There seems to be no consensus concerning taking bladder biopsies during transurethral resection of bladder tumor (TUR-BT). We investigate the clinical significance of bladder biopsy with TUR-BT and the relationship between urinary cytology and the biopsy results.We reviewed a total of 424 patients with non-muscle invasive bladder cancer treated with TUR-BT between 1998 and 2005. Of the total, 293 patients also underwent a bladder biopsy. Biopsies from suspicious-appearing urothelium (N = 59) and those from normal-appearing urothelium (N = 234) were evaluated separately.Bladder cancer was observed in 23 cases (39.0%) who underwent a biopsy of suspicious-appearing urothelium. Among these 23 cases, 9 cases with visible tumor resection had carcinoma in situ (CIS) only in the biopsies from suspicious-appearing urothelium. Urinary cytology was negative in 3 of the 9 cases. Bladder cancer was observed in 26 cases (11.1%) who underwent a biopsy of normal-appearing urothelium. Of them, 5 cases with visible tumors had CIS only in the multiple biopsies from normal-appearing urothelium. Urinary cytology was positive in all of the 5 cases. No upstaging or upgrading cases were found in these patients by the addition of these two types of biopsy. Furthermore, therapy was not altered in these patients. With or without bladder biopsy was not a significant factor for tumor recurrence in either the univariate or multivariate analysis.Based on the results, it is concluded the multiple biopsies from normal-appearing urothelium are not necessary in patients with negative cytology results because of the low detection rate and lack of influence on therapeutic decisions. Meanwhile, biopsy of suspicious-appearing urothelium is needed in patients with negative cytology results in order to detect CIS due to staging properties. This result supports a recent EAU guideline. Bladder cancers are classified as being non-muscle-invasive or muscle invasive according to their histological appearance. Non-muscle-invasive bladder cancers account for about 70 to 80% of all bladder cancers-4. Non-mIn the present study, we evaluated 1) the clinical significance of bladder biopsies from suspicious-appearing urothelium and multiple biopsies from normal-appearing urothelium separately, 2) not only tumor up-staging but also up-grading of these biopsies, 3) the relationship between urinary cytology and biopsy results, 4) its impact on further treatment decision-making, and 5) whether or not additional bladder biopsies promote the intravesical recurrence of bladder carcinoma.Between 1998 and 2005, 424 patients with non-muscle invasive bladder cancer were treated with TUR-BT at Keio University Hospital in Tokyo. The mean age of the patients was 68.7 ± 0.57 (25-92) years old. The patients consisted of 349 men and 75 women. The median follow-up period was 3.6 ± 0.1 years. Bladder biopsies in 59 patients had been performed from reddish or mossy areas apart from the main tumor and edema of the bladder mucosa (biopsy from suspicious-appearing urothelium). Multiple biopsies of apparently normal mucosa were performed systemically in 234 patients  during TUR-BT. Thirty-four patients underwent both a biopsy from suspicious-appearing urothelium and multiple biopsies from normal-appearing urothelium. Thus, a total of 293 patients underwent bladder biopsies. Basically, all patients with reddish or mossy areas apart from the main tumor and edema of the bladder mucosa underwent biopsy from suspicious-appearing urothelium. Meanwhile, multiple biopsies from normal-appearing urothelium were performed based upon the decision of the attending doctor. Multiple biopsies from normal-appearing urothelium were carried out to examine the following 6 cystoscopically normal-appearing bladder mucosal sites: (1) bladder floor, (2) right wall, (3) left wall, (4) dome, (5) posterior wall, and (6) prostatic urethra  or bladder neck .The study was conducted subject to the guidelines of the Declaration of Helsinki and had no influence on the treatment of the patients. Keio university hospitals ethical committee granted ethical approval for the study. We obtained the patients' informed consent to carry out the surgery including their approval for potential use of their anonyms medical data from our data base for research and audit purposes.Patient characteristics, including age, sex, tumor grade, initial number of tumors, pathological stage, the presence of CIS, and preoperative cytology were determined. If the urinary cytology was class III or greater, it was defined as positive.BCG was scheduled for weekly administration for 6 weeks for stage Ta-T1 tumors and for 8 weeks for CIS at a dose of 80 mg of Tokyo strain or 81 mg of Connaught strain of BCG in 40 mL of saline instilled into the bladder by way of an 8 Fr urethral catheter, with retention for 1-2 hours.These patients were assessed by urine cytology and cystoscopy every 3 months for 2 years after the initial treatment, every 6 months for the next 3 years, and then yearly thereafter. Intravenous urography or ultrasonography and/or CT scanning was used to evaluate the bladder and kidneys every 6 months for 5 years after the initial treatment, and then yearly thereafter. Histopathological extensions and grade were classified according to the TNM classification 1997 and WHO The indicators of intravesical recurrence risks due to bladder biopsies were analyzed using univariate and multivariate Cox regression models. Indicators including sex, first or 2 or more TUR, grade, multiplicity, T stage, BCG therapy, presence of CIS, and enforcement of bladder biopsy were also analyzed.The pathological characteristics of the main bladder cancers in the TUR-BT specimens of the 424 patients are listed in Table The pathological results of biopsies from suspicious-appearing urothelium in 59 patients are shown in Table In 34 patients who underwent biopsies from suspicious-appearing urothelium and multiple biopsies from normal-appearing urothelium, no tumor was observed in the biopsies from normal-appearing urothelium.The pathological results for 234 patients who received multiple biopsies from normal-appearing urothelium alone with TUR-BT are summarized in Table We stated the differences in the backgrounds of patients who underwent biopsy and those who did not Table . As showThe clinical significance of multiple biopsies from normal-appearing urothelium is still controversial. Several studies have demonstrated the usefulness of multiple biopsies from normal-appearing urothelium,10,13,20In the present study, cancer tissue was detected in the multiple biopsies from normal-appearing urothelium in 26 patients. No up-staging cases were found. We also focused on tumor grading in this study. In these cases, no up-grading was found. Furthermore, all 5 CIS cases that were only detected in the multiple biopsies from normal-appearing urothelium had positive cytology results. Three cases were recognized as multiple tumors by also performing multiple biopsies from normal-appearing urothelium. These three cases had positive cytology results. In total, 8 cases were recognized as having more malignant features, including CIS and multiplicity, due to the biopsy results. As all of these cases had visible T1 or high grade or multiple tumors in the TUR specimen, they were treated with BCG instillation according to the TUR results. Therefore, therapy was not altered based on the biopsy results. Furthermore, no case with a T2 