2100 system .Small RNAs had linkers ligated to them and bar-coded cDNAs were prepared using the TruSeq Small RNA Sample Prep Kit (Illumina) following the manufacturer's instructions. Individual libraries were analyzed on a Bioanalyzer (Agilent) to detect the presence of linkered cDNA of the appropriate size (135–165 bp), and 11 bar-coded libraries were pooled into one sample by mixing 2 ng of the 135–165 bp peak from each sample, as determined by the Bioanalyzer. The 135–165-bp peak of the pooled cDNAs was purified from the mixed sample by using the Pippin Prep DNA Size Selection System (Sage Science) and confirmed by the Bioanalyzer.Sequencing of the pooled libraries was performed in one lane of the Illumina HiSeq2000 Sequencer at the miRBase v19, and 50-bp single-end reads of the libraries were obtained. After indexing and trimming linker sequences, reads with a minimum length of 15 nt and with <3 terminal mismatches in the sequence were sorted and counted for the following analysis. The deep sequencing experiment was performed by BGI Tech Solutions Co., Ltd. .http://www.geneontology.org/), calculated gene numbers for each term, and then used hypergeometric test to find significantly enriched GO terms in target gene candidates, compared to the reference gene background. The Bonferroni correction for p value was used to obtain a corrected p value. GO terms with a corrected p value of ≤ 0.05 were defined as significantly enriched in the target gene candidates. Pathway analysis from a major public pathway-related database KEGG [First, this method mapped all target gene candidates to GO terms in the database (ase KEGG  was perfhttp://dx.doi.org/10.1155/2015/863192). In total, 7785957, 5978171, 5978171, 5574644, 5988924, and 5975945 high-quality raw reads were obtained from the serum libraries of (F)3d, (F)7d, (F)14d, (N)3d, (N)7d, and (N)14d mice, respectively. After filtering the low-quality sequences, empty adaptors, and single-read sequences, 5458187 (70.1%), 5272112 (88.19%), 5670065 (94.85%), 5199001 (93.26%), 5710818 (95.36%), and 5334192 (89.26%) clean reads, 18–30 nt in length, were selected for further analysis from the serum libraries of (F)3d, (F)7d, (F)14d, (N)3d, (N)7d, and (N)14d mice, respectively (Supplementary File 1: Table S1). Among the selected reads, 2689173, 2853061, 2750572, 2615969, 3120416, and 2574471 sequences from the serum libraries of (F)3d, (F)7d, (F)14d, (N)3d, (N)7d, and (N)14d mice, respectively, mapped perfectly to the mouse genome, amounting to 44.14% and 49.94% of the total reads, respectively; and 2602557, 2763676, 2657576, 2590795, 3088195, and 2555335 reads in the serum libraries of (F)3d, (F)7d, (F)14d, (N)3d, (N)7d, and (N)14d mice, respectively, were found to be similar to miRNAs. The rest of the sequences were found to be other types of RNA, including noncoding RNA, rRNA, scRNA, snRNA, snoRNA, srpRNA, and tRNA. The size distribution of small RNAs (sRNAs) was similar in the two libraries, and majority of them ranged from 21 to 24 nt in length 3d, (F)7d, (F)14d, (N)3d, (N)7d, and (N)14d). The libraries were sequenced by Illumina small RNA deep sequencing technology. The number and proportion of the categories of small RNAs found are supplied in the Supplementary File . The results showed that 507 mature miRNAs were present in these six libraries (Supplementary File 1: Table S2). Among them, there were 147 mature miRNAs with ≥50 sequence reads (Supplementary File 1: Table S3) and nine mature miRNAs with ≥5000 sequence reads in at least one of these six libraries (Supplementary File 1: Table S4). In these libraries, known miRNAs had a broad range of expression levels; some (such as miR-486-5p and miR-3107-5p) were found to have more than hundreds of thousands of sequence reads, while many others had ≤20, indicating that expression varies significantly among different miRNA families. The proportion of different categories of small RNAs often reflects the roles in a particular stage and the associated biological mechanisms during nerve graft allotransplantation. With ≥5,000 reads, the following four miRNAs were dominantly expressed in all these six libraries: miR-25-3p, miR-486-5p, miR-3107-5p, and miR-92a-3p (Supplementary File 1: Table S5). In addition to miR-486-5p and miR-3107-5p, which have ≥200000 sequence reads in all six libraries, miR-92a-3p and miR-25-3p were the third and fourth most abundant miRNA in (F)7d , (F)14d , (N)3d , and (N)14d  samples (Supplementary File 1: Table S6).p < 0.001). According to the changes in relative miRNA abundance between the two serum libraries obtained from mice receiving nerve allograft transplantation with and without FK506 immunosuppression at the same indicated time points , unsupervised hierarchical clustering  as well http://www.microrna.org/microrna/home.do), MirTarget2 (http://mirdb.org/miRDB/), PicTar (http://pictar.mdc-berlin.de/), PITA (http://genie.weizmann.ac.il/index.html), and RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). Only the gene with miRNA target interactions predicted by at least three of these five target prediction programs was chosen (Supplementary File 2). GO enrichment and KEGG pathway analyses were performed to identify the functional modules regulated by these nine miRNAs. The results of GO annotation enrichment analysis regarding biological processes, cellular components, and molecular functions are listed in Supplementary File 3. KEGG pathway annotation showed that 21,703 background genes were annotated for 270 biological functions. KEGG pathway analysis revealed 39 pathways that were significantly enriched with these miRNA targets (Supplementary File 4). Among them, the MAPK signaling pathway, Wnt signaling pathway, pathways involved in cancer, focal adhesion, gap junction formation, and degradation, and axon guidance, calcium signaling pathway, and Notch signaling pathway were ranked among the most enriched pathways. These results suggested that, during early stages, these targets show a high possibility of being regulated by miRNAs in cases of nerve allotransplantation with FK506 immunosuppression, although the possibility of false-positive prediction from the prediction algorithm always exists.To further understand the physiological functions and biological processes involving the nine upregulated miRNAs at 3 d in the sera of mice receiving nerve allotransplantation with FK506 treatment, target prediction was performed by integrating five common public databases: miRanda  identified nine upregulated miRNAs  at 3 d, but none at 7 d or 14 d, suggesting that these upregulated miRNAs impact biological functions, particularly during the early stages after nerve allotransplantation with FK506 immunosuppression.The Illumina deep sequencing platform is efficient for miRNA discovery and is widely used to generate small RNA profiles in various organisms. In this study, we identified the circulating miRNAs in a mouse model of nerve allograft transplantation under FK506 immunosuppression by Illumina small RNA deep sequencing. The sequence analysis showed that the dominant size of small RNAs in circulation after nerve allotransplantation in the presence or absence of FK506 immunosuppression was 22 nt, followed by 23 nt. This result is typical of Dicer-processed small RNAs and was consistent with the known size of ~22 nt for miRNAs. Selection of the differentially expressed miRNAs under the relatively strict conditions , apoptosis , and cell proliferation  has been reported in stimulated human peripheral blood lymphocytes . miR-340Although allograft biopsy is the gold standard for diagnosis of conditions such as acute rejection, disease recurrence, and drug toxicity , 25, it In conclusion, this study identified nine upregulated circulating miRNAs after nerve allotransplantation with FK506 immunosuppression by Illumina small RNA deep sequencing. These miRNAs affect biological functions, in particular during the early stages after nerve allotransplantation under immunosuppression.Expression profile of the miRNAs by Illumina small RNA deep sequencing.


Current research focuses on several aspects of H2S biology such as the biochemical pathways that generate the compound and its functions in human pathology or drug synthesis that block or stimulate its biosynthesis. The present work addresses the knowledge we have to date on H2S production and its biological roles in the general human environment with a special focus on the oral cavity and its involvement in the initiation and development of periodontal diseases.In the past years, biomedical research has recognized hydrogen sulfide (H However, results are controversial.Hydrogen sulfide .Interestingly, H2S production, its biological roles and implications in periodontitis development.This paper reviews the most significant studies concerning H2S synthesis is generally connected to three enzymes: 3-mercaptopyruvate sulfurtransferase (3MST), cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), all three taking part in the cysteine synthesis pathway [In mammalian organisms, including the human body, endogenous H pathway ,11,12  [2S is also caused by the metabolic products of oral sulfate-reducing bacteria [2S is considered a pathogenic factor in bowel inflammatory diseases characterised by inflammatory mucosal lesions like periodontal diseases lesions [In the oral cavity, human periodontal ligament stem cells (PDLSCs) express Hnd (CBS) . CBS maynd (CBS) . H2S is bacteria  that deg lesions .2S is a gaseous mediator with multiple roles depending on the tissue or organ. Thus, H2S is involved in blood vessels dilatation, inflammation, cardiac reaction to ischemic injuries [2S are associated with respiratory affections such as chronic bronchitis, emphysema, pneumonia or diseases related to the cardiovascular system  [2S in the protection against tissue injury, in reducing inflammation, and tissue repair [2S might be both beneficial and harmful in cerebral ischemic injury depending on its concentration [2S synthesis, administered at a low dose has protective effects; but it worsens the ischemic injury at higher concentrations [Hinjuries , nervousinjuries , insulininjuries ,19. In ttension) . Howevere repair . H2S migntration . A recentrations .2S cellular effects is the regulation of intracellular redox homeostasis and post-translational modification of proteins through glutathione (GSH) generation and S-sulfhydration [2S exerts anti-oxidative, anti-inflammatory and cytoprotective effects [2S donor) had interesting effects in the kidneys of uranium-intoxicated rats: it managed to lower malondialdehyde(MDA) accumulation, and to restore GSH levels and anti-oxidative enzymes’ activities like superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione S-transferase (GST) [2S can protect neurons and cardiac muscle from OS and ischemia-reperfusion injury [Among the most studied molecular mechanisms of Hydration . Moreove effects . Sodium se (GST) . Furtherse (GST) . Also, Hn injury , as welln injury .2S  were shown to be significantly elevated in patients with cerebrovascular pathology , as well as increased cholesterol levels, asthma or hepatic affections like cirrhosis [Likewise, other volatile sulfur compounds related to Hirrhosis   [Reactive oxygen species (ROS) are products of normal oxygen metabolism and have beneficial biological effects, in low levels and under normal conditions. Instead, higher concentrations present harmful effects to the body. External environment , behavioural activities  and inflammatory cells (such as activated macrophages and neutrophils release various ROS (Hnd HOCl) ,27,28,29nd HOCl) . At the nd HOCl) . Lipid pnd HOCl) .Several amino acids (such as tyrosine) can also react with ROS, generating a wide range of products, from modified and less active