We compared the immunohistochemical expression of putrescine (PUT), spermine (SPM), ornithine decarboxylase (ODC), and diamine oxidase (DAO) in bioptic samples of canine colonic mucosa with chronic inflammation  or neoplasia. Single and total polyamines levels were significantly higher in neoplastic tissue than in normal samples. Samples with different degrees of inflammation showed a general decrease expression of ODC if compared to controls; SPM was practically not expressed in control samples and very low in samples with chronic-granulomatous inflammation. In carcinomatous samples, the ODC activity was higher with respect to controls and samples with inflammation. This is the first description of polyamines expression in dog colonic mucosa in normal and in different pathological conditions, suggesting that the balance between polyamine degradation and biosynthesis is evidently disengaged during neoplasia. The polyamines (PAs) spermine (SPM) and putrescine (PUT) are intimately involved in regulation of DNA, RNA, and protein synthesis; therefore, they are essential for proliferation of both normal and neoplastic cells. Dysregulation of cellular polyamines is associated with various pathological conditions, including inflammation, and cancer; for this latter association, polyamine pathways have been explored as targets for cancer chemotherapy and chemoprevention , 2. FujiThe enzyme diamine oxidase (DAO) inactivates histamine and other biogenic amines, such as PAs by a reaction of oxidative deamination. DAO is mainly expressed in the intestine, located almost exclusively in the villus tip enterocytes of mammals . DecreasThe present study was undertaken with an objective to evaluate, by means of immunohistochemistry, the localization, the pattern, and the levels of expression of polyamines SPM, PUT, ODC, and DAO in dog. For this purpose, we have compared results obtained from biopsy samples taken from clinically healthy dogs, and dogs with clinical and histological signs of large bowel pathology (ranging from chronic inflammation to neoplasia), to evaluate the significance of polyamines within the proliferative process and their possible role as prognostic markers and target of antiproliferative drugs.Forty dogs participated in the study, after histological evaluation and inclusion in four different groups (control (group 1), granulomatous colitis (group 2), lymphoplasmacytic colitis (group 3), and colonic adenocarcinoma (group 4) consisting of 10 dogs each) .Dogs with histological diagnosis of intestinal lymphoma were excluded from the study, because the completely flat mucosa, showing crypt atrophy or hypertrophy and reduced epithelial cell number and height, with important alterations of epithelium showing colonocytes loss related to lymphocytes and neutrophils infiltration, could reduce the degree of polyamines expression. All enrolled dogs, excluding control animals, showed a long-time diagnosis of IBD according to published criteria  and were Dirofilaria antigen assay performed immediately before or after death. All the owners of the IBD dogs gave informed written consent before enrollment.The control group was composed of dogs hospitalized in urgency and that died for different causes (mainly for severe trauma) but that were free of gastrointestinal signs for at least four months, not presenting gastrointestinal signs before death and in which necropsy did not reveal neoplastic conditions. Control dogs were considered “to be healthy,” based on clinical history, normal results on physical examination (excluding lesions due to trauma), serum biochemistry, urinalysis, fecal examinations, andn = 30, 10 dogs per group of disease). Naturally deceased or humanely suppressed dogs (for the severity of the condition and independent of the study) were sampled directly, during the necropsy procedures, performed immediately after death. In dogs undergoing endoscopy, food administration was suspended around 36–48 hours before endoscopy, and dogs were prepared following standard protocols. Dogs of groups 2, 3, and 4 underwent a colonoscopy under general anesthesia  ); a set of a minimum of 8 biopsies was taken from each enterocolic tract. In dogs of group 3, additional bioptic samples were taken in neoplastic areas. All dogs showed predominantly mixed signs of enterocolitis , and upper and lower endoscopic examinations were performed. Biopsy specimens were obtained directly from mucosal lesions of increased granularity, friability, or erosions as well as areas of normal-appearing mucosa. Tissues for histopathology were fixed in 4% buffered formaldehyde and then paraffin embedded and serial 4 μm thick sections were prepared. Sections were cut, dewaxed, and stained with hematoxylin-eosin (H&E). Adjacent sections were subjected to immunohistochemical analysis (IHC) using a set of polyclonal antibodies. Histopathology was performed by a single pathologist, who was blinded regarding history, clinical signs, or endoscopic observations. A severity score was assigned for each dog, by using a standardized and previously described histologic grading system, based on the extent of architectural disruption and mucosal epithelial changes [After enrollment, multiple (10–15 specimens) mucosal biopsy specimens were procured endoscopically from the large intestine of all dogs , DAO , PUT , and SPM  antigens. All primary antibodies were diluted in Tris-buffered solution (TBS) containing 0.1% crystalline bovine serum albumin (BSA). Tissue sections were incubated overnight in a moist chamber at 4°C with different primary antibodies, diluted (1 : 50). The antigens-antibodies complex was detected by ABC-peroxidase technique using 3-3′-diaminobenzidine-hydrochloride  as chromogen substrate to reveal the immunoreaction, with Meyer Haematoxylin as nuclear counterstain.Paraffin sections were rehydrated and neutralized for endogenous peroxidases with 3% hydrogen peroxide for 5 minutes followed by rinsing for 5 minutes in distilled water. For antigen retrieval, slides were incubated in EDTA buffer, pH 9.0, and processed in a microwave oven at 650 W for two cycles of 10 minutes each to unmask antigens. Slides were then allowed to cool at room temperature for at least 20 minutes before being processed for immunostaining by standard procedures. Tissue sections were incubated overnight in a moist chamber at 4°C with different primary antibodies (Abs): rabbit polyclonal Ab against ODC  before application of the primary antibody. Specific primary antibodies substituted with TBS or nonimmune sera were used as negative controls in immunohistochemical techniques.The antibodies used were not validated for canine tissue, but the specificity of the reaction was assured by the fact that polyamines, consisting in polycationic molecules with multiple amino groups, and their cellular receptor are identical in both prokaryotic and eukaryotic cells . Canine Histopathologic examination of all biopsies was performed by a single pathologist, who graded endoscopic specimens and assigned a lesion severity score for each dog by using a standardized and previously described histologic grading system, based on the extent of architectural disruption and mucosal epithelial changes , 17, 18.Histologic criteria for normal colonic mucosa included detection of none or only a few mononuclear cells scattered in the chorion per HPF, absence of lymphoid aggregates, and none or only a few scattered neutrophils across the intestinal epithelium.In pathological samples, neutrophils were considered as absent (score 0) when there was none or only single sporadic cells per high-power field , mild (score 1) for a few cells (5 to 10 cells) per HPF, moderate (score 2) for several cells (11 to 50 cells) per HPF, or severe (score 3) (50 to 200 cells or more) per HPF. Number of mononuclear cells was considered to be normal when none or only few cells (<5 cells) were visible in each HPF among intestinal glands (score 0). Increasing in number of cells was considered mild for specimens with several cells per HPF (score 1 = 5 to 10 cells), moderate (score 2 = 11 to 30) for specimens with many cells for HPF, or severe (score 3 > 30 cells) for specimens with conspicuous and very conspicuous number of cells per HPF, respectively. Number of lymphocytic aggregates in a specimen was counted.Finally colonic adenocarcinomas were described according to Head et al. , 21 on tIn the second step, a histochemical staining was performed on the same sections previously subjected to the histological and immunohistochemical assay. The histochemical staining was performed with only minor modifications according to Poletti et al. . For Alcμm2). Ten appropriate sites were chosen for each colonic biopsy and arithmetic means were calculated for each colonic area. Results were expressed as IHC positive cells per 62 500 μm2.Cells on the margins of the tissue sections were not considered for evaluation to avoid inflation of positive cell numbers.In immunohistochemical essays, the number of immunoreactive cells for each antibody observed in normal or pathological colonic samples was calculated using a light microscope (Carl Zeiss), a ×40 objective, a ×10 eyepiece, and a square eyepiece graticule  and a pixel intensity threshold was determined to include only those image pixels in immunopositive areas. For evaluation, colonic epithelium was divided into luminal, proximal, and distal gland/crypt regions. Finally, the scoring of colonic molecules expression was calculated as previously described.To assess the intensity of polyamines expression  in biopsies sampled of all groups and to compare data to the polyamines expression in healthy control dogs (group 1) stained tissue sections, ×250 photos were used. Photographs were evaluated by two blinded investigators who scored both the extent and intensity of staining in the lining and crypts epithelium of each picture on a scale of 0–3 (0 = absent and 3 = severe). The score of extent and intensity of staining were added and the mean of the total scores was calculated and used for analysis. The ×400 photographs were used to calculate the amount of immunostaining present in each section. One examiner opened each photograph in ImageJ , while granulomatous colitis biopsies showed significantly increased neutrophils and macrophages (p < 0.001) interspersed in the lamina propria (In chronic-granulomatous colitis (group 2), a general altered morphological status of the epithelial cells was observed, with flattened aspect of some areas of epithelium and epithelial cells loss, particularly in lining superficial epithelium; additionally an evident decrease in mucous goblet cells was found, comparing samples with normal dogs or dogs with LPC. Additionally, more pyknotic and karyorrhectic epithelial cells occurred both in superficial lining and in crypts of granulomatous colitis cases than in LPC. Lymphoplasmacytic colitis (group 3) still manifested histologic evidence of chronic inflammation, with a significant increased number of mononuclear cells in the mucosal chorion of bioptic samples ( propria .p < 0.001).The Alcian PAS stain revealed an alteration of the chemical composition of mucin limited to the groups of adenocarcinoma and granulomatous colitis; a strong cytosolic PAS positivity was in fact observable, indicating a remarkable increase in the mucopolysaccharides amount if compared to controls or LPC samples  difference and a characteristic trend of the expression for every kind of lesion considered.Changes in polyamines cellular expression between groups were compared using Wilcoxon matched pairs tests. Resulting a et al. . No nuclp < 0.01; for both groups) (p < 0.001) in all kinds of lesions  the ODC antigen was “downexpressed” if compared to controls ( groups) . In samp groups) . The exp Figures , withoutThe gastrointestinal (GI) tract is lined by a continuous layer ofepithelial cells which maintain the physical and functional barrier to undesirable luminal antigens . EpithelPolyamine metabolism is involved with a wide variety of GI diseases. Numerous factors may influence polyamine homeostasis; however, the changes seem to be tissue-specific. The altered polyamine metabolism contributes to epithelial cell proliferation and esophageal carcinogenesis in experimental animals , 26. In Our results suggest that, in the adenocarcinoma affected colonic dog mucosa, the balance between biosynthesis and degradation of polyamines may be disengaged. In fact, while ODC is strongly upregulated, DAO follows both an absolute and relative decrease, from a quantitative point of view, if compared, respectively, to control's levels of the same enzyme and to the levels of the other PAs.On the other hand, the balance is conserved in inflammatory disease. Our main hypothesis about the trend of that balance in different class of diseases is based on the pathogenesis of the epithelial Noxa. In case of colitis both the upper and lower part of the cryptae are affected by the damage. The reduction in number of the mature enterocytes justifies the decrease of DAO which is an enzyme usually expressed in well-differentiated cells. The downexpression of ODC and PAs could be explained by the fact that the hyperplasia of the proliferative section does not supply from a quali-quantitative point of view the lack of cells, even if lymphoplasmacytic colitis PAs are higher than controls.The neoplastic tissue is characterized by a population of immature cells, which produces a lower amount of DAO; at the same time, the exceptional rate of proliferation produces a large amount of newborn and highly immature cells that are able to express PAs. This evidence could explain the remarkable overexpression of ODC and PAs. Interestingly, almost all our markers, except SPM, have been found to be downregulated in IBD with respect to controls. Despite the fact that PAs and their acetylated derivatives are a prerequisite for cellular metabolism and considered to be essential for proliferation and differentiation of the rapidly renewing intestinal mucosa, their role during intestinal mucosal inflammation is less clear . In someThe immunohistochemical evaluation of the polyamine's cycle in bioptic samples could be therefore useful in detecting the severity of some “borderline” lesions. A predictive value of these markers could be assessed by evaluating the group of dysplastic lesions, looking for a “cut-off” point in the quantitative expression between preneoplastic and nonneoplastic lesions.The coherence of our results with the findings reported in human beings  could suSimilar to humans, dogs express different intestinal levels of PAs, relative to different pathological conditions. We can hypothesize that contextual evaluation of ODC, DAO, and PAs could be suggestive of the severity of the lesion and in cases of overexpression a possible predictive factor of malignancy. While such a high polyamine supply may be of benefit in nonneoplastic colonic mucosal growth, the role of tissutal and luminal polyamines in colon cancer is a clear concern. The pool of PAs is taken up by neoplastic colonocytes, they are utilized in full to support neoplastic growth, and their uptake is strongly upregulated by the mitogens known to play an important role in colonic carcinogenesis. Inhibition of polyamine synthesis and their uptake, impaired utilization of exogenous polyamines, and enhanced catabolism of polyamines in neoplastic colonocytes may be therefore conceivable future approaches also in the chemoprevention of dog colorectal cancer.