specimen was found in the multiple biopsies from normal-appearing urothelium. Taken together, multiple biopsies from normal-appearing urothelium did not provide any further information to assist with the treatment decision. Thus, we conclude that multiple biopsies from normal-appearing urothelium in negative cytology cases would not be of any real benefit.In our study, biopsies from suspicious-appearing urothelium were performed in 59 cases, and cancer tissue (no up-staging case and up-grading were found) was detected in 23 patients (38.9%) and CIS was detected in 13 patients (22.0%). Nine cases had CIS only in the biopsies from suspicious-appearing urothelium. Of these 9 cases, 3 patients had negative cytology results. Altogether, 13 cases who underwent biopsies from suspicious-appearing urothelium were diagnosed as having more malignant features than that in the TUR specimen alone. However, almost all of these cases had visible T1 or high grade or multiple tumors in the TUR specimen. Since this was the same result as for multiple biopsies from normal-appearing urothelium, a change in clinical treatment based on the biopsies from suspicious-appearing urothelium was not undertaken in these 13 cases in our hospital.The bladder mucosa after intravesically administered BCG or chemotherapy mimics the irregular carcinomatous mucosa. In our population, 6 patients underwent biopsy from suspicious-appearing urothelium within 6 months after BCG therapy. Three of these patients had positive results . Therefore, the other 3 patients had no malignancy in their biopsy specimens. Two patients underwent biopsy of suspicious-appearing urothelium within 6 months after intravesical chemotherapy. These patients had no malignancy in their biopsy specimens.The EAU guideline states that, except for a papillary tumor, if the rest of the bladder mucosa appears normal and if urine cytology is negative, it is not advisable to routinely perform multiple biopsies from normal-appearing urothelium because the likelihood of detecting CIS is extremely low and the choice of adjuvant intravesical therapy is not influenced by the biopsy result. This recommendation of the EAU guideline is based on the EORTC study-4. HowevSeveral reports have suggested that bladder biopsy provokes implantation of tumor cells in biopsy sites,10,14-17There are several limitations to our study. First, the results were analyzed in a retrospective fashion. There is also a lack of reproducibility in the determination of histological aberrations such as dysplasia, metaplasia, and hyperplasia in the biopsy specimens. Therefore, the interpretation of histological abnormalities in the bladder biopsies is somewhat controversial. There is a significant bias in regard to our data taken from non-standardized reports from multiple pathologists. The high inter- and intra-pathologist variation indicates that these alterations are difficult to interpret and provide no reliable clinical value for the urologist. However, at our institution, all pathological and cytological specimens were carefully reviewed by uro-pathologists and well trained cytologists. Another limitation is that a second TUR was not performed in the diagnosis and treatment of bladder tumor in our cases because they were not routinely performed at our institution. No maintenance BCG therapy or immediate intravesical chemotherapy was performed in the study period.Bladder cancer was detected in 26 of 234 cases (11.1%) by performing multiple biopsies from normal-appearing urothelium in addition to TUR-BT. In total, 8 cases were recognized as having more malignant features, including CIS and multiplicity, due to the biopsy results. The 8 cases had all positive cytology results. The therapeutic decision was not altered in these 8 cases. Thus, our findings suggest that multiple biopsies from normal-appearing urothelium would not be of benefit and are therefore unnecessary in patients with negative cytology results. On the other hand, the positive rate for detecting CIS  in biopsies from suspicious-appearing urothelium was relatively high. Meanwhile, 3 patients with CIS only detected in the biopsies from suspicious-appearing urothelium had negative cytology results. In patients with negative cytology results, suspicious-appearing urothelium would be needed in order to detect CIS because of staging properties. This result supported the recent EAU guideline.The authors declare that they have no competing interests.MH, KM and MM formulated database. MM performed the initial analyses and drafted the first manuscript. All authors assisted in the analysis and interpretation of data. EK conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2490/10/12/prepub


LMP-420 is a boronic acid-containing purine nucleoside analogue that transcriptionally inhibits TNF production but is non-cytotoxic to TNF-producing cells.This study investigated the efficacy of LMP-420 as an anti-inflammatory agent in acute and chronic colitis induced by oral administration of dextran sulfate sodium (DSS) to mice and in chronic colitis following piroxicam administration to IL-10-deficient mice. The severity of colon inflammation was assessed histologically. TNF levels were measured by enzyme immunoassay.-/- mice deficient in T and B cells also developed severe ongoing colitis in response to 3 cycles of DSS, but showed marked differences vs. wild type mice in stool TNF and TNF-RII in response to DSS exposure. Systemic and oral LMP-420 treatment for 16 days decreased colonic TNF levels in IL-10-deficient mice with chronic colitis, with a trend to decreased histologic inflammation for oral LMP-420.Administration of DSS for 7 days resulted in severe acute colitis that was associated with a marked increase in stool and colon tissue TNF levels. Initiation of therapy with intraperitoneal (i.p.) LMP-420 on day 4 of DSS exposure decreased colonic TNF to near normal levels on day 7. However, neither i.p. nor oral treatment with LMP-420 affected the development or severity of acute DSS colitis. Initiation of LMP-420 therapy after 3 cycles of DSS administration to establish chronic colitis also had no effect on the severity of chronic colitis. Analysis of colonic TNF combined with longitudinal analysis of TNF and TNF receptor (TNF-RII) levels in stool during the development of chronic DSS colitis demonstrated that the initially elevated colonic TNF levels returned to normal despite intense on-going inflammation in mice with chronic colitis. RAG-2These studies demonstrate that short-term treatment with a transcriptional inhibitor of TNF production can decrease systemic and local colonic levels of TNF but may not decrease the histologic severity of colitis. Longer term studies using colitis models that are more dependent on TNF elevation should be performed to more accurately assess the potential of LMP-420 for therapy of inflammatory bowel disease. Inflammatory bowel diseases such as Crohn's disease (CD) and ulcerative colitis (UC) are hypothesized to result from abnormal immune responses to antigens derived from intestinal microbiota  that in vitro in the presence or absence of complement . Using Stool for cytokine analysis was collected before DSS exposure began (day 0), at the end of each 5 day cycle of DSS administration . Freshly obtained stool was kept on ice until it was homogenized at 100 mg stool/ml buffer in PBS containing 1% bovine serum albumin, 0.1% Kathon , and Protease Inhibitor Cocktail . Insoluble material