enzymes to denatured, non-functioning proteins. Furthermore, mitochondrial DNA are also affected by the ROS attack. HO can react with all components of DNA molecules, damaging both purine and pyrimidine ,32,33,34in vivo [There are many enzymatic antioxidant defence mechanisms in order to protect against ROS effects in vivo : SOD, GPin vivo ,36,37. Sin vivo ,40,41,42in vivo . Other sin vivo ,44.Tissue destruction in periodontal diseases is considered to be the result of an altered inflammatory/immune response to microbial plaque and involves massive release of neutrophils, ROS and enzymes ,46,47,48Therefore, the balance between antioxidant mechanisms and ROS is of utter importance in periodontal pathogenesis. Increased ROS and inhibited antioxidant mechanisms and/or decreased antioxidant capacity might lead to problems of the periodontium. Several authors reported a positive correlation between periodontal tissue damage and high levels of ROS ,55,56,57Furthermore, animals infected with periodontal pathogens presented a five-fold increase in the OS index compared with controls . ClinicaResearchers have also started to consider a possible relationship between periodontitis and systemic diseases. Diabetes mellitus ,62,63, mFurther experiments demonstrated there is an increased amount of ROS not only in serum, but also in the oral fluids of periodontally diseased subjects ,65. GingNew therapies are constantly being developed and could have future uses against OS present in chronic periodontitis: protein transduction treatments , bone tain vitro, being able to partially antagonize the negative effects of hydrogen peroxide [Ascorbic acid, an antioxidant, plays an important role in the maintenance of periodontal health in the elderly . Its useperoxide . Furtherperoxide . Other speroxide  or lipopperoxide .Considering periodontitis a polymicrobial anaerobe infection, researchers focus on the following main mechanisms for explaining its pathogenesis: the production of certain waste products in the proteolytic metabolism, an intense host–inflammatory response and increased OS ,3,29.2S is a bacterial waste product eliminated in the subgingival pocket [2S are causative agents of periodontitis because the toxicities are similar to that of cyanate [2S has also shown antioxidant properties, for example, in elevating endogenous antioxidase, such as SOD [Hl pocket  which, dl pocket ,73,74,75 cyanate . Howeverh as SOD ,77,78.2S can be regarded as a double-faced molecule: on one side, it can promote an antioxidant effect and becomes cytoprotective; while on the other side, it stimulates OS and is cytotoxic.So, H2S is directly linked to the initiation and development of periodontal diseases: the compound inhibits the proliferation process of oral keratinocyte cells [Reports show that Hte cells , decreaste cells .2S and the H2S levels. Low physiological concentrations of H2S have been shown to induce dental pulp cells differentiation towards hepatic or pancreatic cells [2S maintain the proliferation and differentiation of PDLSCs [2S in PDLSCs led to significant reduction in their proliferation rate, as well as decreased osteogenic and adipogenic differentiation [2S-releasing drugs had enhanced anti-inflammatory effects and reduced side effects in tissues [2S -releasing derivative of naproxen, ATB-346, led to significant inhibition of alveolar bone loss and inflammation in periodontal rats [There seems to be a direct relationship between the type of biological effect induced by Hic cells  or to swic cells . Moreovef PDLSCs . Blockinntiation . However tissues . Recentltal rats .2S are demonstrated to induce programmed cell death through different molecular pathways in a number of cell types. These concentrations induced apoptosis through inhibition of SOD in human gingival fibroblasts. This enzyme is paramount in eliminating ROS and leads to damage of DNA structure. The same process was observed in normal keratinocytes [High physiological concentrations of Hinocytes  and kera2S were shown to induce the apoptotic process in several cell types belonging to oral structures. Generally, apoptosis follows well established pathways such as: intrinsic mitochondrial pathway where the inner mitochondrial membrane is depolarized followed by cytochrome c release into cytosol, assembly of the apoptosome that leads to activation of initiator caspase 9 ultimately followed by executioner caspase 3 activation [Volatile sulfur compounds and especially Htivation .2S-exposure, human oral keratinocyte stem cells expressed key p53-related molecules associated with cell death, DNA repair and cell cycle control.This pathway was activated in most studied cell types: oral fibroblasts, oral keratinocytes, oral keratinocyte stem cells, and general keratinocyte stem cells. The extrinsic pathway, or theligand-activated pathway was shown to be responsible for apoptosis induction only in cells isolated from the alveolar bone, osteoblast cells. At the same time, after H2S is its association in the development of physiological and pathological halitosis [Another clinical effect of Halitosis ,49. Halialitosis .2S producing species like Fusobacteriumspp., Parvimonasmicra, Tannerella forsythia or Filifactoralocis [Fusobacteriumspp. acts on substrates such as cysteine [Treponemadenticola [Several studies reported bacterial Hcysteine , homocyscysteine  or GSH [cysteine . Likewisenticola .2S production pathway. Glutamate or glutamine and the dipeptide cysteinylglycine (Cys-Gly), are obtained from GSH. Cys-Gly degradation results Gly and L-Cys. Pyruvate, ammonia, and H2S are the final products of L-Cys degradation. In GSH metabolism are involved three enzymes γ-glutamyltransferase (GGT), cysteinylglycinase, and L-cysteine desulfhydrase  [2S, in the presence of L-cysteine [Three steps has been proposed for the bacterial Htalysin) ,90, the cysteine . 2S -producing capacity is commonly tested with gas chromatography [The Htography , colorimtography , by usintography  or by bltography .in vitro and in vivo to detect the rate and amount of H2S produced by various species and strains. In present more research is needed to be done under various conditions both 2S, Porphyromonas gingivalis, produces several other virulence factors such as proteases (gingipains) [2S (650–1.150 μmol/L), produced by Porphyromonas gingivalis affect IL-8 production in Phorbolmyristateacetat (PMA)-stimulated epithelial cells. In addition to Hgipains) , lipopolgipains) , and hemgipains) . A highe2S (less than 400 μmol/L), did not effect IL-8 production in PMA-stimulated epithelial cells [2S present in blood at concentrations in the range of 30–100 μM [2S in concentrations lower than 400 μmol/L. Another study observed that increased levels of H2S (800 or 1.600 μmol/L) did not stimulate IL-8 production in epithelial cells in the absence of PMA [A lower concentration of Hal cells . H2S pre0–100 μM , epithele of PMA .2S-mediated production of IL-8, the presence of a predisposed inflammatory condition is very important [2S.Additionally, a recent study found that H2S synergistically up regulates Porphyromonas gingivalis LPS-induced expression of IL-6 and IL-8 in gingival fibroblasts and PDLCs, which could further promote the development of periodontitis [2S inhibited LPS which induced synthesis of prostaglandin E2 (PGE2), NO, IL-1β and IL-6 in LPS-treated murine macrophages [2S donor, promoted the synthesis of pro-inflammatory mediators [This means that for Hmportant . This codontitis . Lower crophages ,99. Howeediators ,99. An iediators . Neutropediators .2S, causes an enhancement of local inflammation by recruiting excess numbers of neutrophils. Several studies made in vivo on pancreas, liver and lung, observed that both endogenous H2S and exogenously supplied H2S increased neutrophil migration to the inflammation sites [IL-8 production by epithelial cells is mediated by Hon sites ,103.2S could induce the chemotaxis of neutrophils to the periodontal pocket, becoming this way, a real promoter of local OS, indirectly.These data support the premise that the periodontal bacteria released H2S toxic effects in periodontal tissue has been showed [2S in periodontal tissue physiologic function remains less understood. A recent study involving 43 subjects with moderate or severe periodontal breakdown could not correlate H2Sproduction to periodontal disease severity or to a specific bacterial composition [2Smay be a valuable clinical marker for degradation of proteins in the sub gingival pocket [Exogenous Hn showed , but theposition . It was l pocket .2S, OS and periodontal diseases is controversial, but should not be underestimated. Further research is needed in order to elucidate the exact mechanisms and conditions which cause the H2S molecule to exhibit antioxidant or cytotoxic proprieties in the oral cavity.Altogether, data presented in recent studies suggests that the relationship between H2S biochemistry and its functions in cell biology . In this respect, the field can be further expanded together with the development of tools that could correctly identify and quantify H2S synthesis and catabolism in organs and tissues [2S production together with a better understanding of the chemical pathways responsible for its removal. Overall, a more clear understanding of the biochemistry of H2S in relation to its biological roles is greatly needed.To date, there is no general consensus regarding H tissues . Another


Direct modulation of the molecular pathways concerning these potential macrophage-related targets represents a promising field for new therapeutic alternatives in atherosclerosis and CVD.Cardiovascular disease (CVD) is a global epidemic, currently representing the worldwide leading cause of morbidity and mortality. Atherosclerosis is the fundamental pathophysiologic component of CVD, where the immune system plays an essential role. Monocytes and macrophages are key mediators in this aspect: due to their heterogeneity and plasticity, these cells may act as either pro- or anti-inflammatory mediators. Indeed, monocytes may develop heterogeneous functional phenotypes depending on the predominating pro- or anti-inflammatory microenvironment within the lesion, resulting in classic, intermediate, and non-classic monocytes, each with strikingly differing features. Similarly, macrophages may also adopt heterogeneous profiles being mainly M1 and M2, the former showing a proinflammatory profile while the latter demonstrates anti-inflammatory traits; they are further subdivided in several subtypes with more specialized functions. Furthermore, macrophages may display plasticity by dynamically shifting between phenotypes in response to specific signals. Each of these distinct cell profiles is associated with diverse biomarkers which may be exploited for therapeutic intervention, including IL-10, IL-13, PPAR- Cardiovascular disease (CVD) is currently recognized as the leading cause of morbidity and mortality in the adult population worldwide, with an estimated projection of 23.3 million yearly deceases attributable to these disorders by the year 2030 , 2. Dataα (TNF-α), and growth factors and expresses multiple cell adhesion molecules which allow recruitment and adhesion of monocytes to vessel walls, promoting their differentiation to activated macrophages capable of phagocytizing oxidized low-density lipoprotein (oxLDL) present in the subendothelium , “intermediate” monocytes [CD14++CD16+], and “nonclassic” monocytes [CD14+CD16++]  , 34. Eac+CD16++] , Figure ++CD16−] constitute 80–95% of total circulating monocytes and primarily act as phagocytes, boasting strong peroxidase activity and predominantly releasing Interleukin-10 (IL-10) in response to LPS  represent 2–10% of total circulating monocytes, show minimal peroxidase activity, and secrete large quantities of Interleukin-1β (IL-1β) and TNF-α in response to LPS; thus, their role is preeminently proinflammatory, intensely expressing CXCR-1 and moderate amounts of CCR2  comprise only 2–8% of total circulating monocytes and are considered “patrolling” or “surveillance” cells because they express low levels of CCR2 and high levels of CX3CR1, resulting in great endothelial affinity with a stunted response to chemotaxis [Finally, nonclassic monocytes [CD14emotaxis , 39.Macrophages play a crucial role in immune responses, by actively participating in a myriad of biological processes, such as resolution of infections and repairing of injured tissues, as prompted by numerous signals, which include microbial molecules and proinflammatory cytokines . Followiγ (IFN-γ) to its extracellular heterodimeric receptors IFNGR-1 and IFNGR-2, which initiates JAK1- and JAK2-mediated signaling cascades, culminating in activation of STAT1, which in turn activates the transcriptome for the M1 profile [Macrophages adopt the M1 phenotype following binding of Interferon- profile . These c profile .heterogeneity” was introduced after observations of monocytes undergoing macrophagic differentiation through an alternate route, towards a cellular subpopulation with different characteristics, termed M2 macrophages [The concept of macrophage “rophages , 45. Morrophages , 47, Figrophages . After brophages . IL-10 mrophages , and glurophages .