Mitochondria are critical modulators of cell function and are increasingly recognized as proximal sensors and effectors that ultimately determine the balance between cell survival and cell death. Volatile anesthetics (VA) are long known for their cardioprotective effects, as demonstrated by improved mitochondrial and cellular functions, and by reduced necrotic and apoptotic cell death during cardiac ischemia and reperfusion (IR) injury. The molecular mechanisms by which VA impart cardioprotection are still poorly understood. Because of the emerging role of mitochondria as therapeutic targets in diseases, including ischemic heart disease, it is important to know if VA-induced cytoprotective mechanisms are mediated at the mitochondrial level. In recent years, considerable evidence points to direct effects of VA on mitochondrial channel/transporter protein functions and electron transport chain (ETC) complexes as potential targets in mediating cardioprotection. This review furnishes an integrated overview of targets that VA impart on mitochondrial channels/transporters and ETC proteins that could provide a basis for cation regulation and homeostasis, mitochondrial bioenergetics, and reactive oxygen species (ROS) emission in redox signaling for cardiac cell protection during IR injury. In recent years the mitochondrion has gained recognition as a key factor in the etiology of numerous diseases Duchen, , includim), decreased adenosine triphosphate (ATP) production, impaired Ca2+ homeostasis, increased “bad” reactive oxygen species (ROS) emission, matrix swelling and membrane permeability, and release of cytochrome c and other apoptotic factors leading to cell death  is characterized by a reduced oxygen supply to demand ratio in the hearts of patients at high-risk of coronary artery disease (CAD) or who are undergoing high-risk cardiac surgery. Due to limited blood supply in the manifestation of MI, IR injury leads to a dysfunctional redox imbalance in mitochondria with a concomitant decrease in oxidative phosphorylation (OxPhos) and an overall switch to anaerobic metabolism. Experimental and clinical data have provided several types of mechanical and pharmacological conditioning strategies that lead to reduce IR-induced myocardial dysfunction and cell death as discussed next.Murry et al.  were theClinically, IPC can be mimicked pharmacologically by a variety of substances like aspirin, beta-blockers, alpha 2-adrenoceptor agonists, statins, opioids, and VA . Myocardial protection by the VA enflurane was first demonstrated by Freedman et al.  in the ide novo protein synthesis  produced during IR. VA likely interfere with mitochondrial function by directly or indirectly targeting many, but not all, mitochondrial proteins. Specifically, VA probably directly modulate the function of known targeted proteins that are believed to underlie the mechanism of IPC-induced protection against IR injury. Therefore, APC is of practical importance because administration of a VA could reduce mortality during ischemic heart surgery, and could also safely be given to patients who are at high risk, e.g., during heart transplant procedure , discussed in detail later. VA appear to indirectly relax coronary arteries by altering intracellular Ca2+ regulation in the vascular smooth muscle cell by stimulating mKATP channel opening    contains several enzymes including monomine oxidase and the integral transport proteins, porins, which makes the OMM permeable to small molecules less than 6 kilodaltons (kDa). The voltage-dependent anion channel (VDAC) constitutes the major porin of the OMM and it regulates the metabolic and energetic fluxes across the OMM by transporting metabolites and ions necessary for electron transfer, bioenergetics, and redox potentials for normal mitochondrial function. Mammalian mitochondria have three different VDAC isoforms: 1, 2, and 3 , which activate proteolytic enzymes, e.g., the caspases. VDAC normally exists in the open configuration  and Bcl-2 associated X protein (Bax)  is impermeable to charged substances and so distinct channels, exchangers, and pumps are utilized to transport ions and metabolites to and from the matrix. The IMM also contains the ETC complexes that carry out OxPhos. This bioenergetic process is dependent on an intricate interplay among the supply of substrates, breakdown and elimination of metabolites, and ion fluxes across the IMM. For example, Ca2+], and consequently to mitochondrial Ca2+ loading , suggesting that mKATP channel opening was associated with a decrease in matrix Ca2+ overload possibly via attenuated Ca2+ uptake.Myocardial IR leads to an increase in cytosolic [Ca2+ by the mitochondrial calcium uniporter (mCU) specifically depends on a large ΔΨm gradient  led to a Na+-dependent, but ΔΨm-independent, increase in mitochondrial Ca2+ by attenuating NCE without affecting uptake via the mCU. Moreover, this was consistent with the lack of increase in matrix Ca2+ in the absence of buffer Na+ so that NCE could not be activated  that trigger effectors of several survival signaling pathways involved in pre- and post-conditioning  was identified in the IMM and demonstrated that ROMK channels can localize to mitochondria , and a “small” transient rise in ROS emission  associated with a decrease in mitochondrial Ca2+ load  opening, as reviewed by our group previously  to traverse mitochondrial membranes and enhance ROS emission, mitochondrial swelling and cell death. The molecular identity of the mPTP remains unclear. The mitochondrial matrix protein, cyclophilin D (CyP-D), a member of a family of highly homologous peptidylprolyl cis-trans isomerase (PPIase), is believed to constitute an integral component of the mPTP, and thus to play an important role in regulating the pore  during cardioprotection by anesthetic post-conditioning (APoC) was suggested to prevent mPTP opening  of a complex I inhibitor (rotenone).In our most recent study  radical intermediate via electron transport in complex III  from the matrix to the inter-membrane space generates the ΔΨm necessary for OxPhos. A H+ leak (uncoupling) in the IMM has potential implications in both IR injury and in preconditioning . In turn this leads to an increase in intracellular Ca2+ by activating the sarcolemmal Na+-Ca2+ exchanger (NCE)   agonist G1 improved cardiac function, reduced infarct size, and inhibited mPTP opening by activating ERK signaling in the isolated mouse hearts after IR  of mitochondrial proteins have been proposed to modulate cardioprotection  of mitochondrial proteins involved in bioenergetics and electron transport complexes are implicated in the role of PTMs in regulating mitochondrial function that confers cardioprotection  that are involved in transcriptional and post-transcriptional regulation of gene expression  encodes 13 mitochondrial proteins and the mitochondrial rRNAs and tRNAs needed for translation (Kirby and Thorburn, Improvement in the clinical management of ischemic heart disease remains elusive despite the discovery of many molecular and cellular mechanisms that may be valuable targets to treat against IR injury. The importance of mitochondrial bioenergetics and function in contributing not only to cardiac injury but also to reducing cardiac injury is now well recognized. But there remains a lack of clear understanding of the mitochondrial-cytosolic mechanisms that might lead to more targeted intervention. Hence, we need to identify new targets that could uncover the mechanisms of dysfunction associated with IR injury. With a better understanding of mitochondrial targets as hubs for controlling metabolism and cellular redox signaling pathways that elicit protection, we could better develop novel therapeutic drugs for clinical trials to protect against IR injury. Because VA are lipophilic agents with multi-targeted actions that, together, confer cardioprotection, they give us valuable clues into which potential sites to investigate; these clues may be especially useful to selectively and reversibly target mitochondria to reduce IR injury. Unfortunately, as summarized in this review, there is contradictory evidence with respect to the potentially large number of pathways by which VA might protect the heart. Nonetheless, there are molecules with characteristics of a VA but without anesthetic properties. These could be developed as cardioprotective drugs while obviating the need for inducing anesthesia.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rare variation has a key role in the genetic etiology of complex traits. Genetically isolated populations have been established as a powerful resource for novel locus discovery and they combine advantageous characteristics that can be leveraged to expedite discovery. Genome-wide genotyping approaches coupled with sequencing efforts have transformed the landscape of disease genomics and highlight the potentially significant contribution of studies in founder populations. Genetically isolated or founder populations have recently returned to the fore of genetic association studies as valuable resources for complex trait gene identification . PopulatRecent successes in the literature have highlighted how these advantageous characteristics can help with disease gene mapping. Researchers studying the Icelandic population have, in recent years, pioneered the use of next-generation association studies, a hybrid of genome-wide genotyping and whole genome sequencing (WGS) approaches, for complex disease gene mapping ,3. In IcTBC1D4 gene was found to be strongly associated with postprandial hyperglycemia, impaired glucose tolerance and T2D. These unique insights into the mechanism conferring muscle insulin resistance for this subset of T2D was afforded by studying the small Greenlandic population, which has experienced a dramatic increase in T2D prevalence, and recalling individuals based on their TBC1D4 variant status. This polymorphism is common in Greenland , but vanishingly rare in other global populations . This work elegantly demonstrates the value of combining the genetic characteristics of founder populations with the potential to recontact participants for further follow-up of promising results. Studies in extensively phenotyped founder population cohorts, such as the Amish, have also demonstrated the value of combining unique population characteristics with recall of subjects to increase our understanding of disease etiopathology. The Old Order Amish are a cultural isolate and geographically localized, genetically homogeneous population with extensive genealogical records available. This deeply phenotyped cohort has been the subject of long-term genetic studies. For example, in 2008, Pollin et al. [APOC3 gene and is strongly associated with a cardioprotective phenotype (higher high-density lipoprotein and lower blood triglyceride levels).More recently, novel insights into the biological pathways underpinning T2D were achieved through the study of a Greenlandic founder population . A nonseAPOC3 variant was carried by approximately 4% of the individuals studied and reached genome-wide statistical significance with a sample size of fewer than 1,300. Discovery of the same effect in the general population would have required over 50 times the number of subjects. Large-scale studies of over 110,000 individuals of European descent have recently also established an association of rare variants in the APOC3 locus with protection against high triglyceride levels and coronary artery disease [APOC3 is now becoming a poster child for the power afforded by founder populations and clearly demonstrates the generalizability of findings in isolates into more cosmopolitan populations.Notably, the same missense cardioprotective variant was also found in an independent isolated population from Greece in the HELIC-MANOLIS study . Residen disease . APOC3 iet al. first established that the Finns have fewer variable sites overall but more loss-of-function variants compared with non-Finnish European individuals, and subsequently identified robust associations with key traits of medical relevance. Linkage to national medical records resulted in the demonstration that splice variants in the LPA gene that are associated with low levels of plasma lipoprotein(a) confer protection against cardiovascular disease.A prime example of how founder population characteristics coupled with linkage to medical records can accelerate discovery was recently produced by studying the Finnish population . In a whGoing forward, it is clear that founder populations can provide a unique and powerful resource for the identification of low frequency and rare variants of direct medical consequence. Power to detect association is demonstrably boosted for individual sequence variants that have drifted up in frequency. In addition, power to detect a significant accumulation of rare variants at particular loci is further increased in founder populations as neutral rare variation may be lost from the haplotype pool. In this context, meta-analysis at the locus level across different isolates is posited to be important for establishing burden of proof, although this principle requires empirical substantiation. Historically, the transferability of findings in isolates across to more cosmopolitan populations has been a topic of debate. However, there is an accrual of emerging examples of loci discovered in founder populations that are more widely generalizable, with replication of signals achieved in diverse sample sets ,5,7-9. F