was removed by centrifugation at 4°C at 15,000 × g in a microfuge for 10 min. Stool extracts were filtered at 0.2 μm then stored at -20°C until assayed. Fresh colon samples were homogenized at 100 mg tissue/ml buffer using a PowerGen 125 tissue grinder  and the BioPlex Cell Lysis Kit  according to the manufacturer's instructions. Colon tissue extracts were then frozen at -20°C until analysis. TNF and TNF-RII were quantitated in tissue culture supernatants, stool, and colon tissue extracts by enzyme immunoassay using Duo-Set reagents . Results were expressed as pg/ml for culture supernatants or as pg/100 mg tissue or stool.Statistical comparison of groups was performed using Student's t test or analysis of variance (ANOVA). A value of p ≤ 0.05 was considered to be significant.in vitro to macrophages and lymphocytes, cell types that are present in the colonic lamina propria  by LPS-stimulated murine monocytes in vitro is ~500 nM . The molecular basis for the ~10-fold increased sensitivity of human vs. murine cells to LMP-420 is unclear, but suggests that LMP-420 is likely to have greater anti-inflammatory activity in humans than in mice.LMP-420 very effectively inhibits production of TNF mRNA and protein when applied topically in vitro profile, LMP-420 was hypothesized to be an efficient inhibitor of TNF in vivo. The degree of systemic and colonic TNF blockade that resulted from different LMP-420 in vivo dosing protocols was first assessed in wild type mice using a bacterial lipopolysaccharide (LPS) challenge model. Mice treated with 2 mg (100 mg/kg) LMP-420 given intraperitoneally (i.p.) experienced transient behavioral depression consistent with hypotension, but recovered to normal behavior within 20 minutes after injection. TNF was below the limit of detection (< 10 pg/ml) in the serum of mice not challenged with LPS. As expected, serum TNF levels were markedly increased to 1109 ± 87 pg/ml  upon LPS challenge. However, pre-treatment with i.p. LMP-420 decreased LPS-induced serum TNF levels by 42%  and total colonic TNF levels  compared to mice who were not exposed to DSS  (p = 0.03). DSS-exposed mice that were treated with i.p. LMP-420 demonstrated a significant (85%) decrease in colonic TNF levels , that represented near normalization of their colonic levels of TNF compared with control mice that were not exposed to DSS. TNF was not detected in the serum of either vehicle- or LMP-420-treated DSS-exposed or control mice at this time point.The LPS challenge experiments demonstrated that both i.p. and orally-administered LMP-420 significantly inhibited Despite the efficacy of i.p. LMP-420 in decreasing colonic levels of TNF induced by DSS exposure, there was no difference in clinical or histologic severity of colitis observed on day 7 in mice treated i.p. with vehicle- vs. LMP-420 (data not shown). However, the 3 day treatment period used was likely too short to allow healing. Longer treatments were not possible in the acute DSS model due to severe weight loss that required euthanasia for humane reasons. To determine if LMP-420 treatment could prevent the development of acute DSS colitis, LMP-420 was administered i.p. for 5 days prior to as well as throughout the 7 day exposure to DSS. Oral administration of LMP-420 was not used for these studies, since mice rapidly decrease their food consumption when acute colitis develops and it was not possible to maintain a consistent oral dose using drug mixed with food. Systemic (i.p.) treatment with LMP-420 given prior to and during DSS exposure did not alter the clinical or histologic severity of colitis Figure . The sevNext, we determined whether LMP-420 treatment would influence the severity of chronic colitis initiated by multiple cycles of DSS exposure. Treatment with a dose range of LMP-420 was begun immediately upon discontinuation of the 3d DSS cycle when all mice had severe chronic colitis. The LMP-420 dose range was 0, 5, 15, 45 mg/kg/day for i.p. administration and 0, 26, 63, and 145 mg/kg/day for oral administration. Mice were euthanized after 16 days of treatment to determine colonic TNF content and the histologic severity of colitis. Although gross inflammation  was subjectively decreased in LMP-420-treated mice, there was no difference in histologic scores between treated and untreated mice Figure . DespiteThe effects of LMP-420 on TNF production was profound in acute DSS colitis, but LMP-420 therapy apparently had no effect on inflammation severity in either acute or established chronic DSS colitis. Furthermore, colonic TNF levels were not elevated in untreated control mice with chronic DSS colitis, despite the presence of very severe colonic inflammation . To begin to understand these observations, a longitudinal study of the levels of TNF in the stool was performed. Stool samples were obtained prior to the initial DSS exposure (day 0), at the end of each 5 day cycle of DSS administration , and after 16 days of healing prior to beginning the next cycle of DSS administration . Because levels of soluble TNF receptors increase during inflammation due to receptor shedding after binding TNF and/or increased alternative splicing that generates the soluble form, the levels of TNF-RII  in stool were also measured. In wild type C57BL/6 mice, TNF levels in stool were significantly increased compared to baseline levels at the end of each cycle of DSS administration. Stool TNF then spontaneously decreased to normal levels by the end of each 16 day period of recovery Figure . Stool l-/- mice that lack both T and B cells were exposed to the same regimen of 3 cycles of DSS that induced severe chronic colitis in wild type mice, then euthanized 16 days following the 3rd administration of DSS. All RAG-2-/- mice had severe colitis after exposure to 3 cycles of DSS, with histologic scores of 53 ± 1 . These scores were statistically similar to those observed in wild type mice exposed to a similar regimen of 3 DSS cycles, however the histologic picture was very different. In contrast to the marked mucosal hyperplasia seen in wild type mice, RAG-2-/- mice exhibited minimal mucosal hyperplasia. Mucosal histologic sub-scores were high nonetheless, due to marked squamous metaplasia in the rectum and architectural distortion throughout the colon, manifested primarily by crypt branching and crypt dropout. Inflammatory infiltrates in the RAG-2-/- mice consisted of a small number of mononuclear cells and a moderate to large number of neutrophils, and these infiltrates were more uniformly spread through the tissues. Frank ulcerations and crypt abcesses were present, but less common compared with the wild type mice.Acute DSS colitis has been shown to occur in the absence of T and B lymphocytes , however-/- mice initially developed higher mean levels of TNF in their stool following DSS exposure, compared to wild type mice . The TNF levels in colon tissue measured in a subset of these mice (n = 5) was 72 ± 3 pg/100 mg tissue, which is similar to that seen in control wild type mice without colitis  or oral LMP-420  for 16 days. Effects on colonic TNF and histologic severity of colitis were determined. Colonic levels of TNF were elevated in untreated IL-10-/- mice with colitis compared with wild type mice without colitis  and colonic TNF responses (-67%) to challenge with a lethal dose of LPS. LMP-420 treatment also reduced (-85%) TNF elevations associated with acute DSS colitis to near baseline levels. However, despite its efficacy in