γ, IL-1, IL-6, IL-10, GM-CSF, and TNF-α, have poor phagocytic capacity, and contribute to deposition of extracellular matrix by potentiating synthesis of polyamines, collagen, and Transforming Growth Factor-β (TGF-β); thus, M2a may be described as “tissue-repairing” macrophages [Each kind of M2 macrophage displays distinct biologic profiles. M2a are strongly anti-inflammatory, inhibiting release of INF-rophages . On the rophages . Howeverrophages .α, IL-6, and IL-1 [β, or glucocorticoids, are also capable of abundant IL-10 release but are also able to inhibit production of proinflammatory cytokines, Nitric Oxide (NO), and Reactive Oxygen Species (ROS), outlining an important role in limiting progression of inflammation [On the other hand, transition to M2b may be triggered by exposure to immune complexes, TLR ligands, LPS, lipoteichoic acid, and other intermediaries. These cells boast remarkable IL-10 secretion associated with FoxO1 expression  and veryand IL-1 . Lastly,ammation .2 (PGI2), Endothelins , Thromboxane A2 (TXA2), and angiotensin II [ endothelial dysfunction, sets the stage for the earliest phases of atherosclerosis [Atherosclerosis is a multifactorial, chronic, and progressive inflammatory process which develops from endothelial dysfunction . The endensin II –60. Thesclerosis . This alclerosis , uncontrclerosis , hyperteclerosis , and smoclerosis . These eclerosis . Other rclerosis .+CCR2+) [The natural history of atherosclerotic disease may be studied in 6 continuous stages, from the formation of the lipid core to vascular lumen obliteration . Elevate+CCR2+) , 70. LDL+CCR2+) , and by +CCR2+) . Neverth+CCR2+) .In Stage II , PUFA inScavenger receptors are found in a broad array of cells, remarkably in macrophages, monocytes, platelets, endothelial cells, smooth muscle cells, and epithelial cells , mediatiIn this stage, oxLDL are particularly potent mediators within the subendothelium, given their strong antigenicity and their ability to induce expression of genes involved in the inflammatory response in endothelial cells and macrophages. For instance, they are able to increase endothelial permeability via multiple mechanisms, such as cytoskeletal disorganization through inhibition of myosin light-chain phosphatase, with activation of Rho kinase , and proκB (NF-κB) [κB leads to expression of several cellular adhesion molecules like P-Selectin, Vascular Cell Adhesion Molecule-1 (VCAM-1), and Intercellular Adhesion Molecule-1 (ICAM-1), indispensable for the fixation of immune cells to the endothelium during chemotaxis [α [Nevertheless, one of the most notable roles of oxLDL is the induction of Nuclear Factor- (NF-κB) –86, a ke (NF-κB) , a cruci (NF-κB) . Activatemotaxis , 89. Furemotaxis , is alsotaxis [α . MCP-1 itaxis [α . During taxis [α , chemokitaxis [α . Finallytaxis [α .src kinase) [β, loss of cell polarity, stunting of cell movement, inhibition of autophagy, and, finally, cell death [Although formation of macrophage-derived foam cells requires activity from several scavenger receptors, CD36 from Class B appears to be the main mediator , 79, 92. kinase) , 95, fol kinase) . Platele kinase)  and Toll kinase)  also appll death . Other fll death , thymic ll death , Visfatill death , and TRAll death  through  in vitro models [κB and induction of Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) [Development of foam cells derived from VSMC is another paramount aspect of advanced atherosclerotic lesions, due to their involvement in intimal thickening and stability of the fibrous cap , 104. Seo models , 106. Im (ACAT1) . Further (ACAT1)  and prom (ACAT1) , respect (ACAT1) , 111. Fi (ACAT1) .plasticity.” This mechanism allows adaptation to a myriad of possible changes in dysfunctional endothelium or damaged tissue, including acute oscillations in levels of lipids, ROS, proinflammatory cytokines, microbial products, or oxLDL [In addition to the processes and macrophage subtypes described previously, macrophages may shift between these phenotypes in response to certain signals within their dynamic local microenvironment , a propeor oxLDL . Indeed,or oxLDL : M1 cellor oxLDL , and areor oxLDL . Neitheror oxLDL .γ signaling [ Listeria monocytogenes, Chlamydia ssp., and Salmonella typhimurium [ C. pneumoniae [ Helicobacter pylori [ Cytomegalovirus [α, IL-6, IL-1β, CCL2, CCL5, and CXCL8 [As commented beforehand, the main command for M1 switching is IFN-ignaling , 44, witignaling . Likewisignaling . Intereshimurium . In facteumoniae , 123, Her pylori , and Cytalovirus  have beend CXCL8 .+ M1 macrophages have also been observed in premorbid states, such as prediabetes, where these cells have been reported to share a direct correlation with HbA1C levels [κB [γ, and PPAR-α [Obesity and various obesity-associated disorders, such as DM and metabolic syndrome, have been linked with imbalanced M1/M2 ratios , propitiC levels . One of C levels , a pentavels [κB , 133. Fid PPAR-α . Therefod PPAR-α , 26. in vitro SLE models have described greater cholesterol accumulation in macrophages, along with increased IFN-α signaling and SR-A expression [β2GPI/anti-β2GPI complexes [Other disorders featuring proinflammatory states, such as systemic lupus erythematosus (SLE) , rheumatpression , as wellpression . Likewispression  and macrpression , as wellpression . In APS,omplexes  and partomplexes , 143.κB has been observed to inhibit NF-κB-induced M1 polarization [κB activation in macrophages during lipoprotein endocytosis and processing, lowering release of inflammatory cytokines [α and IL-2Rγ [INK4a, with accompanying reduction of lesion area in animal models [On the other hand, M2 polarization requires attenuation of various M1-promoting intermediaries. In this regard, the p50 subunit of NF-rization . Similarytokines . Other Md IL-2Rγ , and thil models .As can be observed, macrophage polarization within a certain scenario is a game of power, whose outcome is dictated by the prevailing kind of microenvironmental stimuli –150. The in vitro models have shown lysophosphatidic acid and platelet-activating factor to be the main factors involved in their retention within the subendothelium [One of the earliest events in the pathogenesis of the atherosclerotic plaque is the adhesion of circulating monocytes to the vascular endothelium, followed by their migration to the intima. Various adhesion molecules , chemoattractant cytokines, and modified lipoproteins are responsible for monocyte recruitment to the intima , whereasothelium . In thisothelium , highligothelium .In this regard, emigration of monocyte-derived cells from atherosclerotic lesions has been associated with their regression; thus, decreased efflux of these cells may be a key phenomenon for their progression , 155. NeLastly, macrophage proliferation is yet another fundamental finding in atherosclerosis: this mechanism may originate up to 90% of all locally accumulated macrophages . AlthougInflammation is a major component of CVD and a fundamental driving force throughout all phases of atherosclerosis , with maResults from recent prospective studies have shown control of atherosclerosis may be poor, despite optimal risk factor management , and pro in vitro, and many aspects remain incompletely elucidated in humans.This alarming outlook accentuates the importance of assessing the potential benefits of targeting macrophage biomarkers for therapy, by either hindering proinflammatory pathways or enhancing anti-inflammatory mechanisms, while accounting for age and the impact of early intervention. Although many biomarkers have been associated with macrophage activation, their relationship with the different phenotypes remains unclear. Therefore, macrophage heterogeneity may represent a foundation for the study of their products' potential clinical applications, with respect to prediction of atherosclerotic progression and therapeutic modulation of the inflammatory response. This conundrum has already spawned a large body of research –172, Tabγ [γ expression, as well as greater-sized atherosclerotic lesions with significantly higher lipid deposition, lower collagen content, and increased T cell infiltration in comparison to mice with unaltered immune responses in equal conditions [Interventions exploiting anti-inflammatory cytokines, such as IL-10, appear promising . IL-10 hγ . Indeed,nditions . Similarnditions  evidencenditions .IL-13 has also demonstrated possibly exploitable antiatherogenic assets, as described by Cardilo-Reis et al. , who fouγ in macrophages represents another alternative, due to their ability to inhibit expression of many inflammatory mediators [γ in macrophages has been linked with improved insulin sensitivity and M2 polarization, with reduced inflammation and atherosclerotic progression, expanding these agents' well-established therapeutic role in the management of CVD beyond the management of dyslipidemia [β release and inhibition of proinflammatory messengers such as IL-12, TNF-α, and IFN-γ, powerfully inducing M2 polarization in murine models [Modulation of PPAR-ediators . Additioipidemia , 180. Inipidemia . Youssefipidemia  have proe models .In light of these molecular findings, statins have been suggested for use in primary prevention, supported by research with animal –186 and Therefore, currently available evidence is insufficient to recommend statin therapy as a primary preventive strategy , 196. Coα in these cells [β1-induced calcification of VSCM and promote autophagy via blunting of β-catenin signaling [γ and AMPK and inhibition of mTOR [In any manner, statins may impact not only macrophages and endothelial cells, but also VSMC: pitavastatin and fluvastatin have been shown to lower expression of MCP-1 and TNF-se cells . Likewisignaling . Similar of mTOR .Liver X Receptors (LXR) are important nuclear transcription factors in macrophages, with specific genomic targets, such as the LBP (Lipopolysaccharide Binding Protein) gene, involved in foam cell modulation via LXRa expression , 210, anOther drugs, such as metformin, have also been described to intervene in macrophage functionality: this molecule can induce expression of AMPK and ATF-1, increasing heme concentration and thus driving Mhem polarization, ultimately preventing formation of foam cells . MetformIn addition, microRNA (mRNA) molecules, short RNA sequences capable of regulating genetic expression, have recently claimed considerable interest as therapeutic targets in all genome-related diseases , 221, inκB inhibition, and deceleration of foam cell generation and plaque progression.Finally, regulation of NLRP3 inflammasome activation in M1 macrophages represents another novel alternative. In animal models, NLRP3 activation by ATP  and cholThe study of monocyte and macrophage heterogeneity has led to the identification of these cells' distinct functional phenotypes, which play dynamic roles in the onset and progression of atherosclerosis, as these cells may act as pro- or anti-inflammatory mediators, depending on the prevailing cues involved in their activation. A broad range of pre- and postpolarization biomarkers appear throughout this process, posing as valuable subjects for further investigation, in view of their possible applications in CVD.κB and MAPKp38 [As research efforts have advanced concerning macrophage activation and their associated biomarkers, new therapeutic targets have emerged in every stage of the atherosclerotic process. These innovative examples include modulation of macrophage autophagy –230, ind MAPKp38 , and mod MAPKp38 . Indeed,