TNFSF15, which encodes the pro-inflammatory cytokine TNF super-family member 15, but it was unknown whether these disparate diseases were associated with the same genetic variance in 9q32, and how variance within this locus might contribute to pathology. Using genetic data from published studies on CD, PBC and leprosy we revealed that bearing a T allele at rs6478108/rs6478109 (r2 = 1) or rs4979462 was significantly associated with increased risk of CD and decreased risk of leprosy, while the T allele at rs4979462 was associated with significantly increased risk of PBC. In vitro analyses showed that the rs6478109 genotype significantly affected TNFSF15 expression in cells from whole blood of controls, while functional annotation using publicly-available data revealed the broad cell type/tissue-specific regulatory potential of variance at rs6478109 or rs4979462. In summary, we provide evidence that variance within TNFSF15 has the potential to affect cytokine expression across a range of tissues and thereby contribute to protection from infectious diseases such as leprosy, while increasing the risk of immune-mediated diseases including CD and PBC.Genetic polymorphism within the 9q32 locus is linked with increased risk of several diseases, including Crohn’s disease (CD), primary biliary cholangitis (PBC) and leprosy. The most likely disease-causing gene within 9q32 is  TNFSF15 has been identified as the likely candidate mediating the observed disease-associationsTNFSF15 in infectious and autoimmune disease development21TNFSF15 expression was significantly higher in biopsies from inflamed CD lesions than those from uninvolved tissues. A study showing that blockade of TNFSF15 attenuates intestinal inflammation in inflammatory bowel diseasePrevious genome-wide association studies (GWAS) have revealed multiple associations of diverse disease phenotypes on chromosome 9q32, including primary biliary cholangitis  associated with each disease and used in silico functional annotation combined with in vitro experimentation to show that the diverse disease associations within TNFSF15 are probably driven by two independent causal variants that contribute to regulating TNFSF15 expression across a range of tissues and cell types.While PBC, CD and leprosy share some pathological features, such as granuloma formationTNFSF15 locus: two studies of leprosy in Chinese ethnicity populations 1410We selected several previously-published GWAS datasets for fine-mapping analysis of the 2 = 1) with the previously reported SNP, rs49794622 ≥ 0.97, 2 ≥ 0.97), but less so with the top index SNP (rs4979462) of the Japanese PBC (r2 ≤ 0.55). Consistently, both rs6478108 and rs4366152 showed strong associations with CD and leprosy, and conditioning on either SNP abolished the extensive associations within the locus in the two disease datasets . Therefore, both leprosy and CD risk associations map to the same SNP, and are likely driven by the same risk variant within the locus.While imputation of absent data from GWAS studies is a robust and valuable tool, it has the potential to increase analytical errors. Therefore, to confirm the results from the fine mapping analysis above, we genotyped the top five most significantly-associated SNPs from the analysis of published leprosy data (including both rs6478108 and rs4366152) and the most significantly-associated SNP from the analysis of published PBC data, rs4979462, in an independent sample of 2,812 leprosy cases and 5,464 controls from China . This apWe then genotyped rs6478108 (the top SNP for CD and Leprosy) and rs4979462 (top SNP for PBC) in three independent populations: 5,259 leprosy cases and 5,282 controls of Chinese ethnicity , 722 CD cases and 2,124 controls of Korean ethnicity, and 772 PBC cases and 599 controls of Japanese ethnicity. The analysis of the independent validation samples  and the joint analysis of the combined GWAS and validation samples of the three diseases  provided strong supportive evidence for the associations of rs6478108 and rs4979462 with risk of the three diseases .conditional = 2.76 × 10−17, ORconditional = 0.81) and CD  datasets. Conditioning on rs6478108 also reduced the strength of association at rs4979462 in both leprosy and CD, but the association remained genome-wide significant in CD , while becoming suggestive  with significantly reduced OR (from 0.75 to 0.87) in leprosy after conditioning. In contrast, in PBC, conditioning on rs4979462 abolished the risk association at rs6478108 , but not vice versa . Taken together, these results indicate that variance at rs6478108 and rs4979462 has independent effects on risk of CD, and possibly of leprosy, but not in the Japanese PBC dataset where rs4979462 remains the top SNP within the TNFSF15 locus.Interestingly, rs6478108 and rs4979462 showed independent effects on risk of CD and leprosy : conditi−11, −16, OR = 1.58), but the TC haplotype did not , though the samples had sufficient power (>80%) to detect a moderate risk effect of the TC haplotype (OR = 1.30) at a P value of 0.05. Thus rs6478108_T is not an independent risk allele . In the Chinese leprosy samples, both TC and TT haplotypes were associated with significantly protective effect than the CC haplotype  (−6)  . The effly) (−6) . As for ly) (−6) . This suTNFSF15 locus in CD, leprosy and PBC, we used functional annotation to identify the most likely potential causal SNP(s). Since the two index SNPs, rs6478108 and rs4979462, are located within a LD block that contain multiple variants in high LD, we therefore identified all the SNPs and indels that were tagged by them at r2 ≥ 0.8, then carried out functional annotation of these SNPs; we predicted their potential effects on RNA splicing, deoxyribonuclease (DNase) activity, histone modifications, RNA expression (RNA-Seq), and transcription factor (TF) binding (ChIP-Seq) peaks, using data generated in 131 different tissues/cell types from the ENCODE and ROADMAP databases. Alongside, we predicted their impact on expression quantitative trait loci (eQTL) in different blood cells and using published results from functional assays (see Methods).After dissecting the pattern of genetic associations within the TNFSF15, while the remaining 12 were located adjacent to TNFSF15 gene  overlapped with DNase peaks in at least one out of 81 cell types  in CD14+ monocytes  present in stromal and connective tissues, lung and skin fibroblasts, epithelial cancers, epithelial and endothelial cells, heart, muscle, brain and fetal tissues; while rs6478109 predominantly overlapped with DNAse peaks in various immune cells, including primary CD3onocytes . Interesonocytes , suggestTNFSF15 expression (TNFSF8 (data not shown). The SNP rs6478109 was significantly associated to the expression levels of TNFSF15 in independent datasets as determined in whole blood , however this effect was abolished when conditioning on the effect of rs6478109 , while the eQTL effect of rs6478109 remained strong after conditioning on rs4979462 , CD  and PBC ; through conditional and haplotype analyses, we clearly demonstrated that there are two independent associations at both rs6478108/rs6478109 and rs4979462 for risk of CD and leprosy, and a single association at rs4979462 for risk of PBC. While an association between risk of PBC and variance at rs6478108 was observed, this effect was purely due to its LD with rs4979462 and was accordingly abolished by conditioning on rs4979462. Thus rs4979462 plays a role in all three diseases, while variance at rs6478108/rs6478109 has a genetic risk effect in CD and leprosy, but not PBC.Several GWAS studies have hinted at associations between the 9q32 region, and specifically the TNFSF15 locus show opposite effects in leprosy and CD/PBC: the T allele at rs4979462 significantly increases risk of CD and PBC, but significantly decreases risk of leprosy; while at rs6478108, which only showed independent associations with CD and leprosy, the T allele significantly increases risk of CD, but significantly decreases risk of leprosy. A similar phenomenon has been reported in studies of CD risk and norovirus susceptibility, where individuals that carry two copies of the CD risk allele at rs601338 in FUT2 are protected from norovirus infection12SH2B3 might also play a protective role against bacterial infections2728TNFSF15 was not among these 21 loci, and we could not find any evidence of recent selection in the current study. A more thorough analysis for selection signatures is required to understand the evolutionary forces that might have shaped the variance within this locus.Interestingly the disease associations with specific SNPs in the TNFSF15. By intersecting rs6478108 and rs4979462, as well as those SNPs within the TNFSF15 locus in high LD with rs6478108 and rs4979462, with the regulatory information from the ENCODE and ROADMAP Projects, we found that both rs4979462 and rs6478109 (which is in perfect LD with rs6478108) are much more likely to exhibit a regulatory effect on gene expression than other variants within the region. While both SNPs are predicted to exhibit regulatory function in gastrointestinal tissues, rs6478109-related regulatory sequence was likely to be functional in immune cells from blood, whereas rs4979462-related regulatory sequence was likely to be functional in epithelial and connective tissues. The cell and tissue specific regulatory roles of rs6478109 and rs4979462 provide biological insight into the differential associations of these two SNPs with three different diseases: because the regulatory sequences of both SNPs are functional in gastrointestinal cells, we would anticipate that both SNPs would exhibit a risk effect in CD; while the role of rs6478109-related regulatory sequence in immune cells makes sense of the SNP’s association with leprosy, where immunity to the pathogenic agent will play a major role. That rs4979462 is also associated with risk of leprosy suggests that the transcriptional regulation of TNFSF15 in gastrointestinal or epithelial/connective tissues might play a previously-unappreciated role in the development of leprosy. Moreover, the positive association of rs4979462, and the negative association of rs6478108, with risk of PBC, suggest that TNFSF15 may influence the development of PBC predominantly through its function in epithelial and connective tissues.In order to evaluate the likelihood that rs6478108 and rs4979462 were directly linked with disease causation, we estimated their potential for regulatory effects upon TNFSF15 expression in whole blood and in isolated monocytes. This indicates that TNFSF15, and not other genes within the 9q32 locus, such as TNFSF8, is likely to be the causative pathogenic disease gene of the locus. Interestingly, the A allele of rs6478109 was associated with higher expression of TNFSF15 in blood; given that the same allele was associated with increased risk of CD, and decreased risk of leprosy, it follows that high expression of TNFSF15 will increase CD risk, but decrease leprosy risk. Although there was no evidence for an eQTL effect of rs4979462 here, we previously showed that increased expression of TNFSF15 may be driven by the PBC-susceptible allele of rs4979462, as a result of the generation of a novel NF-1 binding site by the A allele in human T-cell Jurkat and bile duct HuCCT1 cell linesWe also assessed the likely effects of rs6478108 and rs4979462 on eQTLs and found that rs6478109 