reducing TNF levels in vivo, LMP-420 had no effect on the histologic severity of colonic inflammation in response to either acute or chronic DSS administration. A trend toward decreased inflammation (p = 0.06) was observed in IL-10-/- mice treated orally with LMP-420, which correlated with decreased colonic TNF levels. Histologic healing of colonic inflammatory lesions is known to lag behind clinical remission and endoscopic healing. Thus, it is possible that LMP-420 treatments of longer durations might result in beneficial clinical effects that were not observed in this pilot study using short treatment periods.The novel small molecule drug LMP-420 is highly effective in inhibiting TNF production both in vivo. Spohn et al. recently showed that anti-TNF antibodies that bound to both membrane-bound and soluble forms of TNF had a greater anti-inflammatory effect in a murine model of rheumatoid arthritis than antibodies reactive only with soluble TNF [The availability of LMP-420, a small-molecule, orally-active inhibitor of TNF production, provided us an attractive opportunity to define the potential role of TNF in the murine model of DSS-induced colitis, a commonly-used model of inflammatory bowel disease. Although we were able to demonstrate significant inhibition of colon TNF by LMP-420 in this model, we had no effect on the pathological inflammation. However, in contrast to the biological TNF antagonists currently used in humans which are capable of "neutralizing" essentially all circulating TNF, LMP-420 allowed ~20% of colon TNF to be produced in our model. Thus, while our data might be interpreted to suggest that TNF does not play a significant role in this model, we cannot at this time rule out the possibility that the small amount of TNF produced was sufficient to induce pathogenesis. Alternatively, prevention of TNF synthesis without toxicity to TNF-producing cells may not be sufficient to stop the inflammatory cascade uble TNF . Furtheruble TNF . Thus, iuble TNF  despite et al, who previously reported that TNF was not detectable in colon tissue or plasma of CBA/J mice with acute DSS colitis, and that a polyclonal anti-TNF antiserum had no effect on disease severity [et al showed that TNF-deficient mice have increased intestinal inflammation in response to DSS compared with wild type mice [Levels of TNF excreted in the stool correlated well with levels of TNF measured in colon tissues harvested at the time of euthanasia, providing a method to follow colonic TNF levels non-invasively. Longitudinal measurements of TNF in the stool of mice subjected to multiple cycles of DSS demonstrated that, although TNF levels are elevated in acute DSS colitis, these levels decrease spontaneously and eventually return to baseline despite ongoing severe inflammation. Taken together with the lack of efficacy of LMP-420 in acute or chronic DSS colitis despite its ability to significantly lower colonic TNF levels, these data suggest that elevated levels of TNF are not required for the initiation and maintenance of colonic inflammation in this murine model. Our data thus is similar to that of Olson severity . Howeverype mice . These c-/- mice, in contrast to the markedly higher TNF-RII levels observed in stool of wild type mice subjected to multiple cycles of DSS. The manufacturer reports that the presence of TNF-RII does not affect the ability of its TNF ELISA assay to quantitiate TNF. Thus additional mechanisms beyond simple shedding of receptor that has bound TNF likely account for the marked and sustained increase in stool TNF-RII during chronic DSS colitis in wild type mice. The induction of severe chronic colitis following repeated DSS administration to RAG-2-/- mice that lack T and B cells suggests that innate immunity is sufficient to drive the development of chronic colitis in the DSS model. Use of other models that are TNF-dependent or driven by induced T cell responses will be necessary to determine if LMP-420 may have efficacy in maintaining remission of chronic colitis driven by TNF or T cells.Our studies showed that levels of TNF and TNF-RII in the stool are highly correlated for RAG-2-/- mice. The presence of squamous mucosa is typically limited to the anus in both humans and mice. The simple columnar epithelium that is normally present in the terminal colon and rectum functions to absorb fluid from stool as well as to secrete mucus to lubricate its passage. These functions would be missing from metaplastic squamous epithelium. By analogy with other organs, it is possible that mice with extensive squamous metaplasia of the colon might be at increased long-term risk for development of squamous carcinomas of the colon rather than the adenocarcinomas that typically develop at this site. Longer term studies will be needed to address this possibility.The induction of extensive squamous metaplasia in the terminal colon and rectum by multiple cycles of DSS administration is an interesting pathologic observation that is of uncertain clinical significance. Metaplasia has been observed in a range of organs and is thought to occur in response to chronic irritation . T and B-/- model of murine colitis. As we saw for acute DSS colitis, LMP-420 treatment  significantly decreased colonic TNF levels in a setting where TNF was elevated in the colons of mice that did not receive this drug. However, again we saw no statistically significant decreases in severity of colitis as measured histologically, although there was a trend to decreased histologic inflammation for mice that received 41 mg/kg oral LMP-420. Increased potential effect of oral as compared with systemically administered drug suggests that LMP-420 may exhibit local anti-inflammatory activity as it passes through the gastrointestinal tract. Lack of TNF lowering in the IL-10-/- model at higher LMP-420 doses indicates a complex dose-response profile that may reflect dual activity of LMP-420 in competing inflammatory/anti-inflammatory pathways.The effect of LMP-420 was additionally studied in the IL-10-/- mice. A detailed dose-response study and longer treatment durations using other colitis models that are more dependent on TNF elevation should be performed to more accurately assess the potential of LMP-420 for therapy of inflammatory bowel disease.Taken together, these studies demonstrate that short-term treatment with a transcriptional inhibitor of TNF production does not decrease the severity of acute and chronic DSS colitis or piroxicam-accelerated colitis in IL-10BSA: bovine serum albumin; CD: Crohn's disease; DSS: dextran sulfate sodium; FBS: fetal bovine serum; i.p.: intraperitoneal; IBD: inflammatory bowel disease; IFN: interferon; IL: interleukin; LPS: lipopolysaccharide; MCP: monocyte chemoattractant protein; PBS: phosphate buffered saline; SD: standard deviation; SEM: standard error of the mean; TNF: tumor necrosis factor; TNF-RII: tumor necrosis factor receptor, type II; UC: ulcerative colitis.LPH has no competing interests. GJC also has no competing interests, but discloses that he was a co-discoverer of LMP-420 and was associated with LeukoMed Inc (the company that holds the license for LMP-420) until October 2005.LPH conceived of and designed the studies, obtained funding, performed the pathologic and data analyses, prepared figures, and drafted the manuscript. GJC assisted in study design, reviewed data, prepared figures, and helped to draft the manuscript. All authors read and approved the final manuscript.