PTPN11, RAF1, MEK2, or SHOC2) were more rarely described. Here we report, a syndromic familial case of a 12p duplication encompassing the dosage sensitive gene KRAS, whose phenotype overlapped with rasopathies. The patient was referred because of a history of mild learning disabilities, small size, facial dysmorphy, and pigmentation abnormalities . This phenotype was reminiscent of rasopathies. No mutation was identified in the most common genes associated with Noonan, cardio-facio-cutaneous, Legius, and Costello syndromes, as well as neurofibromatosis type 1. The patient constitutional DNA exhibited a ~10.5 Mb duplication at 12p, including the KRAS gene. The index case’s mother carried the same chromosome abnormality and also showed development delay with short stature, and numerous café-au-lait spots. Duplication of the KRAS gene may participate in the propositus phenotype, in particular of the specific pigmentation abnormalities. Array-CGH or some other assessment of gene/exon CNVs of RAS/MAPK pathway genes should be considered in the evaluation of individuals with rasopathies.RAS/MAPK pathway germline mutations were described in Rasopathies, a class of rare genetic syndromes combining facial abnormalities, heart defects, short stature, skin and genital abnormalities, and mental retardation. The majority of the mutations identified in the Rasopathies are point mutations which increase RAS/MAPK pathway signaling. Duplications encompassing RAS/MAPK pathway genes (The online version of this article (doi:10.1186/s13023-016-0479-y) contains supplementary material, which is available to authorized users. NF1 [PTPN11, RAF1, MEK2, or SHOC2) were more rarely described [KRAS, whose phenotype overlapped with RASopathies.Rasopathies are a class of genetic syndromes caused by germline mutations in the RAS/mitogen-activated protein kinase (RAS/MAPK) cascade , better NF1  and duplescribed –8. HowevWe report a patient who was evaluated in our clinic at age 12 and 17 years because of a history of mild learning disabilities (late of 2 years at school), small size (1.35 m as adult = −4 SD), and pigmentation abnormalities: nine café-au-lait spots all over body (the biggest one 3 cm of size), 14 achromic spots, and axillar lentigines Fig. . We did NRAS, PTPN11, RAF1, SHOC2, SOS1, SOS2, RIT1, RASA2, LZTR1, RRAS, BRAF, KRAS, MAP2K1, MAPK2K2, NF1, and HRAS genes was performed using targeted-capture next generation sequencing (NGS) as previously described [The most common genes associated with Noonan, cardio-facio-cutaneous, Legius, and Costello syndromes, as well as neurofibromatosis type 1 were sequenced in the propositus. The coding exons sequencing of the escribed . This geKRAS gene.Genome-wide array-CGH was performed as previously described  to identKRAS lead to increase in RAS signalling. These mutations are responsible for less than 5 % of PTPN11 mutation negative Noonan patients or of patients with CFC [de novo rearrangements was previously discussed [BRAF or KRAS genes. However, although many of the activating mutations are similar to activating somatic mutations seen in cancer, on the whole, they tend to be not as strongly activating in rasopathies. For example, the most common oncogenic BRAF mutation, p.Val600Glu, does not occur in CFC syndrome and the specific KRAS mutations associated with Noonan syndrome are not the same as the known recurrent somatic mutations associated with cancer. It is likely that the strongly activating oncogenic mutations cannot be tolerated as constitutional mutations [RASopathy-associated constitutional activating mutations in with CFC , 12. Theiscussed . Authorsutations .PTPN11, MAP2K2, or RAF1 constitutional duplications [KRAS gene may participate in the propositus phenotype, in particular of the specific pigmentation abnormalities. The RAS/MAPK pathway was identified as crucial for controlling pigmentation [KRAS may partly result in the observed syndrome phenotype. Array-CGH or some other assessment of gene/exon CNVs of RAS/MAPK pathway genes should be considered in the evaluation of individuals with rasopathies with no point mutation identified by sequencing.Rasopathy-specific phenotypic traits associated were sometime lacking in previous reported ications , 7. Our entation  and someentation –18. We carray-CGH, array-comparative genomic hybridization; CNV, copy number variation; CFCS, cardio-facio-cutaneous syndrome; CS, Costello syndrome; LS, Legius syndrome; NF1, neurofibromatosis type 1; NS, Noonan syndrome; OMIM, Online Mendelian Inheritance in Man; RAS/MAPK, RAS/mitogen-activated protein kinase


AbstractCyclopinidae G.O. Sars, 1913 is described from male and female specimens collected at Laguna de Términos, a large coastal lagoon system in the southern Gulf of Mexico. Mexiclopinacampechanagen. et sp. n. cannot be adequately placed in any extant genus within the family. It differs from other cyclopinid genera in having a unique combination of characters including: 1) absence of modified brush-like seta on the mandibular exopod; 2) maxillule exopod with stout setal elements and brush-like setae absent; 3) basis of mandible with one seta; 4) presence of a modified seta on endopod of fourth leg; 5) fifth leg exopod unsegmented, armed with three elements in the female and five in the male; 6) intercoxal sclerite of first swimming leg with two medial spiniform processes on distal margin. The new genus is monotypic and appears to be most closely related to Cyclopina Claus, 1863 and Heptnerina Ivanenko & Defaye, 2004; the new species was compared with species of Cyclopina and it resembles Cyclopinaamericana Herbst, 1982 and Cyclopinacaissara Lotufo, 1994. This is the second record of a species of Cyclopinidae in Mexico and the first in the Gulf of Mexico; the number of cyclopinid species recorded from the Americas is now 13.A new, monotypic genus of the interstitial marine cyclopoid copepod family  Cyclopinidae G.O. Sars, 1913 is one of the most diverse and successful among the benthic marine poecilostomatoid/cyclopoid copepods. It contains 12 valid genera  is one of the largest lagoon estuarine ecosystems of the gulf; it has a significant ecological and economic importance in southeastern Mexico because of its permanent connection to the sea and high productivity and diverse fish fauna . Copepod18°44'29.3"N; 91°29'44.6"W). Water temperature was 25 °C, salinity 28psu, and pH slightly alkaline (7.5). Trawls followed a parallel course with respect to the coastline. Samples were placed in a bucket with 5 liters of water; copepods were isolated alive 5 hours after collection, they were later on fixed in 4% formaldehyde buffered with borax  and kept in a 5% glycerin/ 70% ethanol solution. More than 35 male and female specimens were PageBreaktaxonomically examined in the laboratory; specimens were processed, dissected and examined following ECOSUR in Tapachula, Mexico. The process included dehydration of specimens in progressively higher ethanol solutions , critical point drying, and gold-palladium coating (20 nm) following usual methods. This hitherto unknown genus and species was described and illustrated following the current standards for the taxonomic study of the group (El Colegio de la Frontera Sur (ECO-CH-Z), in Chetumal, Mexico and in the National Museum of Natural History, Smithsonian Institution, Washington, D.C. (USNM). Original zooplankton samples containing more non-type specimens remain in the helminth collection of the Universidad Autónoma Metropolitana-Xochimilco, Mexico (CHUX), maintained by the co-author (RJAA).Night zooplankton samples were obtained on February 13, 2015 with three hand nets (two of 100 and one of 200 µm) in shallow areas (depth: 60–120 cm) of the lagoonal system, particularly at Isla Tortuga ; male fifth leg exopod unsegmented, armed with five elements, three setae, two spines. Sixth leg with two setae in female, and with two setae plus short spiniform process in male.First pedigerous somite free, posterolateral margins of second and third pedigerous somites slightly produced. Caudal ramus with six setae, seta I absent. Female antennule 10-segmented, 6PageBreakTaxon classificationAnimaliaCyclopoidaCyclopinidaehttp://zoobank.org/D569F55D-9C92-41E5-842C-C711103C014D18°44'29.3"N;91°29'44.6"W), collected February 13, 2015 by R. J. Almeyda-Artigas, C. Lara-Bautista, and C. Chamorro-García. Allotype male, dissected, same site, date, and collectors (ECO-CHZ-09299). Paratypes. Two adult females, dissected, slides (ECO-CHZ-09300), 6 adult females, undissected, ethanol-preserved, vial (ECO-CHZ-09301); 3 adult males, undissected, ethanol-preserved, vial (ECO-CHZ-09302). One female, one male, specimens undissected, ethanol-preserved, same locality and collectors (USNM-1283307). One female and 2 males, all used for SEM analysis. Other material examined included +25 undissected adult and juvenile specimens, deposited at CHUX .Holotype. Adult female, dissected, mounted in glycerin sealed with Entellan (ECO-CHZ-09298), Laguna de Términos, Campeche, Mexico (n=11) 350–400 µm, average: 372 µm. Body cyclopiform  of type specimens : 313–328 µm, average 321 µm. Body cyclopiform, smaller than female and slightly narrower , state of Campeche, Mexico, southern Gulf of Mexico.Laguna de Términos , mangrove areas and zones with no vegetation. It is a shallow system, (average depth = 2.5 m). The lagoon receives freshwater input from several rivers. Most of its bottom is covered by sediments of sand, silt and clay with a high content of calcium carbonate mainly in the vicinity of Boca de Puerto Real (between 50 and 70%).The lagoon has a length of 70 km and 30 km at its widest sector. It has extense coverage of seagrass beds  absence of modified brush-like seta on the 4th mandibular exopodal segment; 2) maxillule exopod with stout setal elements and no brush-like setae; 3) presence of modified seta on the fourth leg endopod; 4) fifth leg exopod armed with three elements in the female and five in the male; 5) outer exopodal spine of leg 5 blunt in both sexes; 6) male sixth leg with two outer slender setae and inner spiniform process; 7) intercoxal sclerite of first swimming leg with two medial spiniform processes on distal margin. The new genus diverges from Heptnerina in the lack of an endopodal lobe in leg 5, in the presence of a single antennary exopodal seta vs. two setae present in Heptnerinaconfusa  the short, stout distal setae of the exopodal segment of the maxillule  the ptnerina , fig. 1Eptnerina , not desPageBreakCyclopina, we performed a comparison with the most closely related species of this genus. Only a few species of Cyclopina have a female leg5 with the inner spine of the exopodal segment less than half the length of the outer spine, the latter being longer than the segment itself  in the new species.The new species differs from aesilis , fig. 2BMexiclopinacampechana sp. n. differs from Cyclopinaamericana in body shape, with the third and fourth pedigerous somites strongly produced posteriorly, the process of the fourth somite reaching well beyond the posterior margin of the fifth pedigerous somite , the caudal rami are clearly longer than the anal somite and have a length/width ratio of 2.6 , whereas these spines are distributed on segments 9-12 in the new species , Cyclopinaarenosa Lotufo, 1994, Cyclopinacaissara Lotufo, 1994, Cyclopinacaroli Lotufo, 1994, Cyclopinamediterranea Steuer, 1940, Cyclopinadorae Lotufo, 1994, Cyclopinayutimaete Lotufo, 1994, and a species of the new genus, Mexiclopinacampechana. The new species is the first cyclopinid described from Mexico, and represents the first record of the family in the Gulf of Mexico (see Cyclopinacaissara in the Mexican Pacific coast (This work increases the number of species of cyclopinids known from the Americas . Recordsxico see . After tic coast , it is t