had a significant and specific eQTL effect on TNFSF15 locus in leprosy, CD and PBC. Two independent associations have been discovered within the locus, with complex patterns of genetic effects across the three diseases: one variant shows pleiotropic effect across the three diseases, and the other plays a role in CD and Leprosy but not PBC. For both associations, the genetic effect is opposite in the autoimmune/inflammatory diseases and infection. Further regulatory function annotation and eQTL analysis both confirmed TNFSF15 as the most likely pathogenic gene of the locus, and revealed strong evidence for the regulatory functionality of both associations in a cell/tissue-specific fashion. Our study highlights the potentially diverse function of TNFSF15 in immune regulation, gastrointestinal cells and epithelial/connective tissues, and indicates that increased expression of TNFSF15 may simultaneously confer protection against infections, such as leprosy, while increasing the risk for immune and inflammatory diseases.In summary, we have performed a fine-mapping and functional annotation analysis of the The Chinese leprosy GWAS dataset consisted of 1,548 leprosy cases and 2,150 healthy controls that were derived from 2 previously published studies14The Korean CD dataset was derived from 2 previously published studies10The discovery dataset of Japanese PBC included 509 cases and 493 controls: an additional 313 cases and 437 controls were involved in the first replication study, with a further 772 cases and 599 controls involved in the second replication study. These sets were derived from our previously-published GWASThe collection of blood samples and clinical information from all subjects was undertaken in accordance with the Declaration of Helsinki.TNFSF15 locus was performed in each of the five previously-published GWAS datasets separately: two leprosy datasets from Chinese ethnicity individuals1410Imputation analysis of the SNP validation was performed by genotyping using Sequenom MassArray platform or running TaqMan assays; details in Unpublished dataset). RNA was isolated from blood collected in Tempus tubes  and gene expression was estimated by analysing on the Illumina HumanHT-12-v4 Expression Bead Chip . The genotypes were determined using the Illumina Human Omni5Quad chip. Only Illumina probes free of any SNPs were used to determine the expression level of the genes to avoid allele-specific artefacts. Probes used for analysis included the following: ILMN_1759501 (TNFSF15), ILMN_1761778 (TNFSF8).We associated gene expression data obtained from whole blood mRNA from 206 Chinese ethnicity volunteers in the context of their genotypes for the SNPs under investigation in this report.  with rs6478108 and rs4979462 using the LD information provided by HaploReg v2TNFSF15 transcript abundance, using the SPANR web-based toolWe first determined the critical region of the We also evaluated the cis-eQTL effect of the 18 SNPs (P < 0.01) on genes located within 1 Mb of these SNPs using data from multiple cell types, including B-cells, monocytes, T-effector, T-regulator cells264445How to cite this article: Sun, Y. et al. Fine-mapping analysis revealed complex pleiotropic effect and tissue-specific regulatory mechanism of TNFSF15 in primary biliary cholangitis, Crohn’s disease and leprosy. Sci. Rep.6, 31429; doi: 10.1038/srep31429 (2016).


Chemotherapy and targeted therapy have opened new avenues in clinical oncology. However, there is a lack of response in a substantial percentage of cancer patients and diseases frequently relapse in those who even initially respond. Resistance is, at present, the major barrier to conquering cancer, the most lethal age-related pathology. Identification of mechanisms underlying resistance and development of effective strategies to circumvent treatment pitfalls thereby improving clinical outcomes remain overarching tasks for scientists and clinicians. Growing bodies of data indicate that stromal cells within the genetically stable but metabolically dynamic tumor microenvironment confer acquired resistance against anticancer therapies. Further, treatment itself activates the microenvironment by damaging a large population of benign cells, which can drastically exacerbate disease conditions in a cell nonautonomous manner, and such off-target effects should be well taken into account when establishing future therapeutic rationale. In this review, we highlight relevant biological mechanisms through which the tumor microenvironment drives development of resistance. We discuss some unsolved issues related to the preclinical and clinical trial paradigms that need to be carefully devised, and provide implications for personalized medicine. In the long run, an insightful and accurate understanding of the intricate signaling networks of the tumor microenvironment in pathological settings will guide the design of new clinical interventions particularly combinatorial therapies, and it might help overcome, or at least prevent, the onset of acquired resistance. Cancers evolve in complex tissue environments, where they obtain support for expansion, invasion, and metastasis. The past decade has seen significant and accelerated progress in the design, improvement, and application of anticancer therapies; however, most clinical regimens including chemotherapy and targeted therapy ultimately fail to cure patients. Even cancers that show dramatic initial responses to treatments frequently relapse as resistant malignancies, and disease recurrence remains a critical challenge in clinical oncology. The resistance force can arise as a consequence of cell intrinsic changes including upregulation of drug efflux pumps, activation of detoxifying enzymes, increased drug metabolism, loss of specific oncogenes, enhancement of DNA repair machineries including translesion polymerase upregulation, disruption of calcium homeostasis, emergence of apoptotic defects, epigenetic abnormalities, tumor heterogeneity, or plasticity of cancer stemness.As a major part of the microenvironment, the stroma is a loosely organized scaffold composed of diverse cell types intertwined in an extracellular matrix (ECM), which generates spatial support and mediates cell signaling. In normal tissue the stroma also functions as the physiological barrier against tumorigenesis; however, transformed tumor cells initiate significant changes that can convert the adjacent microenvironment into a TME that supports pathological progression.As a representative and predominant cell type in the tumor stroma, fibroblasts mainly reside in connective tissue of solid organs, regulating tissue remodeling during wound healing and development.30CAFs are specialized myofibroblasts that exist in pathological settings intimately connected with cancer progression,35Endothelial cells, pericytes, and smooth muscle cells are stromal lineages that play active roles in angiogenesis and neovasculature development. In the course of tumorigenesis, majority of proangiogenic cytokines are produced by stromal and epithelial cell populations at early neoplastic stages or as a consequence of immune response or local inflammation, while enhanced angiogenesis usually takes place as a late event of tumor progression.39Under both normal and neoplastic conditions, inflammation involves overall orchestration of multiple cell lineages from the innate and adaptive immune systems. Neutrophils, in most cases, are the first to be attracted to local sites of acute inflammation.43Macrophages, in particular, can also regulate the inflammatory microenvironment in some pathological settings by generating metabolic products such as reactive oxygen species (ROS), reactive nitrogen species (RNS), cyclooxygenase 2, and prostaglandins, synthesis of which can be targeted pharmacologically.47XL, abnormal epigenetic and posttranslational modifications, enhanced DNA damage repair activities, and implication of key prosurvival signaling molecules such as the transmembrane receptor Notch and the nuclear factor-κB (NF-κB).As an important part of the cell populations that have malignant potential, CSCs are highly resistant to multiple types of anticancer therapies owing to manifold inherent features, including enhanced aldehyde dehydrogenase activity, assembly of ATP-binding cassette transporter complexes, overexpression of antiapoptotic proteins such as Bcl-2 and Bcl-In nature, CSCs are regulated by multiple TME-derived factors including those active in inflammatory cytokine networks, such as IL-1, IL-6, and IL-8.56Therapeutic resistance, local recurrence, and distant metastasis remain the major causes of cancer mortality. Dynamic interactions between CSCs and stromal cells represent one of the main mechanisms of treatment resistance and cancer recurrence. In both follicular lymphoma and colorectal cancer, drug-resistant CSCs interact with follicular dendritic cells via CXCL12/chemokine (C-X-C) ligand receptor 4 (CXCR4) signaling to provoke tumorigenicity, and such CSCs are enriched by chemotherapy in the presence of stromal cells.The structurally dense ECM in tumors has a couple of functions. First, it acts as a physical shield to restrict drug transport, thereby reducing local drug efficacy.62On the other hand, the ECM augments drug resistance by providing attachment sites for cancer cells. ECM–cell interactions have consequences for both the cell and the ECM, as binding of cells to ECM not only renders the local ECM stiffer, but dramatically changes the activity of anchored cells.69Integrins are a family of heterodimeric transmembrane receptors, each consisting of an α and a β polypetide chain and bridging for cell–cell and cell–ECM interactions. Once triggered by physical or biochemical stimulations integrins can engage signal transduction pathways most of which eventually induce biological responses such as cell cycle regulation, cell shape recovery, and motility adjustment.73In addition, integrins are involved in signal transduction activities by coordinating the intracellular pathways of transmembrane protein kinases such as receptor tyrosine kinases (RTKs). Although the interplay between integrin and RTKs use to be thought of as unidirectional and supportive, new data indicate that integrins have additional and diverse roles in mammalian cells by regulation of RTK axes via engaging specific adaptors to the cell surface. For instance, β1c integrin recruits Gab1/Shp2 and presents Shp2 to insulin growth factor 1 receptor (IGF-1R), resulting in dephosphorylation of the receptor.75Within the TME, autocrine and paracrine activation of oncogenic signaling by a large pool of bioactive soluble factors including cytokines, chemokines, proteases, and growth factors derived from the benign stroma or cancer cells have critical roles in generating resistance to both chemotherapy and targeted therapies. As such, soluble factors work as extracellular or intracellular effectors that are able to both trigger and maintain the activation of various survival signaling pathways of cancer, a primary and indispensable step toward development of more malignant phenotypes.Growth factors bind to RTKs and stimulate both the Ras/Raf/MEK/ERK and PI3K pathways. Stromal secretion of HGF causes phosphorylation of receptor c-Met, engagement of the mitogen-activated protein kinase (MAPK), and implication of PI3K signaling pathways, which culminate in prominent resistance to RAF inhibition.Similarly, HGF and c-Met have been linked to drug resistance of Her2-positive, estrogen receptor α-positive, and triple-negative breast cancers (TNBCs). In MCF-7 and T47D breast cancer cells, the acquisition of fulvestrant resistance was accompanied by an increase in c-Met expression, which promoted the response to CAF-secreted HGF.78It is important to pay attention to another group of proteins secreted by stroma: the cytokines. For example, transforming growth factor (TGF) β has a dual function in cancer biology as it possesses both tumor-suppressive and tumor-permissive potentials. The tumor-promoting effect of TGFβ is mostly seen in the later stages of epithelial cancers, as mainly mediating the induction of epithelial-mesenchymal transition (EMT), which is one of the major driving forces of cancer resistance.Stromal cell derived growth factor 1 (SDF-1), or CXCL12, is frequently expressed by TME of multiple tumor types. Specifically, SDF-1 promotes breast cancer growth by binding to its cognate receptor, CXCR4 on cancer