Targeting tumor angiogenesis and vasculature is a promising strategy for the inhibition of tumor growth and dissemination. Evidence suggests that tumor vasculature expresses unique markers that distinguish it from normal vasculature. Our efforts focused on the molecular characterization of endothelial cells (EC) in the search for selective markers of tumor vasculature that might be helpful for the development of effective therapeutic approaches.In vitro experiments confirmed the higher expression of the selected genes in tumor-derived endothelium with no expression in tumor cells. In vivo investigation by in situ hybridization established that ADAM23, GPNMB and PRSS3 expression is localized on blood vessels of human cancer specimens.We investigated by microarray analysis the gene expression profiles of EC purified and cultured from tumor (ovarian carcinoma [HOC-EC]) and normal  tissue specimens. We found distinct transcriptional features characterizing the EC of different origin, and identified 158 transcripts highly expressed by HOC-EC. We analyzed four of these genes, ADAM23, FAP, GPNMB and PRSS3, which were not previously known to be expressed by endothelium. These findings elucidate some of the molecular features of the tumor endothelium. Comparative transcriptomic analysis allowed us to determine molecular differences of tumor and normal tissue-derived endothelium and to identify novel markers that might be exploited to selectively target tumor vasculature. Vascula® . Experim® . Moreove® .Tumor-induced vessels are ultrastructurally abnormal and differ from normal vasculature. They lack functional pericytes and are unusually permeable . These ain vivo, isolated from ex vivo specimens or cultured in vitro. The in vivo phage display technology was used to identify molecules selectively expressed on tumor endothelium [in vivo proteomic analysis to detect antigens accessible from the vasculature [In recent years an increasing body of evidence suggests that tumor vasculature expresses unique markers that distinguish it from normal vasculature. Numerous efforts have been aimed at the molecular characterization of tumor associated endothelial cells (EC) in the search for differences between tumor and normal tissue EC. A variety of techniques have been applied to study EC othelium ,9, whileculature ,11. Thesex vivo human cancer and normal specimens, analyzed by means of Serial Analysis of Gene Expression (SAGE) or microarray, revealed differences at the transcriptional level in tumor and normal tissues [In vitro models have been widely used to identify the specific functions of the endothelium and to understand the molecular modifications that might occur during angiogenesis. In these studies EC were exposed to different microenvironmental conditions, such as angiogenic growth factors and matrix proteins, shear stress, hypoxia, or tumor conditioned media [in vitro models have used cultures of immortalized EC and/or primary EC from human umbilical vein (HUVEC).Gene expression of EC isolated from  tissues -15. In ved media -20. All in vitro maintain several of the features described for tumor vasculature and that they differ from EC isolated from normal tissue  [We developed a method to isolate and culture EC from tumor specimens (TdEC), by which we were able to show that TdEC d HA-EC) . These fin vitro. We successfully identified molecules that are expressed selectively by the tumor vasculature in vivo, thus providing distinctive features to be exploited to selectively target tumor vasculature.Here we describe the isolation of EC from human specimens and the characterization of the transcriptional profiles of EC derived from ovarian carcinoma (HOC-EC) and HA-EC by means of microarray technology. Through the comparison of HOC-EC and HA-EC gene expression we were able to demonstrate that tumor-associated EC differ from those in normal tissues, thus proving that molecular differences are maintained Endothelial cells were reproducibly isolated from human tissue specimens: HOC-EC from ovarian carcinoma, HKC-EC from kidney carcinoma, HA-EC from adrenal gland and HSk-EC from skin.Before use, their endothelial origin was established. Endothelial cell cultures typically created a homogeneous monolayer of elongated cells in close contact and maintained an uniform morphology after few passages in culture; they formed capillary-like cord structures when plated on matrigel (data not shown and ). Their The purity of the isolated EC was comparable to that of EC from umbilical vein (HUVEC), which were used as the reference population for EC Table .Microarray results, performed on HA-EC and HOC-EC, confirmed the endothelial origin of the isolated cells Table . The typin vitro. According to our selection criteria, 158 gene transcripts, corresponding to 179 probe sets, were more expressed by HOC-EC than by HA-EC. The genes belong to the different functional classes that are listed in Table Comparison of the transcriptional profiles of HOC-EC and HA-EC  showed that EC from tumor and normal tissue have distinct and characteristic expression patterns that are maintained Microarray results reveal an alteration in cell-cell and cell-matrix interaction as shown by the enhanced expression of molecules such as desmoglein 2, N- and OB-cadherin, protocadherin-9 and -17 and P-selectin Table . In keepThe peptidase family is the most represented among the enzymatic classes of genes expressed by HOC-EC Table . It inclMost of the listed genes are involved in signaling and comprise molecules previously shown to be involved in angiogenesis and other vascular functions, such as adrenomedullin receptor, CD44, endothelin-1 and -2, endothelin receptor-A and tissue factor Table .Consistent with the notion that the vascular and nervous systems share similar signals and principles , 3 membeFour members of the Solute Carrier Family  were over-expressed in HOC-EC, suggesting an alteration in the extra-intracellular exchange of metabolites Table . Among tHigher expression also affected genes involved in transcriptional response, such as members of the zinc finger protein family  and of the homeobox family  Table .SMAD7 Table  and BAMBAmong the class of cell cycle regulators, the high expression of the anti-apoptotic protein BCL2 is noteworthy Table .In order to identify novel markers of tumor endothelium our attention focused on transcripts that are highly expressed by tumor-derived endothelium , but whose products and functions were not previously reported as being involved in tumor angiogenesis or associated to vascular functions. Among them we distinguished four genes on the basis of their structural or functional characteristics, such as the presence of transmembrane domains and/or enzymatic activities: disintegrin and metalloproteinase domain 23 (ADAM23), fibroblast activation protein alpha (FAP), transmembrane glycoprotein nmb (GPNMB), and serine protease 3 or mesotrypsin (PRSS3). The relative expression in HOC-EC compared to HA-EC was 2.6 for ADAM23, 4.1 for FAP, 4.6 for GPNMB and 2.5 for PRSS3, as calculated from microarray data. These results were validated by real-time PCR in EC isolated from six neoplastic tissues (five ovarian and one kidney carcinoma specimens) and from four non-neoplastic tissues  different from those investigated by microarray. These tumor or normal tissue derived EC are described in Table in vivo – either subjected to persistent stimulation imposed by the tumor cells or not, EC were investigated