Purpose. The aim of this study was to evaluate the 30-year progress of research on exhaled breath condensate in a disease-based approach. Methods. We searched PubMed/Medline, ScienceDirect, and Google Scholar using the following keywords: exhaled breath condensate (EBC), biomarkers, pH, asthma, gastroesophageal reflux (GERD), smoking, COPD, lung cancer, NSCLC, mechanical ventilation, cystic fibrosis, pulmonary arterial hypertension (PAH), idiopathic pulmonary fibrosis, interstitial lung diseases, obstructive sleep apnea (OSA), and drugs. Results. We found 12600 related articles in total in Google Scholar, 1807 in ScienceDirect, and 1081 in PubMed/Medline, published from 1980 to October 2014. 228 original investigation and review articles were eligible. Conclusions. There is rapidly increasing number of innovative articles, covering all the areas of modern respiratory medicine and expanding EBC potential clinical applications to other fields of internal medicine. However, the majority of published papers represent the results of small-scale studies and thus current knowledge must be further evaluated in large cohorts. In regard to the potential clinical use of EBC-analysis, several limitations must be pointed out, including poor reproducibility of biomarkers and absence of large surveys towards determination of reference-normal values. In conclusion, contemporary EBC-analysis is an intriguing achievement, but still in early stage when it comes to its application in clinical practice. Exhaled breath condensate (EBC) analysis is a recent, noninvasive method of detecting biomarkers, mainly coming from the lower respiratory tract. EBC is collected during quiet breathing, as a product of cooling and condensation of the exhaled aerosol . It is aThere is detailed literature focused on detection of inflammatory markers, which reflect the state of chronic airways diseases such as chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis (CF) . HoweverThere is undoubtedly an increasing trend of biomedical research towards development of noninvasive, subject-friendly respiratory function testing. Furthermore, there is rapidly increasing number of original research articles regarding potential clinical applications of EBC-analysis, using various methodologies. Therefore, we aimed to present a systematic review of the literature describing the 30-year progress of research on exhaled breath condensate in a disease-based approach, with special reference to the potential clinical applications and limitations of the contemporary EBC-analysis.We searched the PubMed/Medline, ScienceDirect, and Google Scholar databases using the following keywords: exhaled breath condensate, EBC, biomarkers, pH, asthma, gastroesophageal reflux, GERD, chronic obstructive pulmonary disease, smoking, COPD, lung cancer, mechanical ventilation, non small lung cancer, NSCLC, cystic fibrosis, pulmonary arterial hypertension, PAH, idiopathic pulmonary fibrosis, interstitial lung diseases, obstructive sleep apnea, OSA, and drugs.We found 12600 related articles in total in Google Scholar, 1807 in ScienceDirect, and 1081 in PubMed/Medline, published from 1980 to October 2014. We used the following as exclusion criteria: (i) studies available only in languages other than English, (ii) studies published only in the form of abstract, (iii) studies published in journals without impact factor, and (iv) studies applying EBC-analysis in veterinary medicine. Furthermore, we preferably used original investigation articles instead of review articles, when possible.207 original investigation papers and 21 review articles where eligible after applying the abovementioned criteria. 47 of them refer to methodological and technical aspects and 181 refer to the diagnostic aspects of EBC-analysis.EBC collection is a simple, noninvasive technique, requiring only quiet breathing of the subject, via a system of exhaled air cooling. After applying a nose-clip, the subject breaths quietly for 10 minutes through a special mouthpiece with a salivary trap and a single-way valve attached, diverting the exhaled airflow through a Teflon or polypropylene tube inside a cooling container. There, exhaled air in the form of droplets is converted to exhaled breath condensate (EBC) 4 have alith COPD  and thus2 (PGE2), have also been studied in asthmatic populations. 8-isoprostane levels have been found increased in adult and pediatric populations with stable asthma, but they were not significantly reduced after inhaled or oral corticosteroid treatment [2 concentration was not increased significantly among both children and adult nonsmokers with stable asthma [2 have been found significantly increased in adult smokers with stable asthma [2 possibly reflects smoking status in asthmatic patients.EBC-concentrations of other eicosanoids, such as 8-isoprostane and prostaglandin Ereatment , 94, 95.e asthma , 83, 95.e asthma . Therefoγ were identified among pediatric asthmatic population, changes indicative of Th2-mediated inflammation.There is also detailed literature regarding EBC-interleukins (ILs) concentration in patients with asthma, detecting increased levels of IL-4 –102, IL-α, TGF-β, MIP1α , MIP1β , and RANTES was identified among adult steroid-naïve asthmatic patients. Furthermore, EBC-RANTES increase was significantly correlated with both FEV1 reduction and airways resistance increase, while increased levels of EBC-TNF-α and TGF-β were significantly correlated with nonspecific bronchial hyperresponsiveness .Another interesting biomarker involved in bronchoconstriction mechanism is RANTES. It is a chemotactic agent for eosinophils, T-lymphocytes, and mast cells, which induce histamine and Cys-LTs secretion from mast cells and eosinophilic cationic protein from eosinophils, causing bronchospasm in patients with asthma . In a stIn other studies by Zietkowski et al. , 108 sigExtensive proteomic analysis of EBC has also revealed other biomarkers related with pathophysiological mechanisms involved in asthma. In a study of Bloemen et al. , signifiAsymmetric Dimethylarginine (ADMA) is an L-arginine analog, which inhibits nitric oxide (NO) synthesis, contributing to asthma pathogenesis . In a study of Carraro et al. , signifiEotaxin-1 (CCL11) is a chemokine for the eosinophils, inducing both their accumulation to the airway wall and their activation and degranulation. In a study by Zietkowski et al. , signifiIn another study by Zietkowski et al. , signifiEBC genomic analysis is also becoming an intriguing area of research, especially when combined with proteomic analysis. In a prospective study of Klaassen et al. , interceEBC has been also studied in asthmatic patients with GERD  in order to assess the airway acidification. When the acid refluxes up to the pharynx, it enters the airways causing epithelial damage due to acid injury and therefore worsening of asthma symptoms. In a study investigating the determinants of EBC-pH in a large population with asthma, GERD symptoms were associated with low EBC-pH . Howeverp = 0.006). Moreover, both asthmatic and GERD children had lower total magnesium  with the later demonstrating a negative correlation  between magnesium levels and pH values. Additionally, in a study by Fitzpatrick et al. [Banovic et al.  demonstrk et al.  EBC-pH fk et al. , patientx (nitrites and nitrates) levels were lower in GERD patients compared with non-GERD patients (13.7 μmol/L versus 21 μmol/L). A negative correlation between reflux index and NOx levels in EBC was also demonstrated . This finding indicates that longer periods of reflux may cause more epithelial damage and subsequently a drop in NOx levels. Based on the above, there is no specific EBC biomarker indicative of airway acidification due to GERD in asthmatic patients.Pepsin was also used as a marker of GER-related aspiration in patients with GERD and GERD-induced respiratory problems. Pepsin is normally present in gastric secretions but not in bronchial tree, so an increase of EBC-pepsin would be expected in GERD patients. However, in a study by Soyer et al. , pepsin Summarizing EBC alterations in asthma, it can be observed that the main problem of existing studies is the poor reproducibility of results. This could be attributable to the lack of specific analysis of the different inflammatory pathways in the vast majority of studies. Asthmatics with neutrophilic pattern of inflammation would possibly have mediators in their EBC different from asthmatics with an eosinophilic pattern and probably would have a different response to therapy. Therefore, existing knowledge is insufficient regarding the detection of specific markers for asthma.An additional methodological issue is that, in the great part of studies, biomarkers have been studied only in stable asthma . More stImmediate effects of smoking a single cigarette (CS) have been extensively studied. Some early studies detected nonsignificant changes of EBC-pH among healthy smokers , 122, whμM to 29,8 μM) [μS/cm to 90 μS/cm) [Significant increase of EBC-nitrates concentration  , EBC-8-i29,8 μM) , and EBC0 μS/cm)  immediat0 μS/cm) .In regard to EBC-nitrites, S-nitrosothiols, nitrotyrosine , ammoniuγ, due to either decreased production or decreased delivery, as an effect of high levels of carbon monoxide (CO), which is a potent anti-inflammatory agent.A recent study investigating the immediate effects of 30-minute waterpipe smoking (WPS) on EBC  detectedIt has been shown that there is no significant difference in EBC-pH among mild healthy smokers (<10 pack-years) and healthy never-smokers . Contrarily, EBC-pH of asymptomatic smokers with cigarette consumption higher than 10 pack-years has been found significantly reduced, presenting values close to the ones of COPD patients  , 126. Fu4 [α [α levels are significantly correlated with Forced Vital Capacity (FVC) and FEV1 values [4 levels [Inflammatory agents, including EBC-IL-6, LTB4 , IL-8 [14 , 128, an4 [α , have be1 values , 129, wh4 levels , suggest2O2 were identified at 120 minutes after exposure and remained increased for the next 120 minutes.Little is known regarding SHS effects on EBC. In a recent study , signifiThere are still a limited number of studies investigating the effects of cigarette smoking on EBC. However, it has been proved that even a single cigarette smoking causes significant alterations involving inflammatory pathways. Furthermore, there is a different inflammatory response (both short- and long-term) of the respiratory system to cigarette smoking among smokers with and without allergic rhinitis. This finding