cells, rendering ERα-positive breast cancer cells refractory to fulvestrant.86Chemokine CCL2 has also recently received attention concerning its involvement in the recruitment of infiltrating leukocytes. CCL2 produced by stroma or tumor cells recruits cytotoxic lymphocytes to the TME to suppress or stimulate tumor growth.88For many cancers, increased IL-6 expression plays a central role in augmented chemoresistance. Bone marrow stroma-derived cytokines protect Janus-activated kinase 2-V617F (JAK2(V617F)) mutant cells from the effects of atiprimod, a strong JAK2 inhibitor.Altogether, the structural and functional presence of various cell types in the TME and their pathophysiologic signals released into TME niches exert comprehensive influences to the biological behaviors of tumors including the priming activities of treatment resistance Fig..Increasing lines of evidence have recognized noncancerous cells of the TME as major determinants of cancer treatment in a large number of cases, though cancer cell responses are still subject to their intrinsic characteristics. Importantly, data also suggest that novel combination treatments could eventually overcome such “damaged TME” conferred resistance.Upregulation of WNT16B by neoadjuvant or regular chemotherapy was found in the TME of prostate, breast, and ovarian cancer patients. Intriguingly, NF-κB was identified as a key signaling node that actively mediates WNT16B production. Cell culture experiments and tumor xenograft models proved the protective effect of fibroblast-derived WNT16B, indicating that WNT16B secreted by stroma attenuates cancer cell apoptosis induced by genotoxicity, and compromises drug response through activation of a DNA damage secretory program (DDSP)So far studies substantiated the first evidence of WNT16B, supporting that signaling initiated by stroma is actually a critical regulator of cancer response to chemotherapy, targeted therapy, and even endocrine treatment.97Years ago, a well-established mouse model of Burkitt's lymphoma was employed to elucidate the mechanism through which paracrine factors in the TME influence lymphoma cell survival following genotoxic chemotherapy. Both IL-6 and tissue inhibitor of metalloproteinase 1 (Timp1) were remarkably released in the thymus upon doxorubicin treatment, a step leading to the establishment of a “chemoresistant niche,” which in turn allowed the survival of residual lymphoma cells as a minimal residue disease and ultimately caused disease relapse.98The growth and maintenance of blood vessels is regulated by multiple pathways, including those mediated by proangiogenic factors secreted by both tumor and stromal cells.Experimental data of extrinsic factors support that TME dictates the development of chemoresistance by multiple facets including regulation of drug distribution and inflammatory responses. Monitoring tumor status in real time at cellular resolution revealed that myeloid cell infiltration is increased in human breast tumors after chemotherapy and the cellular composition is a strong predictor of overall survival.Although the stromal components of the TME exert profound impacts on disease progression and pose substantial challenges for the advancement of effective therapies, studies addressed the fact that such alterations are indeed creating new opportunities for innovative cancer treatments.To date there are several herald datasets presenting therapeutic opportunities oriented toward eliminating TEM-induced cancer resistance. In particular, cytokines as well as growth and survival factors released by the TME represent legitimate therapeutic targets. As a major growth stimulator and resistance enhancer by counteracting genotoxic chemotherapy and glucocorticoid-involved treatments typically applied in multiple myeloma, IL-6 is also considered as a therapeutic target in Castleman's disease and several epithelial malignancies including ovarian, prostate, and breast cancer.Adhesion molecules across plasma membrane of cancer cells and/or the underlying stromal cells also make valid target candidates for sensitizing cancer cells to therapeutic agents, provided that treatments by antibodies, peptides, conjugates, or other toxins does not affect normal cells dependent on such stromal cells or surface markers for regular turnover and physiological activities.Strategies that effectively inhibit the resistance acquired from the microenvironments confronted by either chemotherapy or targeted therapies have the potential to improve overall therapeutic outcome. Pathological influence of microenvironment on cancer cell survival and subsequent repopulation is usually mediated through the activation of signaling networks that provoke the development of a secretory phenotype and activation of tumor-stroma crosstalk. So far many agents have been developed to inhibit these pathways, including small-molecule inhibitors against specific regulators of key signal pathways, including the p38MAPK cascade, NF-κB complex, CCAAT/enhancer-binding protein components, mTOR molecules, and even DNA damage response moieties; or cytostatic antibodies with the ability to neutralize major soluble factors that play significant roles in advanced cancer phenotypes, such as those targeting IL-6, IL-8, WNT16B, SFRP2, and SPINK1.Among the previously unrecognized implications of tumor–microenvironment interactions, a major one is that development of molecular markers for personalized medicine and their prospective application in clinical oncology will be significantly dependent on the manner and consequence of such “in-milieu” crosstalk. These markers are usually identified and established by linking the cancer cell response to a given therapy with the engagement of oncogenic signaling networks, but new strategies that allow to improve existing therapies and to open new therapeutic avenues by identifying more predictive biomarkers are still expected. As the local microenvironment can influence both the biological profile and treatment responsiveness of cancer cells, it should be integrated into in vitro assays and preclinical studies to identify candidate markers of therapy resistance with the aim to avoid confounding effects while improving their potential use for molecular diagnostics in clinical practice.120To date majority of the proposed molecules for individualized cancer treatments are based on mutations found in particular subtypes of human malignancies, such as TMPRSS2-ERG gene fusions in prostate cancer patients. Many such genomic alterations involve constitutively activated oncogenes or pathways that can be targeted by small-molecule inhibitors available in clinics. For most of these markers, the favorable outcome is usually associated with a higher response rate to a certain treatment, although a large number of cancer patients still failed to respond, indicating the complexity of clinical intervention. Unfortunately, even advanced and exhaustive high-resolution genetic approaches including deep sequencing techniques for a large body of examples were not able to discover additional genomic abnormalities that account for such unresponsiveness. Recent data suggested that in majority of these cases the incompatible genotypes observed against the clinical responses to chemotherapies and/or targeted therapies may be ascribed to stroma-related factors or other TME influences, which cannot be exclusively deduced from the cancer cell genotype alone.+ T cells, specifically kill the CAFs, and increase intratumoral drug uptake.One of the open issues in realizing the new concept of incorporating stroma-derived factors into clinical diagnosis is which markers can be utilized as a gold standard for individual evaluation. It is always the case that patients have response heterogeneity upon treatments, and those who previously underwent anticancer regimens may not show continued clinical indexes, partially due to the chronic phenotypes of damaged cells including senescence, a fine-tuned cellular activity through biological evolution.The functional role of the TME as an incrementally recognized modulator of treatment responsiveness is emerging just in recent decade. The design and development of novel anticancer therapies will require a comprehensive and insightful appreciation of the pathological significance of the TME as well as an appropriate evaluation of new agents in relevant microenvironment systems that can recapitulate the disease progression in patients. Besides, these systems will accelerate the paces to adapt specific regimens to patient subgroups, which cannot be determined exclusively by the cancer cell innate features but also by biomarkers presenting the characteristics of their surrounding TME milieu. The “orthogonal” large-scale assessment of the molecular features and the therapeutic responsiveness of cancer cells in the TME will be the upcoming frontier for prospective novel therapy identification and their rapid translation to improve clinical outcomes across multiple malignancies.Current treatment strategies are focused on targeting the cancer cells, but mostly ignore the TME. Multiple types of stromal cells compose a pathologically active and metabolically dynamic niche essential for the development of resistant cancer cells, frequently through paracrine signaling with cancer cells. Despite the daunting challenge of acquired resistance and the complexities caused by tumor–microenvironment interplays, one should admit that targeted therapies and chemotherapeutics are effective in many pathological settings, significantly prolonging the lifespan of patients, or even producing cures in certain cases. Nevertheless, more lessons should be learned from experiences with first waves of conventional cytotoxic treatments and targeted drugs to employ the increasing arsenal of anticancer therapies. Excellent anticancer agent combinations are often proposed by recognition of in vitro and in vivo synergy between these pharmaceuticals; however, dampening a single pathway at multiple points or on separate signaling sections may sometimes provide a relatively simple “escape route” for the cancer, probably the most “cunning” disease that claims numerous lives per year. In contrast, orthogonal therapies that target oncogenic signaling pathways of cancer cells while manipulating released factors or key signal transduction nodes of the activated TME may provide an ideal and optimal option to open the avenues for advanced drug development and clinical administration to thoroughly abrogate cancer resistance in clinical settings. Most importantly, it is necessary to stratify patients according to the likelihood that they may respond to a specific singular treatment or certain combinatorial therapy. It is imaginable that powerful high-throughput techniques such as RNA-Seq, ChIP-Seq, miRNA chips, lncRNA profiling, and next-generation sequencing conveys a large pool of data that can be used to identify predictive biomarkers for therapeutic strategy per patient. On the other hand, a large array of human cell lines is used worldwide but whether they represent the best platform to establish clinically meaningful biomarkers and to appraise compound combinations is a matter of debate. Instead, patient-derived xenograft models that faithfully model patient tumor progression are increasingly applied to allow accurate assessment of disease resistance, evaluation of potential drug combinations, and determination of biomarker values in disease therapeutics. In a long run, preclinical studies should be translated into to the clinics for advanced evaluation, which necessitates the development of “intelligent” practical trials involving cutting-edge-front techniques of molecular pathology and large-scale protein arrays.This is an exciting era to study how TME distorts cancer therapy indexes in clinically relevant backdrops. With the rapid discovery of multiple new pathways and a myriad of functional components there remain many more questions than answers. Clearly, understanding the biological influence of microenvironments on disease evolution, and the existence of additional pathological factors associated with these conditions is of high public interest and holds research priority. With the use of a combination of neoadjuvant agents and targeted therapies together with more traditional discovery platforms, establishment and advancement of the long-sought preventive, precise, and personalized medicine finally seems within reach.