both in the presence , chosen as an internal control of a gene expressed to a much lesser extent by HOC-EC, confirmed the microarray results Figures  and 1B.Having shown that ADAM23, FAP, GPNMB, and PRSS3 were expressed by a panel of EC, with the highest expression found in TdEC, we next investigated their expression in other cell types. The gene expression analysis revealed that they could be expressed to different extents also by other stromal cells, such as fibroblasts  and smooth muscle cells (UASMC), but in general not by tumor cells  array: a panel of human mRNAs in dot-blot format from 58 normal adult tissues, seven fetal tissues, and eight cell lines. As shown in Figure Data from RefExA  and litein vivo localization of ADAM23, GPNMB and PRSS3, human tumor tissues were analyzed by in situ hybridization.In order to investigate the in vitro results, tumor cells did not express the transcripts, as shown by the absence of staining in the parenchyma of the tissues  and adrenal gland (HA-EC) revealed the higher expression of 158 gene transcripts by HOC-EC. The finding that tumor and normal tissue EC have distinct gene expression patterns concurs with other investigations, which describe genes that are preferentially expressed by endothelium from malignant cancer -15. In toperties . Our resfindings  and agrefindings ,33 showiin vivo . These results suggest that the transcriptional differences are not culture condition artifacts, but, rather, true differences that distinguish EC of different origin .Here we describe for the first time a much higher expression of ADAM23, FAP, GNPNB and PRSS3 in EC derived from six tumor samples than in EC from four normal tissue specimens examined by real-time PCR, whose origin differed from that investigated by microarray analysis. The differences we detected between TdEC and NdEC were maintained whether or not they were exposed to a combination of pro-angiogenic factors that presumably mimic the different environmental conditions under which EC might be embedded Despite having mainly utilized EC from ovarian cancer and from adrenal glands as the non-neoplastic counterpart, the results hold true for EC isolated from kidney cancer and skin specimens . These observations, would seem to indicate that the EC characteristics we observed are not organ-specific, but, rather, exclusive of TdEC. This is in agreement with a recent report  that comin vivo. To this end, in situ hybridization results confirmed ADAM23, GNPNB and PRSS3 expression associated to blood vessels in tumor tissues, but the lack of their expression in cancer cells .Cultures of EC are valuable tools for biochemical and functional analysis, but they do not reflect the physiological situation entirely. All the information should be considered critically from the standpoint of what might occur in blood vessels in vitro are likely to have in vivo relevance, and bear out the validity of our experimental setting designed to reproduce situations that might occur in vivo.Overall, these findings support the premise that endothelial markers identified in situ hybridization finding  contradicted the latter, which implies that the expression of ADAM23 might indeed be much higher in tumor EC, including blood vessels in medulloblastoma, a highly malignant brain tumor, than in the brain. Further studies, aimed to produce antibodies to ADAM23, are necessary to prove its suitability as a tumor vascular target. Our results also revealed that ADAM23 was not expressed in cancer cells. This observation is consistent with the data available from RefExA and other recent reports [ADAM23 belongs to the cellular disintegrins, a family of membrane-anchored proteins that are potential regulators of cell-to-cell and cell-to-matrix interactions  and for  reports , which d reports .in vivo model of inflammation, and these effects could be inhibited by an NK1 receptor antagonist [in vitro EC proliferation [in vivo also contributes to vascular permeability, plasma extravasation, and edema [in vivo SP effects on EC [GPNMB is a type I membrane protein identical to the hematopoietic growth factor inducible neurokinin-1 type (HGFIN)  sharing tagonist . The acttagonist , and botferation . Substannd edema , all feats on EC .The PRSS3 gene encodes for a trypsinogen whose functions have not yet been fully elucidated . PRSS3 tin vivo expression of FAP on blood vessels was recently demonstrated by Ghersi et al. in invasive breast ductal carcinoma [Our results show that the cell surface protease FAP was also expressed at significantly higher levels by TdEC than by normal EC. FAP was originally reported to be an antigen recognized by F19 antibody in cultured fibroblasts . Accordiarcinoma .in vivo and to colocalize with microvessels in the stroma of non-small cell lung cancer [Many of the transcripts we found to be expressed at a high level by EC from neoplastic tissues were previously reported as being involved in tumor angiogenesis or expressed by tumor EC. In this regard, it is worth listing the various types of matrix proteins that we detected, namely: collagens alpha-2 type I, alpha-1 type III, V, XIII and XV, glypican-1, laminin alpha3, and tenascin C. Our results confirm similar findings from other laboratories ,13,15 ang cancer .The expression of cell adhesion proteins was also altered in TdEC. This may be explained by the fact that migrating and proliferating EC must "loosen up", that is, detach and then create new interactions. Our results implicate CD44 and cadherins in this process, and confirm previous reports showing N-cadherin over-expression on proliferative state EC , and enhInterestingly, ABCA2 and ABCB1 (MDR-1/p-glycoprotein 1) were also more abundantly expressed by TdEC, reflecting the previously reported higher expression of p-glycoprotein 1 in EC from glioma than from normal brain . The ATPMolecules such as SMAD7, BAMBI and BCL2 are listed among the transcripts that are more highly expressed by HOC-EC than by HA-EC. SMAD7 and BAMBI interfere with the TGFβ pathway and may potentially block the anti-proliferative action of TGFβ . Their eEmerging studies suggest that the vascular and the nervous systems share the same molecular signals in their development. The involvement of netrins, semaphorins, Robo/Slit and VEGF families in both angiogenesis and neurogenesis has been demonstrated in recent years . In keepTargeting angiogenesis and vasculature represents a promising option to control tumor growth and dissemination. To exploit this opportunity, however, selective targets in tumor associated vasculature need to be identified.in vitro cultured endothelial cells isolated from human normal and neoplastic specimens that maintained their respective molecular phenotype and enabled us to determine molecular differences between tumor and normal tissue derived EC. Specifically, we identified four transcripts whose expression is much higher in endothelial cells from tumors than from normal tissue. The in vivo expression in cancer-associated blood vessels was confirmed by in situ hybridization of human specimens.We describe a model consisting of This work evinces some of the distinctive features of tumor-derived endothelium, and identifies potential markers that may provide the groundwork for novel therapeutic strategies. Future studies are needed to clarify the functions and roles of these molecules in tumor progression and vascularization.The method for isolation of EC has been described in detail by ,21. BrieHuman umbilical vein EC (HUVEC) were obtained following the procedure described by Jaffe  and cultTo confirm their endothelial origin, EC cultures were analyzed by immunohistochemical methods to evaluate the expression of von Willebrand Factor (vWF), platelet-endothelial cell adhesion molecule-1 (CD31/PECAM-1), Vascular E-cadherin (VE-cadherin), and alpha-smooth muscle Actin , and by fluorescence activated cell sorter (FACS) to evaluate low-density lipoprotein (LDL) uptake and CD31/PECAM-1 antigen expression, as previously described .HT29 colon carcinoma cells (ATCC-American Type Culture Collection) were grown in minimum essential medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS).1A9 (a subclone of the A2780 cell line ) and SKOUmbilical artery smooth muscle cells (UASMC) were purchased from Clonetics  and cultured following the manufacturer's protocol in SmBM Growth Medium.