indicates that there is a high possibility that the same could apply to smokers with and without asthma. Carefully designed studies are needed in order to investigate this possibility and the susceptibility of these patients to develop COPD in the future , cytokines  were identified as the dominant protein type (62%), but INF-α, INF-γ, TNF-α, and C3 complement fraction were detected. In healthy controls, levels of the abovementioned inflammatory mediators were negligible and INF-α and IL-12 were not detected. Interestingly though, haemoglobin β-chain was detected in healthy smokers, probably reflecting haemolysis due to smoking induced oxidative stress. Additionally, considerable amount of enzymes (8%) which were identified in healthy controls, was absent in COPD patients. Furthermore, COPD versus A1AD comparative EBC proteomic analysis, demonstrated significant differences. In A1AD-patients, α1-antitrypsin was not detected, but IL-1α and lysozyme-C, which were undetectable in the other COPD patients, were identified. Other types of proteins, including surfactant proteins  and S100 calgranulin A and calgranulin B , were equally identified in both healthy controls and COPD patients, but not in A1AD-patients. The abovementioned differences in proteomic profile may prove to be a useful tool in regard to identification of pathogenetic mechanisms involved in COPD.A special characteristic of airway inflammation in COPD is its mediation by neutrophils, activated through interaction with cytokines, especially interleukins (ILs). Indeed, significantly increased levels of IL1ble COPD . Furtherble COPD , where p4 [2O2 have been detected, followed by significant decrease after antibiotic treatment with cephalosporins and macrolides [EBC-pH has been found significantly reduced during AECOPD (5.57 ± 0.07) and significant increase following treatment has also been identified (6.04 ± 0.08) . Additio4 , 147, 8-crolides .β, IL-8, IL-10, IL-12, TNF-α [In regard to classic inflammatory markers, significantly increased levels of EBC-IL-6 , 148, IL2, TNF-α , and EBC2, TNF-α  have beeα1-antitrypsin-antiprotease  [4 was found significantly increased in patients with both stable COPD and AECOPD, but EBC-GRO-α (growth related oncogene-α) was found significantly increased only in AECOPD patients. Moreover, EBC-LTB4 and EBC-GRO-α reduced significantly after recovery from AECOPD. Based on the above, GRO-α, a strong neutrophil chemoattractant, may be pathogenetically involved in AECOPD and further investigation is necessary in order to evaluate its role in the follow-up of such patients.EBC proteomic analysis in patients with infectious AECOPD revealed significant increase of le COPD) , metallole COPD) , reflectle COPD)  which enOther agents have been also proposed as prognostic factors of AECOPD, including increased levels of solute-HLA-1 and solute-CD95 . ContrarEBC genomic analysis has also given interesting results. Nucleic acids of both viral and bacterial agents, including influenza virus and respiratory syncytial virus, but also Legionella pneumophila, have been detected in EBC of AECOPD patients, while EBC analysis has been proved more sensitive in detection of Legionella pneumophila compared to induced sputum .Summarizing current knowledge in COPD, there are numerous studies investigating a variety of EBC biomarkers in both stable and exacerbated COPD, without promising results regarding a reliable predictor of exacerbation. Furthermore, no study demonstrates specific inflammatory pathways that can differentiate between and ideally predict COPD clinical phenotypes . Further research is needed in order to identify the specific fingerprint of each phenotype which would lead to different treatment approaches  and all the abovementioned biomarkers were significantly correlated with the tumor diameter, estimated through CT. Additionally, Carpagnano et al. [α and VEGF in patients with T3 and T4 NSCLC.In regard to inflammatory markers, Carpagnano et al.  first deo et al. , increaso et al.  have also et al.  demonstrHowever, many of the abovementioned inflammatory markers have also been found in increased levels among patients with COPD and asthma, as previously mentioned.Kullmann et al.  demonstrβ, IL-6, IL-8, TNF-α, and sICAM-1, have been measured on postoperative days  in patients who have undergone lobectomy or pneumonectomy [α remained increased from day 1 to day 7, IL-8 peaked on day 3, IL-6 peaked on day 1, and IL-1β levels increased after day 1. There was no significant association, however, between levels of EBC-IL-1β, IL-6, IL-8, TNF-α, and sICAM-1 and the extent of lung cancer resection (lobectomy versus pneumonectomy) [Furthermore, significantly increased levels of proinflammatory mediators, including EBC-IL-1onectomy . sICAM-1nectomy) .In a study by Gessner et al. , signifiFurther EBC proteomic analysis has given numerous promising markers, still requiring further evaluation. Carpagnano et al.  suggesteIn another study by Carpagnano et al.  high conCheng et al.  first idZou et al.  suggeste p53 gene in patients with lung cancer, which were undetectable in healthy controls. Carpagnano et al. [ 3p chromosome , but also loss of heterozygosity, both in healthy smokers and NSCLC patients. In another study conducted by the same team [Increasing number of studies demonstrated impressive results after applying genomic analysis of EBC. Gessner et al.  detectedo et al.  describeame team , comparaame team , the sam D3S1289 demonstrated negative prognostic value for lung adenocarcinoma, but it was also related with poor prognosis of patients with NSCLC, regardless of the stage of the disease. Moreover, microsatellite instability and loss of heterozygosity in D3S2338 were associated with poor survival of patients with squamous cell lung cancer [An additional conclusion of the same authors was that the amount of detected 3p mutations was of negative prognostic value, since it was associated with poor survival of patients with lung cancer. More specifically, loss of heterozygosity ing cancer . 19q, resulting in ERCC-1 and ERCC-2 genes, which were present in 25% of NSCLC patients and 16% of smoking subjects, but they were undetectable in nonsmokers [Microsatellite EBC-DNA mutations have also been detected in chromosomensmokers . miRNA-21 and particularly reduced levels of miRNA-486 in patients with NSCLC and proposed their use as first-line screening test for diagnosis of NSCLC.Mozzoni et al.  detected KRAS oncogene (mutation located in codon 12) were significantly reduced in EBC of patients with NSCLC after tumor resection, by 1.3 times at 7th postoperative day and by 3,7 times at 30th postoperative day. In 10 patients the specific KRAS mutation was undetectable at 30th postoperative day.Another impressive finding regarding lung cancer pathogenesis has been identified in a study of Kordiak et al. . Specifi RASSF-1A gene compared to current smokers and nonsmokers and methylation of the DAPK and PAX5β genes was significantly correlated with NSCLC. In a latter study, Xiao et al. [ p16 gene in EBC of patients with NSCLC.DNA methylation is a common biochemical process, essential for normal development through various genomic functions, including X-chromosome inactivation, but also suppression of repetitive elements. However, aberrant DNA methylation patterns (hyper and hypomethylation) have been associated with numerous human malignancies. In a study of Han et al. , ex-smoko et al.  detected Aspergillus niger, A. Ochraceus or Penicillium spp., whereas fungal colonization was absent in EBC of the healthy control population [HPV (human papillomavirus) infection has been proposed as the main infectious agent involved in the pathogenesis of lung cancer, but it has been demonstrated that fungal infections could also be involved, through production of carcinogenic mycotoxins. In a study by Carpagnano et al. , HPV genpulation .In regard to lung cancer, there is rapidly increasing number of studies on EBC biomarkers applying contemporary techniques, especially proteomic and genomic analysis. The main goal of these studies was to demonstrate biomarkers for early diagnosis of LC. The most promising results came from genomic analysis in studies enrolling heavy smokers and NSCLC patients in which common DNA alterations were identified. This finding is promising and indicates that such alterations may be of prognostic value. However, methodological issues should be addressed: current knowledge is based on studies enrolling patients with NSCLC and there is a relative lack of studies enrolling patients with small cell lung carcinoma, which is also a prevalent disease. Additionally, large scale screening and prospective studies are needed, enrolling asymptomatic patients with all histological types of LC, in order to evaluate if EBC markers could identify a preclinical stage of the disease  patients as a noninvasive alternative to evaluate airway inflammation, predict the development of VAP (ventilator associated pneumonia), and assess the ventilator associated damage and lung distention. EBC biomarkers have been studied not only in MV patients with ALI/ARDS and VAP but also in MV patients without respiratory failure and ALI/ARDS .r = −0.636, p = 0.048) supporting the hypothesis that positive-pressure ventilation induces pulmonary inflammation and physical disruption of tissues and cells, resulting in airway acidification [Acidic pH has been found in MV patients both with , 181 andfication . Additiofication  and retufication . Howeverfication , EBC pH 2a, was found significantly higher in MV patients with ALI/ARDS (87 ± 28 pg/mL) compared to healthy subjects intubated during minor surgical procedures (7 ± 4 pg/mL) [2a was described after salbutamol inhalation along with a tendency to decrease nitrosative species [A compound formed by in vivo peroxidation of arachidonic acid membrane, 8-iso-PGF4 pg/mL) . Additio species . These f species  8-isopro2O2 (1.68 ± 0.35 mumol/L) compared to others who did not (0.34 ± 0.08 mumol/L) [β, IL-10, IL-12p70, and TNF-α, are also elevated in EBC of MV patients both with and without ALI/ARDS, revealing the airway inflammation in such patients [2O2 have been found significantly elevated in MV patients who underwent lobectomy, but this change was not significant in MV patients after coronary artery by-pass graft surgery, probably due to the higher risk of ALI development after lobectomy [2− increased linearly with VT and correlated well with expiratory minute volume, possibly due to mechanical stress of the remaining open lung units in injured lungs.The hypothesis that neutrophil activation and oxidant production are involved in the pathogenesis of ARDS is supported by several studies. MV patients who eventually developed ARDS demonstrated higher levels of Hmumol/L) . Other ipatients , 189, wiobectomy . In a stobectomy  EBC-NO22−/NO3− were found elevated in MV patients without ALI (mean 66.22 μM) compared to healthy subjects (mean 15.06 μM) and their levels had negative correlation with dynamic compliance  and positive correlation with respiratory rate  [NO= 0.029) . This fiCytokeratins 2, 9, and 10 were also detected in EBC of MV patients with respiratory failure. PEEP, PIP, and LISS (lung injury score) were positively correlated with the levels of EBC-cytokeratins , revealing a relationship between these biomarkers and the ventilator associated damage to the lung parenchyma . sTREM-12O2, 3-nitrotyrosine, and nitric oxides (NOx) in patients with IPF, compared to healthy controls.Chow et al.  demonstrAdditionally, in a study by Montesi et al. , 9 diffeIn a study by Tate et al. , signifiA significant increase of EBC-nitrotyrosine was identified in patients with stable CF but there was no significant correlation between FeNO and EBC-nitrotyrosine levels . HoweverIn a study by Horak et al. , increas4 were identified during CF exacerbation, especially when it was induced by Pseudomonas aeruginosa infection. Moreover, significant reduction of the abovementioned inflammatory markers was detected after treatment with antibiotics [γ concentration among asthmatic and CF paediatric populations, whereas EBC-IL-5 and TNF-α were detectable only in the CF-population.In regard to classic inflammatory markers, Cunningham et al.  demonstribiotics , 204. Inibiotics , no signα1 were detected. Additionally, He et al. [In a study by Warwick et al. , signifie et al.  demonstrIn a study by Carratu et al. , signifiMansoor et al.  applied Piotrowski et al.  applied β1 were identified in patients with sarcoidosis compared to healthy controls, whereas no significant difference in levels of EBC-angiotensin converting enzyme was detected between the two populations.In a study by Ahmadzai et al. , signifi4 and Cys-LTs were found significantly increased in children with Apnea-Hypopnea Index (AHI) > 5/h and they were associated with severity of the disease [2O2 levels were found in pediatric population with severe OSA. This finding is indicative of the intense, night long oxidative stress [Studies in pediatric populations with OSA  detected significantly increased EBC-IL-6 and 8-isoprostane concentrations compared to healthy controls. The abovementioned biomarkers were significantly correlated with clinical score and cardiac dysfunction . Additio disease . Furthere stress , which ie stress .