Iron is essential for life, while also being potentially harmful. Therefore, its level is strictly monitored and complex pathways have evolved to keep iron safely bound to transport or storage proteins, thereby maintaining homeostasis at the cellular and systemic levels. These sequestration mechanisms ensure that mildly reactive oxygen species like anion superoxide and hydrogen peroxide, which are continuously generated in cells living under aerobic conditions, keep their physiologic role in cell signaling while escaping iron-catalyzed transformation in the highly toxic hydroxyl radical. In this review, we describe the multifaceted systems regulating cellular and body iron homeostasis and discuss how altered iron balance may lead to oxidative damage in some pathophysiological settings. Metal-catalyzed oxidation of H2O2 can then originate hydroxyl radicals (HO∙), the most reactive ROS. Redox balance, that is, regulated production of ROS, is essential for normal cellular physiology, as deregulation in the production of oxidative species, that is, oxidative stress, causes DNA damage, lipid peroxidation, and aberrant posttranslational modification of proteins, thus leading to injury, cell death, and disease [2− and H2O2, particularly H2O2 that is more stable than O2− and can cross membranes, are considered signaling molecules because their levels under normal conditions remain under a physiological threshold and their synthesis is enzymatically regulated, whereas HO∙ is deemed to be a highly toxic reactant leading to permanent modifications of target molecules that can impact cellular function and life. In the cell, rapid and efficient conversion of H2O2 to HO∙ through the Fenton reaction requires the presence of transition metals, that is, copper and iron. While copper is a more efficient catalyst, iron is much more abundant and thus it is the actual key player in the so-called metal-catalyzed oxidative reactions [In eukaryotes, oxygen utilization by the mitochondria, NADPH oxidase enzymes, cytochromes, and so forth leads to the generation of reactive oxygen species (ROS), unstable and reactive molecules formed by one-electron transfers from a redox donor to molecular oxygen. The first of such products is anion superoxide , uptake (transferrin receptor (TfR1) and divalent metal transporter (DMT1)), storage (H and L ferritin subunits), and export (ferroportin) [The challenging task of maintaining intracellular iron levels sufficient for essential cellular functions, including ROS-dependent cell signaling, but as low as possible to avoid ROS-mediated injury, is controlled at multiple steps but primarily accomplished by iron regulatory proteins (IRP1 and IRP2), which strictly control intracellular iron metabolism by posttranscriptionally regulating the coordinated expression of proteins involved in iron utilization  , 16  , 16.μM range bound to low molecular weight compounds like citrate or glutathione [The activity of IRP1 and IRP2 is dictated by the size of the cellular labile iron pool (LIP), a pool of iron in the low tathione , which itathione , 16  as a cofactor, but only IRP1 can assemble a cluster when sufficient iron is available, thus functioning as cytosolic aconitase, which is the prevailing form in most cells. Conversely, in conditions of iron deficiency, the cluster is disassembled and the IRP1 apoform binds IRE , 16. IRPα (HIF2α) [The respective role and importance of IRPs have been revealed by studies involving gene deletion , 22. Abl (HIF2α) .2O2, which may be a phenomenon more correlated to the signaling role of H2O2 rather than to oxidative stress, it appears that IRP1 inactivation occurs in response to ROS both in vitro and in vivo [Although iron is the major regulator of the IRE/IRP network, other signals and conditions can modulate the activity of both IRPs, including oxygen tension and nitric oxide (reviewed in ). Not su in vivo , 16. Ove in vivo ). The ro in vivo .Readers can refer to recent excellent and comprehensive reviews for specific aspects of the IRE/IRP regulatory network , 27–29.Abundant evidence shows that a number of neurodegenerative disorders are characterized by regional iron accumulation in particular areas of the central and/or peripheral nervous systems , 31. ThiFriedrich's ataxia (FRDA) is a paradigmatic example because the disruption of iron homeostasis in this disease has been well defined . FRDA, wAlthough several studies provided evidence that ROS generated through Fenton reaction play a role in FRDA, the primary involvement and the importance of ROS in the pathophysiology of FRDA are still debated . HoweverThe role of iron in anthracycline cardiotoxicity is another illuminating example of the complex interplay and synergism between iron and free radicals as causative factors of apoptosis or other forms of cell damage. Doxorubicin (DOX) is an antineoplastic drug of the anthracyclines family, which plays a recognized key role in the chemotherapy for several types of cancer. However, anthracyclines have established risks of cardiotoxicity, as their chronic administration induces cardiomyopathy and congestive heart failure . This do2− and its dismutation product H2O2, which can then be transformed into the more potent HO∙ by reactions catalyzed by iron. In turn, HO∙ can damage DNA and proteins and initiate membrane lipid peroxidation, thus leading to cardiomyocyte death [The involvement of iron in DOX-induced cardiac damage is well established, and cardiotoxicity induced by DOX may occur at lower cumulative doses under conditions of iron overload . The advte death , 43.However, since antioxidants did not offer protection in clinical settings, the apparently obvious explanation for the aggravating role of iron in DOX cardiotoxicity based on increased iron-catalyzed ROS formation has been called into question , 41. In We also showed that anthracycline cardiotoxicity is related to ROS-dependent and ROS-independent disruption of cardiac iron homeostasis due to targeted interaction of DOX with IRP1, which leads to a “null” IRP1 devoid of both its functions and hence it is unable to sense iron levels and to regulate iron homeostasis. Moreover, DOX triggers IRP2 degradation, which may serve as a protective role by favoring iron sequestration in newly formed ferritin , 46. IndOn the other hand, recent results regarding mitochondrial ferritin (FtMt), a ferritin type particularly expressed in mitochondria-rich tissues, including the heart, where it prevents iron-mediated oxidative damage , reinfor∙.The importance of methodological aspects introduces some cautionary issues that should be taken into account when considering the discrepancies reported above regarding the pathophysiological relevance of iron-mediated ROS production in DOX toxicity. In fact, one should keep in mind that the model systems in which the mechanisms of DOX cardiotoxicity have been characterized have inherent limitations in representing the human chronic cardiomyopathy. Moreover, the dual role of ROS in signaling events and cell damage should be considered when evaluating if iron contributes to chronic cardiomyopathy by mechanisms not related to its ability to generate HOGiven the dual role of iron, elegant control mechanisms have evolved to maintain appropriate body iron levels by means of a complex network of transporters, storage molecules, and regulators. Intestinal iron absorption and iron recycling in reticuloendothelial cells are coordinately orchestrated in order to maintain iron levels in the circulation adequate for the needs of the various tissues and organs but insufficient to activate dangerous ROS production , 14.The task of keeping circulating iron in a safe but readily available form is performed by transferrin (Tf), a protein synthesized and secreted by the liver that binds up to two ferric iron atoms with high affinity and is the major iron transport protein. Under physiological conditions, Tf-bound iron is the main source of iron for the majority of tissues, primarily for bone marrow erythroid precursors, which consume iron for hemoglobin biosynthesis .Tf incorporates iron coming from two major sources: dietary iron (both inorganic and heme iron) absorbed in the duodenum to compensate for daily iron loss and iron derived from destruction of old and effete erythrocytes by reticuloendothelial cells in the spleen and liver . After rSpleen and liver macrophages specialized in recycling iron obtained from the phagocytosis and destruction of senescent erythrocytes are the main iron supplier for hemoglobin synthesis. The major pathway of heme iron recycling involves hemoglobin degradation by cytosolic heme oxygenase-1 and export of heme-derived iron into the circulation by ferroportin .Recent studies in mice with disrupted IRP1 and/or IRP2 in the entire organism or specific tissues have shown that IRPs are important regulators also of systemic homeostasis , but hep∙ production. However, under conditions of heavy systemic iron overload (primarily due to insufficient hepcidin production), the iron-binding capacity of Tf is exceeded, and non-transferrin-bound iron (NTBI) is formed (2+ channels, either L-type or T-type, particularly in cardiomyocytes [∙, which then cause lipid peroxidation and organelle damage and ultimately cell death (∙ production. In fact, it has been demonstrated that mitochondria rapidly acquire NTBI, and this, along with their high generation of ROS [Two mechanisms protect the cells from the damaging effects of excess body iron: the partial saturation of Tf at the systemic level and the IRP-dependent regulated expression of TfR1 at the cellular level. Under normal conditions, Tf saturation is around 30% and the protein can therefore bind excess iron entering the circulation, thus functioning as a protective shield against HOs formed . Despitemyocytes . NTBI toll death . In partn of ROS , props un of ROS . Althougn of ROS .The most important clinical conditions involving primary and secondary iron overload leading to iron-mediated tissue damage are genetic hemochromatosis and transfusional siderosis, respectively. Hereditary hemochromatosis linked to mutations in HFE, a MHC class I-like protein that is a necessary component of the iron-sensing machinery controlling hepcidin expression, is the most common genetic disease in Caucasians and presents a multisystem involvement: although iron overload first affects the liver, in hemochromatosis patients endocrine abnormalities, cardiac problems, and arthropathy are also common . Loss ofβ thalassemia, which is the secondary iron overload condition more closely linked to tissue iron overload [Secondary iron overload is mainly observed in association with transfusion-dependent diseases . Since ooverload , 72. Howβ thalassemia hepcidin levels are paradoxically low because erythropoiesis-dependent downregulation prevails over the upregulation associated with body iron levels; therefore, high intestinal iron absorption contributes to iron overload [With continued transfusion, reticuloendothelial cells can no longer safely store all the excess iron, which thus enters the circulation in amounts that exceed the binding capacity of Tf, and NTBI develops . It shouoverload , 62. Whioverload . Iron ch2O2, which is relatively