® (GIBCO-Invitrogen) or RNeasy Mini Kit  following the manufacturer's protocols. Potential genomic DNA contaminations were removed by DNAse  treatment followed by RNA CleanUp with RNeasy Mini Kit. The purity and integrity of the RNA was checked by gel electrophoresis, and concentration was determined spettrophotometrically.Total RNA was isolated from cultured cells using Trizol2 fibronectin  in EBM plus 5% FBS supplemented with and 10 ng/ml Epidermal Growth Factor (EGF), 10 ng/ml Vascular Endothelial Growth Factor (VEGF), 2 ng/ml Fibroblast Growth Factor (FGF-2) ; from here on respectively called "unexposed" or "exposed" to a tumor/angiogenic environment.We are aware that culture conditions alter gene expression. To this end, all the EC under investigation were analyzed under two different conditions: either seeded onto type I collagen coated tissue culture plastic  in endothelial cell basal medium  plus 5% FBS or onto BioCoat plasticware additionally coated with 1 μg/cmTo maximize the likelihood of detecting the intrinsic differences between HOC-EC and HA-EC by minimizing the influence of the artificial environment upon them, we opted to select only those genes that were differentially expressed regardless of the culture conditions.® Human Genome U95A Arrays  that monitor the expression of approximately 12,000 genes were utilized to analyze the transcriptional profiles of 3 independent HOC-EC together with 2 HA-EC populations, under both "exposed" and "unexposed" culture conditions. Raw Affymetrix data were then processed for each of the 10 GeneChip® arrays.GeneChipFor each probeset, the mean of the average difference values for the two "exposed" HA-EC samples was calculated only if both were greater than 20 arbitrary units. Similarly, the mean of at least two average difference values for the three "exposed" HOC-EC samples was calculated only if each of them was greater than 20 arbitrary units. The same was done for the "unexposed" samples. Then, the log2 of the HOC-EC versus HA-EC ratios was calculated only for the probesets having both means available. Probesets with log-ratios greater than 1.3 (corresponding to a fold change of 2.5) for both "exposed" and "unexposed" samples were considered. Such a threshold was obtained by fitting the distributions of the "exposed" and "unexposed" log2-ratios with a normal distribution and calculating upper bound of the 99% confidence interval.the probesets expressed to a greater extent in HOC-EC than in HA-EC were divided into functional classes using the publicly available Est Annotation and the Keyword Clustering Machines  and were® were found by keyword search within the complete probeset list annotated using the publicly available SOURCE batch search [Endothelial markers represented on the Affymetrix GeneChiph search ,61. The Additional information on gene expression was obtained from the publicly available Reference database for gene Expression Analysis (RefExA) of the Laboratory for System Biology and Medicine (LSBM)  .Two micrograms of total cellular RNA were reverse transcribed (RT) using the Taqman Reverse Transcription Reagents and Random Hexamer primers  according to the manufacturer's protocols. To check for DNA contamination, a control RT reaction was set up for each sample without the addition of reverse transcriptase.Quantitative real-time PCR was performed in 25 μl-reactions on the GeneAmp 5700 Sequence Detection System (Applied Biosystems) using either SYBR Green or TaqMan chemistry. Primer sets for PCR amplifications are listed in supplementary table 1 [see Additional file All assays were performed using duplicate samples of each cDNA synthesis. Reverse transcription reactions and real-time PCR analyses were carried out at least twice for each gene transcript to determine consistency of results.targetgene - Ct 18s). Statistical analysis with Student's t-test (unpaired) on ΔCt values was performed to compare the expression levels of target genes in EC samples from normal and neoplastic tissues.Gene expression of all transcripts was normalized to the endogenous control gene human 18s rRNA and for each population the ΔCt value was calculated .32P-labeled cDNA probe. Filter was washed with SSC2x/SDS0.1% at 65°C, followed by washes at 55°C with SSC0.1x/SDS 0.5%. Because the panel of human mRNAs loaded onto the MTE Arrays in dot-blot format are normalized using eight different housekeeping genes, the hybridization results can be attributed to actual differences in target mRNA abundance.For the analysis of tissue distribution, the MTE Array 2  was hybridized following manufacturer's instructions. Briefly, the filter was pre-hybridized for 30 min at 65°C with ExpressHyb (Clontech). Hybridization was carried out O.N. at 65°C in ExpressHyb containing 130 μg/ml fish sperm DNA, 6 μg/ml Cot -1 DNA and the Densitometric analysis of the dots was performed with Gel-Pro Analyzer Software . Optical Density (OD) values are reported in Figure cDNA probe for ADAM23 corresponding to nt 2241–2743 of GeneBank:NM_003812, and cDNA probe for GPNMB corresponding to nt 416–984 of GeneBank:NM_002510 were PCR amplified and gel purified.In situ hybridization experiments were carried out with a mixture of specific biotin-labeled oligonucleotide antisense (or sense) probes for ADAM23, GPNMB and PRSS3  listed in the supplementary Table 2 [see Additional file After removal of paraffin, the tissue sections were rehydrated and heat-treated in 0.1 M sodium citrate (pH = 6), washed for 5 min with water and then with 2× SSC, and air dried. Sections were allowed to hybridize overnight at room temperature in 15% formamide, 5× SSC, 10% dextran sulphate, 1× Denhardt's solution  and 400 μg/ml fish sperm DNA. Probe concentration was 10 nM for each oligonucleotide (corresponding to 10 fmol/μl). Approximately 100 μl of hybridization solution was applied to each slide.After hybridization, sections were washed for 15 min in 2× SSC and for 10 min in water at room temperature. They were rinsed three times in PBS/0.1% Triton, and incubated for 20 min with blocking buffer . Bound probes were detected using a mouse anti-biotin antibody . Amplification of the signal was achieved with the Vectastain elite ABC Kit  according to the manufacturer's protocol. Colorimetric detection was completed with the AEC Substrate kit (Vector Laboratories). Finally, the tissue sections were counterstained with hematoxylin and embedded on an aqueous mounting medium.Endothelial cells have been isolated from biopsies collected at the San Gerardo Hospital  from patients undergoing therapeutic surgery from September 1998 to March 