α, IL-6 [2O2 [2O2 levels have been identified after 6 weeks of CPAP therapy (from 450 ± 130 nmol/L to 294 ± 110 nmol/L) [Studies enrolling adult population with OSA have also detected a significant raise of EBC markers of oxidative stress. More specifically, significantly increased levels of 8-isoprostane –217, TNFα, IL-6 , 217, H2L-6 [2O2 , and redL-6 [2O2 . In addi nmol/L) .It must be pointed out that EBC-ICAM-1 and IL-8 have been found equally increased both in healthy obese adults and in obese adults with OSA , implyinNielepkowicz-Goździńska et al. detected significantly increased IL-8 , IL-6, a2O2 concentration has been demonstrated in patients with severe uremia who have undergone haemodialysis compared to healthy controls [Significantly increased EBC-pH, nitrites, and nitrates have been demonstrated in patients with end stage CRD. Moreover, levels of the abovementioned biomarkers decreased after haemodialysis . Additiocontrols .EBC proteomic analysis has also been applied in toxicology, for detection of several compounds, including drugs. More specifically, EBC analysis through mass spectrometry can potentially be used for detection of drug metabolites concentrations during patient follow-up . A well-This study is a systematic review of the literature, presenting both the theoretical principles and the potential clinical applications and limitations of the contemporary EBC analysis.Bibliographic investigation detected a prominent research activity on that field, resulting in rapidly increasing number of innovative papers published every day, concerning almost all the areas of modern respiratory medicine.Study of exhaled air has been an area that has garnered interest since the beginning of medical science and investigation. From the Hippocratic medicine (uremic breath)  to the mEBC collection and analysis are the most complete product of that long-term research. It is undoubtedly an advantageous technique, when noninvasive, effortless, and painless collection of respiratory biological material is concerned. Furthermore, it is innovative, since EBC is proved to be a well manageable matrix for application of all the technologically advanced methods of contemporary biomedical research. In this way, EBC-analysis produces findings expressing a considerable part of proteomic, metabolic, and genomic fingerprint.However, in order for a new technique to be introduced in clinical practice, high quality of standardization is required, both in sampling and in interpretation. In regard to sampling and EBC collection procedures, there are reliable guidelines , which hIn regard to standardization of EBC-analysis, it must be pointed out that there is absence of large cohorts towards determination of normal-reference values of EBC-biomarkers  and therThere is also a lack of screening and prospective studies enrolling asymptomatic patients, in order to evaluate if EBC markers could identify a preclinical lung disease condition. Such studies, demonstrating results of clinical significance, could potentially become the brink of a major breakthrough in respiratory medicine. Such results, combined with existing portable EBC collection equipment and the easy, noninvasive sampling, could make EBC an exceptionally promising screening method for early detection of lung disease.Summarizing, it seems that EBC-analysis is an intriguing achievement of biomedical research, but still in early stage when it comes to its application in clinical practice. Current knowledge, however, creates optimism regarding the potential of EBC-analysis to reveal disease-specific fingerprints, as the sum of many mediators evaluated with new technologies, able to characterize a disease or its exacerbation.


Chronic obstructive pulmonary disease (COPD) and lung cancer are leading causes of deaths worldwide which are associated with chronic inflammation and oxidative stress. Lung cancer, in particular, has a very high mortality rate due to the characteristically late diagnosis. As such, identification of novel biomarkers which allow for early diagnosis of these diseases could improve outcome and survival rate. Markers of oxidative stress in exhaled breath condensate (EBC) are examples of potential diagnostic markers for both COPD and non-small-cell lung cancer (NSCLC). They may even be useful in monitoring treatment response. In the serum, S100A8, S100A9, and S100A12 of the S100 proteins are proinflammatory markers. They have been indicated in several inflammatory diseases and cancers including secondary metastasis into the lung. It is highly likely that they not only have the potential to be diagnostic biomarkers for NSCLC but also prognostic indicators and therapeutic targets. Chronic obstructive pulmonary disease (COPD) and lung cancer are the leading causes of deaths worldwide which are associated with cigarette smoking. COPD is a preventable and treatable disease characterised by progressive, irreversible airflow obstruction resulting from chronic airway inflammation –3. It isDespite significant advances in 5-year survival rates of other cancers, that of lung cancer remains low at 15.6%  . Even moMuch research has thus been directed towards the hope of finding new, simple, and minimally invasive biomarkers of early diagnosis or screening for COPD and lung cancer. Exhaled breath condensate and serum samples are two such examples.It is well established that both COPD and lung cancer are usually due to tobacco smoking –22. The α 1-antitrypsin deficient carriers) also carries an elevated risk of lung cancer by 2.4-fold [Apart from smoking, COPD is itself an independent risk factor , 18, 24 2.4-fold . It is also known that COPD patients are at increased risk of developing squamous cell carcinoma with a worse prognosis as they not only develop higher grade tumours but also suffer from a higher rate of recurrence , 27, 28.COPD and lung cancer are both associated with chronic inflammation and oxidative stress, , 29, 30 15 oxidants particles and approximately 4700 different compounds [Oxidants can be generated exogenously or endogenously. Exogenous sources of oxidants include tobacco smoke, infections, and pollutants (such as ozone and nitrogen dioxide) , 32. Of ompounds , 31, 33.ompounds –37. They•O2−), hydroxyl radicals (•OH), and hydrogen peroxide (H2O2) while RNS includes nitric oxide (•NO), nitrogen dioxide, and peroxynitrite (ONOO−) [2−) and nitrate (NO3−).Under normal physiological conditions, oxidants have a role in growth regulation, intracellular signaling, and host defence (inflammation) against infection . They co (ONOO−) . SuperoxReactive species are very unstable and potentially damaging as their unpaired electrons can exert injurious effects by oxidising DNA, proteins, and lipids , 39.κB (NF-κB) and activator protein 1 (AP-1) in airway epithelial cells and macrophages [α (TNF-α) [The introduction of oxidants into the lung from tobacco smoking activates the innate immune cells such as lung epithelial cells whereby damage-associated molecular patterns (DAMPs) are released from injured cells . Followirophages , 45. The (TNF-α) , 45–47.  (TNF-α) , 32, 45. (TNF-α) , 29.α-1-protease-inhibitor (α-1-PI) and antileukoprotease [Following recruitment, neutrophils and macrophages release neutrophil elastase and matrix metalloproteinases-9 which are proteases that degrade lung matrix elastin and collagen , 36, 44.protease  are inacprotease , leadingprotease , 29. In addition, injuries during inflammation also lead to goblet cell hyperplasia and squamous metaplasia. This impairs mucociliary clearance, and inflammatory mediators accumulate in the airways as a result, which again amplifies inflammation . The actApart from initiating inflammation, oxidants also readily attack polyunsaturated fatty acids of cell membranes to form lipid peroxidation products (LPPs) such as hydroperoxides, endoperoxides, and aldehydes including ethane, pentane, isoprostanoids, malondialdehyde, and 4-hydroxy-2nonenal which are even more reactive , 46, 48.Under normal conditions, oxidants are counterbalanced by antioxidants which consist of enzymes  and nonenzymatic free radical scavengers  . In response to elevated levels of oxidants, local antioxidants such as superoxide dismutase, catalase, glutathione associated enzymes, and manganese superoxide dismutase may increase in an attempt to counter the insult –51. The Chronic inflammation increases cell turnover and replication errors , 53–55. Proofing mechanisms of DNA may attempt to repair or remove the damaged DNA via direct repair, double-strand break repair, cross-link repair, nucleotide excision, and base excision . When daApart from direct DNA damage, oxidants also promote tumorigenesis by direct reaction with proteins (protein peroxidation) to impair DNA reparative enzymes such as DNA polymerase . Exhaled breath condensate (EBC) is the cooling of exhaled gas to gain insight into the composition of extracellular lining fluid (ELF) and soluble exhaled gases , 66–68. EBC has several advantages as an investigational technique. It is noninvasive , inexpensive, easy to collect, and also easily repeatable without causing airway inflammation or dysfunction  , 72, 73.EBC has the potential to be employed in the screening and diagnosis of COPD and lung cancer, disease phenotyping, exacerbations, and treatment response monitoring as well as disease severity measuring and prognosis indicating , 72, 77.EBC, however, has a number of limitations which include dilution by water vapour, nonsite specificity, saliva contamination and variable reproducibility. With >99.9% of EBC comprising water vapour , concentAs a result of the collection pathway, EBC also consists of nebulised fluid droplets from the alveoli, bronchi, and mouth, each with an unknown relative contribution . This noWhile the volume of EBC is reproducible, levels of biomarkers in EBC may vary, and this gives rise to problems in repeatability and reproducibility , 81, 83.A range of biomarkers have been studied in EBC of COPD and lung cancer patients. The results are as shown in α-I-antitrypsin can aid in distinguishing between healthy subjects or COPD patients [In addition to EBC, the serum protein profile is another easily collected yet cost-effective tool in detecting and monitoring