stable and diffusible, is indeed a mild oxidant but is suitable for signaling. However, in the presence of ferrous iron, H2O2 can generate the highly reactive and toxic HO∙. Therefore, we propose that increased availability of iron not bound to proteins specifically evolved to transport or store this essential metal can make the critical difference between the two opposite functions of ROS. The view that iron plays a main role in the scenario leading ROS to become signal or stress agents is supported by the sophisticated systems regulating intracellular and systemic iron homeostasis that we have summarized in this review. Additional evidence indicating the contribution of iron emerges from the number of studies showing the occurrence of ROS-mediated tissue damage whenever the control of iron metabolism is disrupted, conditions of which we selectively highlighted a few illustrative examples. Improved understanding of the complex interplay between iron metabolism and redox homeostasis will clarify these pathways and their relevance in pathophysiology. In consideration of the disappointing results of antioxidant therapy for a variety of diseases [Presently, it is not completely clear as to why in some conditions ROS are associated with cell damage and scavenging high ROS levels improves metabolic homeostasis, whereas in other settings ROS exert signaling functions important for essential cell activities. Possible explanations for these distinct biological specificities of ROS action include differences in the amount, sources, duration, and localization of ROS production; in addition, certainly, the higher ROS reactivity is, the greater toxicity is, while signaling capacity is diminished. Therefore, Hdiseases , targeti


Congenital renal malformations are a major cause of childhood and adult onset chronic kidney disease. Identifying the etiology of these renal defects is often challenging since disruptions in the processes that drive kidney development can result from disruptions in environmental, genetic, or epigenetic cues. β-catenin is an intracellular molecule involved in cell adhesion, cell signaling, and regulation of gene transcription. It plays essential roles in kidney development and in the pathogenesis of renal dysplasia. Here, we review the function of β-catenin during kidney development and in the genesis of renal dysplasia. Renal dysplasia is a developmental disorder of the kidney that affects 1 in 250 live births  9.5 in the mouse when epithelial cells emerge from the intermediate mesoderm (termed the Wolffian duct) and migrate caudally toward the urogenital sinus Figure . At E10.Simultaneously, the metanephric mesenchyme receives signals from the ureteric epithelium, and responds by tightly clustering around the ureteric bud tips. These clusters of condensed mesenchyme form the nephrogenic progenitors, which are populations of cells destined to form the nephron Figure . DependiShortly after the invasion of the ureteric bud into the metanephric mesenchyme a third cell population, termed the renal stroma, is observed surrounding the condensed mesenchyme Figure . The renThe impairment of the processes that guide branching morphogenesis and nephrogenesis during kidney development can result in reduced nephron number and abnormal collecting duct formation, which can increase the risk of developing childhood renal failure or adult onset chronic renal insufficiency.β-catenin is an evolutionarily conserved multi-functional protein involved in various cellular activities depending on its intracellular localization  and the formation of pre-tubular aggregates  in the nephrogenic progenitor cells. These data suggest that β-catenin in the cortical stroma controls gene expression in neighboring cell populations possibly through direct cell-cell interactions or by β-catenin regulating secreted factors from the renal stroma. These possibilities are supported by studies that demonstrated renal stromal cells intermingle with the condensed mesenchymal cells and make direct contacts with the ureteric epithelium , Paired Box gene 2 (Pax2), and Transcription factor 2 (TCF2) to the genesis of renal dysplasia , Kidney Foundation of Canada, and CIHR (142264).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The detailed analysis of leaf growth dynamics, when coupled with transcriptomic research, can facilitate the discovery of genes required for leaf elongation.http://www.genomebiology.com/2015/16/1/168Please see related Research article:  The past two decades of plant science have been increasingly dominated by new and powerful ‘-omics’ technologies. Phenomics entails analyses of the entire spectrum of phenotypic traits in an organism, and is an essential component to understanding how genotype translates into phenotype. Compared to the relative ease with which the genome and transcriptome of a plant or plant organ can be analyzed, plant phenomics is more challenging owing to the sheer abundance of plant phenotypic characters and the inherent difficulties in studying phenotypic dynamics throughout the entire plant life cycle (ontogeny).Genome Biology from the Dirk Inzé laboratory [Most plant phenomics studies to date have been limited to macroscopic imaging of seedling stage or mature-stage phenotypes . X-ray tboratory , a novelLeaf organogenesis comprises three main stages . FirPrevious mutant screens have identified a number of interesting ‘master regulator’ genes required for leaf development, for which loss of function mutations result in extreme mutant phenotypes (reviewed in ). HoweveIn their high-throughput phenotypic study of maize leaf development, Baute et al.  used a dIn their study, they used an F12 population of 103 RILs derived from the maize inbred parental lines B73 and H99, and obtained detailed phenotypic analyses of multiple traits that correlated with the final size of leaf 4. Specifically, they assayed the mature-stage phenotypes of leaf 4: leaf length, width, area, and weight. The phenotypic data were complemented by high-throughput phenotypic analyses of the dynamics of leaf 4 growth over developmental time. These traits included the DZ size of leaf 4 at steady-state growth, time of leaf emergence from the whorl, leaf elongation rate (LER), time point of maximal LER, time point when leaf 4 reached its final length, and leaf elongation duration. Taken together, these data provide estimates of the length of time needed to fully expand leaf 4. An important conclusion stemming from this work is that final leaf size is strongly correlated with both rate and duration of leaf elongation, and more interestingly, that the mechanisms controlling the rate and duration of elongation are only partially shared.Baute et al.  then extArabidopsis genes implicated in cell division and/or leaf growth. These included maize homologs of growth-regulating factors 1 (GRF1) and 2 (GRF2), the TIR1/AFB auxin receptor protein, the brassinosteroid receptor DWARF1/DIMINUTO, several cytokinin-binding receptors (histidine kinases), and a number of genes predicted to function during protein ubiquitination  that show defects in mature leaf size when mutated in Arabidopsis.In the past decade, as RNA-seq has emerged at the forefront of transcriptomic research, the consensus is that these experiments are especially powerful when sampling is focused on discrete cells and tissues of isogenic plants, and during specific ontogenic time points . Baute eThis study has shown the power of phenomic analyses that focus on a discrete organ, in this case leaf 4, during an ontogenetic window encompassing leaf elongation to leaf maturity. The exciting discovery that the size of the DZ is correlated with mature-stage leaf 4 traits enabled the authors to extend their correlation analyses to include gene expression within the DZ. By focusing their phenomic and transcriptomic analyses on a discrete organ and developmental stage, the authors reduced the distracting background noise that can often contaminate a less focused analysis. This study has generated a rich and precise transcriptomic data set, and a model for leaf size determination that implicates separate pathways regulating the rate and duration of leaf elongation. This model is eminently testable. Genetic analyses of the differentially expressed gene transcripts identified in this study can now be combined with the focused and multidimensional phenotypic analyses described by Baute et al.  to advan


The sequencing of the human genome has opened doors for global gene expression profiling, and the immense amount of data will lay an important ground for future studies of normal and diseased tissues. The Human Protein Atlas project aims to systematically map the human gene and protein expression landscape in a multitude of normal healthy tissues as well as cancers, enabling the characterization of both housekeeping genes and genes that display a tissue-specific expression pattern. This article focuses on identifying and describing genes with an elevated expression in four lymphohematopoietic tissue types , based on the Human Protein Atlas-strategy that combines high throughput transcriptomics with affinity-based proteomics.C19ORF59.An enriched or enhanced expression in one or more of the lymphohematopoietic tissues, compared to other tissue-types, was seen for 693 out of 20,050 genes, and the highest levels of expression were found in bone marrow for neutrophilic and erythrocytic genes. A majority of these genes were found to constitute well-characterized genes with known functions in lymphatic or hematopoietic cells, while others are not previously studied, as exemplified by in situ affinity-based proteomics in order to identify and describe new gene targets for further research on lymphatic or hematopoietic cells and tissues. The results constitute lists of genes with enriched or enhanced expression in the four lymphohematopoietic tissues, exemplified also on protein level with immunohistochemical images.In this paper we present a strategy of combining next generation RNA-sequencing with  The effort is based on a unique set-up of high throughput generation of affinity-purified polyclonal antibodies for Mapping the human proteome is challenging, as evidence on protein level is missing for more than 30% of the human protein coding genes In this article we explore the gene expression profiles in bone marrow, spleen, lymph node and appendix using transcriptomics analysis using next generation deep sequencing (RNAseq). These four tissues, differing in both histology and function, were selected because they have in common the feature of harboring a major cell population of hematopoietic origin, i.e. lymphoid cells. Bone marrow is considered a primary lymphoid organ, and displays the widest range of hematopoietic cells, as it also constitutes the site for hematopoiesis. Secondary lymphoid organs lymph node, appendix and spleen harbor a pronounced population of lymphoid cells, as these are primary sites for e.g. antigen-driven affinity maturation, somatic hypermutation and immunoglobulin class switch of bone marrow derived naïve B-lymphocytes in germinal centers, and also proliferation. In appendix, the mucosa-associated lymphoid tissue (MALT) contains areas of lymphoid tissue encountering antigens passing through the glandular epithelium. As such, appendix is expected to harbor a wide range of cell types, since although the lymphoid tissue consists mostly of B- and T- lymphocytes, it also includes a glandular epithelium and smooth muscle.The aim of this study was to define the expression profiles of four lymphohematopoietic tissues with a common component of lymphoid cells, and to potentially identify previously uncharacterized proteins within these tissues. The results do not by themselves provide deeper understanding of gene functions, but offer an important foundation for insight into possible mechanisms of identified proteins.RNAseq analysis of 20,050 protein-coding genes was performed using Illumina HiSeq2000 and 2500 (Illumina) on 95 samples representing 27 different tissue types This article focuses on the expression profile in four tissues  harboring a major component of cells of hematopoietic and lymphatic origin. In these four tissues, FPKM values ranged from 0.1 to 25,947, and the maximum FPKM value for each tissue type is indicated in Three to five samples for each tissue type were analyzed, and the correlation between biological replicates was high , is a glycoprotein and is used as a diagnostic biomarker for several cancer types, e.g. breast and colon cancer GAL codes for a small neuropeptide that functions as a cellular messenger within the central and peripheral nervous systems GAL was confirmed by IHC staining of ganglion in e.g. colon  is low both for bone marrow, 137 (0.7%), and secondary lymphoid tissues, 104 (0.5%), suggesting that a large portion of the transcriptome is shared among several tissues. The corresponding numbers of enhanced genes  were 164 (0.8%) in bone marrow and 340  in the secondary lymphoid organs.DEFA1, DEFA1B, DEFA3, DEFA4, CTSG and MPO) and erythrocytes , both of which reach maturity in bone marrow and are released into the blood stream as effector cells, equipped with necessary proteins for their specialized functions. Consequently, a high level of transcription of these genes takes place in bone marrow, explaining the high FPKM-values. This is also evident in When taking into account the number of detected transcripts from each gene , it becoAltogether 215 genes were found to be tissue enriched or group enriched in at least one of the four tissues of lymphoid or hematological origin. 149 of these were included in a network analysis performed using Cytoscape 3.0 Using RNAseq to identify genes with a unique expression in these four lymphohematopoietic tissues is complicated by the fact that immune cells, of both lymphocytic and myelocytic origin, are also naturally occurring in many other peripheral tissues. This circumstance is particularly evident for secondary lymphoid tissues, for which very few genes with a distinct tissue-characteristic expression were identified.CD19, CD20 (MS4A1) or constitute well known cytokines, whereas some are less described  or uncharacterized (C19ORF59 and AC233263.1). The protein expressions of above-mentioned genes are illustrated in The analyses on tissue homogenates still poses a valuable strategy for large scale determination of expression patterns, and in combination with IHC it offers the possibility to identify immune-specific gene expressions. Proteomic data generated using IHC within the Human Protein Atlas initiative in situ information generated by IHC, which is critical when analyzing differentially expressed genes within complex tissues. One prerequisite for inclusion of a protein in the manuscript was therefore the availability of an IHC staining using a stringently validated antibody that supports the RNA-data. The specific examples have been chosen to illustrate i) well-characterized proteins, with known functions in lymphohematopoietic cell types, to validate the set up of the study, as well as ii) previously uncharacterized proteins, to exemplify the potential of the set up to pick up potential proteins of interest for further studies.Due to the large amount of data generated, the following section aims to merely exemplify proteins with an enriched or enhanced expression in lymphohematopoietic tissues identified with the large scale set-up described in this paper. The examples aim to visualize the additional FAM129C, TMC8 and RASGRP2, were found to be group enriched in the four tissues , TNFRSF13C and FCRLA, which are all known to be expressed by B-lymphocytes Sixteen genes were found to be group enriched in appendix, lymph node and spleen, e.g. Bone marrow was the tissue with the highest number of enriched genes. Twenty highly and 60 moderately tissues enriched genes, as well as 57 group enriched genes were found for bone marrow. Most of these are previously well-studied genes, but several uncharacterized genes were also found. Furthermore, 164 genes were found to have an enhanced expression in bone marrow. The relatively great number of genes with an enriched or enhanced expression in bone marrow may reflect the fact that bone marrow represents the sole tissue for generation of all hematopoietic cell linages. It thereby displays the widest repertoire of genes regulating hematopoiesis, especially those genes expressed in the prominent erythro- and myelopoeses.CTSG and DEFA4 are two of the genes with the highest expression in bone marrow , and they were also categorized as highly enriched. The protein cathepsin G is found in the azurophil granules of neutrophils and has bactericidal properties CTSG and DEFA4 show strong staining of granulocytes  was HEMGN and SPATC1. The protein Hemogen is fairly well characterized, and appears to be primarily expressed in immature precursors, and to be involved in hematopoietic differentiation HEMGN has been shown to be a transcription factor specifically involved in sex determination HEMGN in bone marrow and testis  among the tissues/group enriched and enhanced genes for lymph node.The protein CCL21 is expressed in secondary lymphoid organs (e.g. lymph node) and is known to attract T- and B-cells and dendritic cells via their chemokine receptor (CCR7) TCL1A displayed an enhanced expression in lymph node, and IHC staining using antibody HPA016604 supports the RNA-data, showing positivity in lymphocytes mainly in B-cell areas of the lymph node , about which nothing is known. Transcript data displays enhanced expression also in colon, appendix and urinary bladder, and immunohistochemical data (antibody HPA047702) supports this with distinct staining in a subset of immune cells in these tissues. Also enhanced in lymph node was gene RCSD1, enhanced in lymph node, has recently been identified as a novel gene fusion partner of the ABL1 gene in B-lymphoblastic leukemia RCSD1 is expressed in all four lymphohematopoietic tissues. Six genes were defined as moderately tissue enriched in spleen, 57 were group enriched, and 129 genes showed an enhanced gene expression in spleen. Several of the spleen characteristic genes are previously uncharacterized.GPR182 was moderately enriched in spleen, suggesting a function in the immune system. However, IHC showed expression of the protein in sinus endothelium, with the strongest staining seen in spleen , 56 were group enriched, and 73 genes displayed an enhanced expression. A few uncharacterized genes were found in these categories.FPR1 (FPKM 131), which is categorized as enhanced in appendix as well as in bone marrow (FPKM 100). FPR1 codes for a G protein-coupled receptor protein expressed by e.g. neutrophils, and plays a role in chemotaxis, phagocytosis and generation of reactive oxygen species One of the most highly expressed genes found in appendix was Transcriptomics of high quality human tissue samples, evaluated by pathologists for representativity, were analyzed to identify expression of tissue-specific genes in 27 different tissues Fresh frozen material from 27 different morphologically normal tissues was included in this study . TissuesNext generation mRNA-sequencing was performed as earlier described Raw reads obtained from the sequencing system were trimmed for low quality ends using a software called Sickle In the present global study, we observed approximately 0.1% of misidentified reads for samples that were sequenced in a multiplexed way. This has been previously observed For each tissue, the average FPKM value for all replicates was used as a measure of gene expression level. A cut-off value of FPKM = 1 was set as the limit of detection. Each of the 20,050 genes was classified into one of seven categories based on the expression levels over all 27 tissues .Large-scale protein profiling within the Human Protein Atlas project has been previously described The data set supporting the results of this article is available in the version 13 of the Human Protein Atlas, and the primary data (reads) are available through the Array Express Archive S1 FigImmunohistochemical staining of CEACAM5 and GAL. The staining patterns of CEACAM5 in colon and colorectal cancer, and of GAL in colon are shown. CRC: colorectal cancer.(TIF)Click here for additional data file.S2 FigPie charts comparing the gene expression between the lymphohematopoietic tissues. The percentages of genes expressed within the different categories of expression are shown for appendix, bone marrow, lymph node and spleen.(TIF)Click here for additional data file.S1 TableFresh frozen material from 27 different morphologically normal* tissues was included in this study. * All cases of appendix represent cases of appendicitis.(XLSX)Click here for additional data file.S2 TableAll genes with elevated expression in the four lymphohematopoietic tissues . Genes in bold are exemplified in the paper and shown in (XLSX)Click here for additional data file.


Stopping the succession of events that lead to development of Parkinson’s disease (PD) is a principal challenge that requires a brain protective approach in early diagnosed patients. Although PD ethiopathology may not have a single causative factor, information from sporadic and familial cases together with chemical and genetic animal models strongly suggests that low-grade chronic inflammation and oxidative stress play a critical role. Our team is studying the relevance of the transcription factor NRF2 (Nuclear factor (erythroid-derived 2)-like 2), a master regulator of oxidant and inflammatory defense, as a new therapeutic target in PD. The pro-inflammatory activation of microglia and astroglia in response to LPS, MPTP and human α-synuclein over-expression is exacerbated in Nrf2-deficient mice, thus demonstrating an immunomodulatory role of this protein. In PD patients, some evidence gathered from epidemiological, genetic and anatomopathologic studies also support a protective role of NRF2. Several compounds activate NRF2 and provide an immunomodulator and cytoprotective response in preclinical animal models of PD. At this time a crucial point to translate these promising results to the clinic is the discovery of a drug with good pharmacokinetics and pharmacodynamics that fulfill criteria of safety, tolerability and efficacy. The use of repurposing drugs, such as dimethyl fumarate used nowadays for treatment of remitting relapsing multiple sclerosis, may provide excellent candidates.