lung cancer , 84, 85.patients . As suchThe S100 proteins are a family of more than 20 low molecular weight acidic proteins of 10–12 kDa which are calcium-binding, and they belong to the EF hand proteins subfamily –92. TheyS100A8 is also known as calgranulin A or myeloid-related protein 8 while S100A9 is also known as calgranulin B or myeloid-related protein 14. While much of the literature suggests that the S100A8 and S100A9 are proinflammatory, a body of research presents an opposing view. It is possible that the opposing effects of the calgranulins are concentration dependent, being proinflammatory at low concentrations and anti-inflammatory at high concentrations , 99. A100A8 and S100A9 are believed to be anti-inflammatory by being preferentially oxidized, thereby scavenging ROS/RNS. Oxidative modifications by ROS/RNS and posttranslational modifications such as S-nitrosylation and S-glutathionylation are proposed to be the regulatory switches which activate such anti-inflammatory properties , 99. κB transcription pathway, subsequent generation of downstream proinflammatory cytokines, and recruitment of inflammatory mediators such as neutrophils and monocytes in a positive feedback loop  , the S10 S100A8 and S100A9 are expressed by cells of myeloid origin, making up 40%–50% of their cytosolic content. Cells expressing S100A8 and S100A9 include granulocytes , monocytes, and early differentiation stages of macrophages , 110. S1γ) and high levels of ROS, peroxynitrite, and nitric oxide [MDSCs are precursors of macrophages, granulocytes, and dendritic cells  which inic oxide . β, interleukin-6, prostaglandin E2), chemokines, tumour-derived growth factors, and myeloid-related proteins such as S100A9 and S100A8 [κB pathway and mitogen-activated protein kinase [In tumorigenesis, MDSCs are attracted from bone marrow to peripheral blood by inflammatory cytokines (e.g., interleukin-1d S100A8 , 117. Thn kinase . Hence, n kinase , 123.S100A8/A9-positive myeloid cells are not only early infiltrating cells in the inflammatory process  but are In lung cancer, a recent study found that the expression of S100A8 and S100A9 is increased in patients with NSCLC . NSCLC pβ, and TNF-α by primary tumours before metastasis occurs [κB pathway in promoting CD11b+ myeloid cell accumulation [Lastly, S100A8 and S100A9 also play a role in cell proliferation and metastasis of primary tumours into the lung . Their es occurs , 132. S1s occurs , 110. Thmulation . In addimulation , 131. ThEarly Diagnosis, Predicting Prognosis, and Personalised Medicine. EBC and serum are noninvasive and minimally invasive techniques which are cost effective and easily sampled. If EBC markers of oxidative stress and serum proinflammatory S100 proteins or other candidate entities are diagnostic for COPD and NSCLC, it could greatly improve survival outcome by allowing early diagnosis and thus treatment. As many NSCLC patients do not behave as predicted based on tumour staging, new markers are also needed to more accurately predict prognosis . PrognosFurthermore, it will be beneficial to discover more specific and sensitive serum biomarkers for lung cancer as well as to personalise anticancer therapies. For instance, knowing the reduced overall survival of patients with an overexpression of S100A9 may not only identify patients who are at high risk of a poor outcome  but alsoThe S100 proteins have a great potential to be the new diagnostic tumour markers, prognostic predictor, and possibly therapeutic targets for NSCLC.


We found higher serum sCD154 levels (p < 0.001) in non-surviving (n = 26) than in surviving MMCAI patients (n = 24). Multiple binomial logistic regression analysis showed that serum sCD154 levels >1.41 ng/mmL were associated with 30-day mortality . Conclusions: The new more important finding of our study was that serum sCD154 levels in MMCAI patients were associated with mortality.Background: CD154 and its soluble counterpart (sCD154) are proteins of the tumor necrosis factor (TNF) family and exhibit proinflamatory and procoagulant properties. Higher circulating sCD154 levels have been found in ischemic stroke patients than in controls. However, the association between circulating sCD154 levels and mortality in ischemic stroke patients has not been reported, and was the focus of this study. Methods: This was a multicenter, observational and prospective study carried out in six Spanish Intensive Care Units. We measured serum sCD154 from 50 patients with severe malignant middle cerebral artery infarction (MMCAI), defined as Glasgow Coma Scale (GCS) lower than 9, at the moment of the severe MMCAI diagnosis and from 50 healthy controls. The end-point of the study was 30-day mortality. Results: We found higher serum sCD154 levels in patients with severe MMCAI than in healthy controls ( Ischemic stroke is an important cause of disability, mortality and resources consumption . CD154 iHigher CD154 platelet expression in ischemic stroke patients than in controls ,11,12,13This is a multicenter, observational, prospective study carried out in six Intensive Care Units of Spain. The study was approved by the Institutional Review Board of the six participant hospitals: Hospital Universitario de Canarias , Hospital Universitario Nuestra Señora de Candelaria , Hospital General de La Palma , Hospital Clínico Universitario de Valencia , Hospital Insular , and Hospital Universitario Dr. Negrín . Written informed consent from the patients or from their legal guardians was obtained.We included 50 patients with severe malignant middle cerebral artery infarction (MMCAI) and 50 healthy volunteer control subjects. Severity of MMCAI was classified according to Glasgow Coma Scale (GCS) , and we 2), PaO2/pressure of arterial oxygen/fraction inspired oxygen (FI02) ratio, bilirubin, creatinine, hemoglobin, GCS, lactic acid, platelets, international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen, Acute Physiology and Chronic Health Evaluation II (APACHE II) score [The following variables were recorded for each patient: sex, fibrinolityc therapy, decompressive craniectomy, age, temperature, sodium, glycemia, leukocytes, pressure of arterial oxygen (PaOI) score . The endWe recollected blood samples from 50 patients with severe MMCAI at the moment of the diagnosis to measure serum sCD154 levels, serum TNF-alpha levels, and plasma tissue factor (TF) levels. In addition, there were recollected blood samples from 50 healthy controls to measure serum sCD154 levels. To avoid the possible dispersion of serum level results, all the samples were processed at the same time and in the same laboratory, at the end of the recruitment process.g for 15 min. The serum and plasma were removed and frozen at −80 °C until measurement. The determination of serum sCD154 and TNF-alpha levels, and plasma TF levels were centralized in the Laboratory Department of the Hospital Universitario de Canarias .Venous blood samples were collected in serum separator tubes (SST) for determination of serum sCD154 and TNF-alpha levels, and in citrate tubes to determine plasma TF levels. Blood samples were centrifuged within 30 min at 1000× n = 8) and 6.8% (n = 8) respectively; and detection limits for the assays was 0.06 ng/mL.Serum sCD154 levels were assayed by specific ELISA . The intra-assay and inter-assay coefficients of variation (CV) were 4% . The intra-assay and inter-assay CV were <3.6% (n = 20) and <6.5% (n = 20) respectively; and detection limits for the assays was 1.7 pg/mL.Serum TNF-alpha levels were measured by a solid-phase, chemiluminiscents immunometrics assays kit . The intra-assay and inter-assay CV were <7.2% (n = 20) and <8% (n = 20) respectively; and detection limits for the assays was 10 pg/mL.Plasma TF levels were assayed by specific ELISA  and NCSS 2000  and LogXact 4.1 .Receiver operating characteristic (ROC) analysis was carried out to determine the goodness-of-fit of the of serum sCD154 levels to predict 30-day mortality. Kaplan-Meier analysis of survival at 30 days and comparisons by log-rank test were carried out using serum sCD154 levels lower/higher than 1.41 ng/mL as the independent variable and survival at 30 days as the dependent variable. The association between continuous variables was carried out using Spearman’s rank correlation coefficient. A n = 50) and healthy controls (n = 50). There were no significant differences between patients with severe MMCAI and healthy controls groups on age and sex. However, we found higher serum sCD154 levels in patients with severe MMCAI patients than in healthy controls (p < 0.001); in addition, serum sCD154 levels were higher in surviving (p = 0.03) and non-surviving patients with severe MMCAI patients (p < 0.001) compared to healthy controls (controls .n = 26) and surviving (n = 24) MMCAI patients. There were no significant differences between non-surviving and surviving patients in temperature, sodium, PaO2, PaO2/FI02 ratio, lymphocytes, leukocytes, lactic acid, INR, hemoglobin, glycemia, GCS score, sex, fibrinogen, creatinine, bilirubin, aPTT, APACHE-II score and age. We found that non-surviving showed compared to surviving patients lower platelet count, and higher circulating levels of TNF-alpha, TF, and sCD154 (p < 0.001). In addition, non-surviving patients had a lower ICU stay duration than surviving (4 (2–9) vs. 23 (16–40) days; p < 0.001).p = 0.002) controlling for GCS and age . Diagnostic goodness-of-fit for serum CD154 levels of 1.41 ng/mL were the following: sensitivity = 81% (95% CI = 61%–93%), specificity = 63% (95% CI = 41%–81%).p = 0.006).p = 0.04), TF  and TNF-alpha .We found an association of circulating levels of sCD154 with GCS  non-surviving severe MMCAI patients had higher serum sCD154 levels than surviving patients; (b) there is an association between circulating levels of sCD154, GCS, TF and TNF-alpha in patients with severe MMCAI; (c) serum sCD154 levels could be used as prognostic biomarker in patients with severe MMCAI.We found higher serum sCD154 in MMCAI patients than in controls, and previously, higher CD154 platelet expression in ischemic stroke patients than in controls were reported ,11,12,13Previously higher CD154 platelet expression in ischemic stroke patients with poor functional outcome ,15 was rCirculating sCD154 levels in MMCAI patients could play a physiological role by its proinflammatory ,23 and pet al. [Another interesting findings of our study were that we found lower platelet count in non-surviving MMCAI patients than in surviving patients. These findings are in agreement with those of the study by D’Erasmo et al. . In thatThe administration of modulators of sCD154 activity could have a potential beneficial effect in MMCAI patients. In several studies the use of statins has been found to decrease circulating sCD154 levels in patients with coronary artery disease ,33,34. ISome limitations of our study should be recognized. First, we did not report data of circulating sCD154 during follow-up. Second, the determination of other inflammatory cytokines and coagulation biomarker could be interesting. Third, we found that serum sCD154 levels were associated with mortality controlling for GCS score and age; however other factors could have played a role in mortality, and thus, larger series of patients including more variables in a single multiple logistic regression analysis are needed to confirm our findings.We believe, that according to the results of our study, the determination of serum sCD154 levels at the moment of the MMCAI diagnosis could be used to prediction the outcome and could generate interest for research into the use of modulator agents of sCD154 activity in these patients.The novel and most important finding of our study is that serum sCD154 levels in MMCAI patients are associated with mortality.