2004. The collection and use of the tissue samples was approved by the Local Scientific Ethical Committee in compliance with the principles enunciated in the Helsinki Declaration . Tissue ADAM23: a disintegrin and metalloproteinase domain 23; Alpha-SMA: alpha-smooth muscle actin; CD31/PECAM-1: platelet-endothelial cell adhesion molecule-1; CNS: central nervous system; EBM: endothelial cell basal medium; EC: endothelial cells; EGF: epidermal growth factor; FAP: fibroblast activation protein; FBS: fetal bovine serum; FDA: food and drug administration; FGF-2: fibroblast growth factor-2 (basic); GPNMB: glycoprotein nmb; HA-EC: human adrenal gland endothelial cells; HKC-EC: human kidney carcinoma endothelial cells; HOC-EC: human ovarian carcinoma endothelial cells; HSk-EC: human skin derived endothelial cells; HUVEC: human umbilical vein endothelial cells; LDL: low density lipoproteins; MEM: minimum essential medium; MTE Array: multiple tissue expression array; NdEC: non-neoplastic derived endothelial cells; NK1: neurokinin-1; OD, optical density; PAR-1: protease activated receptor-1; PCR: polymerase chain reaction; PRSS3: serine protease 3; SAGE: serial analysis of gene expression; SCGE: sarcoglycan epsilon; SmBM: smooth muscle basal medium; SP: substance P; TdEC: tumor derived endothelial cells; uPA: urokinase-plasminogen activator; uPAR: urokinase-plasminogen activator receptor; VEGF: vascular endothelial growth factor; VE-cadherin: vascular endothelial-cadherin; VWF: von Willebrand factor.in situ hybridization experiments. RG participated in the design of the study and preparation of the manuscript. MRB conceived of the study and participated in its design and coordination and help to draft the manuscript. All authors read and approved the final manuscript.CG carried out the gene expression studies and drafted the manuscript. GC performed the mathematical and statistical analysis. RD participated in endothelial cell isolation. ZN participated in CGhilardi_Supplementary information. This file contains: Isolation and culture of endothelial cells, Supplementary table 1 – Primers and Assay-on-Demand used in quantitative real-time PCR-, Supplementary table 2 – Biotin-labelled oligonucleotides used for In Situ Hybridization -.Click here for file


Work related stress is associated with a range of debilitating health outcomes. However, no unanimously accepted assessment tool exists for the early identification of individuals suffering from chronic job stress. The psychological concept of self-perceived stress reactivity refers to the individual disposition of a person to answer stressors with immediate as well as long lasting stress reactions, and it could be a valid indicator of current as well as prospective adverse health outcomes. The aim of this study was to determine the extent to which perceived stress reactivity correlates with various parameters of psychosocial health, cardiovascular risk factors, and parameters of chronic stress and job stress in a sample of middle-aged industrial employees in a so-called "sandwich-position".In this cross-sectional study, a total of 174 industrial employees were assessed for psychosocial and biological stress parameters. Differences between groups with high and low stress reactivity were analysed. Logistic regression models were applied to identify which parameters allow to predict perceived high versus low stress reactivity.In our sample various parameters of psychosocial stress like chronic stress and effort-reward imbalance were significantly increased in comparison to the normal population. Compared to employees with perceived low stress reactivity, those with perceived high stress reactivity showed poorer results in health-related complaints, depression, anxiety, sports behaviour, chronic stress, and effort-reward imbalance. The educational status of employees with perceived low stress reactivity is higher. Education, cardiovascular complaints, chronic stress, and effort-reward imbalance were moderate predictors for perceived stress reactivity. However, no relationship was found between stress reactivity and cardiovascular risk factors in our sample.Job stress is a major burden in a relevant subgroup of industrial employees in a middle management position. Self-perceived stress reactivity seems to be an appropriate concept to identify employees who experience psychosocial stress and associated psychological problems at the workplace. Organisational restructuring, downsizing, and increasing pressure on management in keeping up with performance figures have radically transformed the nature of work . ChronicSo far, no unanimously accepted psychometric or biological indicator exists for the early identification and evaluation of individuals suffering from chronic job stress -26. In hGiven the very small empirical basis of self-perceived stress reactivity and the complete lack of studies in workplace samples, there is a great need to better understand the correlations and interactions between self-perceived stress reactivity and different measures of chronic stress, job stress, somatic parameters for stress, cardiovascular risk factors, and psychosocial health outcomes. Industrial employees in a middle management ("sandwich") position seem to be an especially interesting group to test for this concept ,3.The present study therefore presents data of a work-health promotion study in a large sample of middle-aged industrial employees with leadership responsibility in a typical middle management position of a large international manufacturing company. The primary aim of this study was to analyse the relationship of dispositional self-perceived stress reactivity with various psychological as well as biological indicators of stress and health. Therefore, we hypothesised that high stress reactive employees have a significantly worse status of health and stress-related phenomena than for low stress reactive employees (H1). The secondary aim of this study was to identify potential key variables that explain part of the observed variance in the self-perceived stress reactivity. We hypothesised that heightened stress reactivity can be predicted by psychosocial as well as biological parameters of stress and health (H2).Participants were recruited from an international manufacturing company. At the time of the study 262 employees were deemed eligible for the study. Following two information sessions for this group, a total of 189 employees agreed to participate voluntarily in the study. 15 of the 189 interested employees failed to fulfill inclusion criteria. Thus 174 employees (92.1%) were finally included into the study. All participants were invited to an 1 1/2 hour medical examination including blood sampling and were asked to provide saliva the following working day to determine the cortisol level. Moreover, the participants were required to complete an assessment battery of questionnaires. The inclusion criteria of the study were: 1) Industrial employees in the production line with leadership responsibility ("sandwich-position"); 2) age between 18 and 65 years with more than 2 years until retirement. Application to early retirement, surgery, or other severe diseases potentially leading to more than 30 sick leave days led to exclusion. Written informed consent was obtained. Information was provided according to the Declaration of Helsinki , systolic and diastolic blood pressure, 24 hours ECG, saliva cortisol and alpha-amylase levels, levels of serum low-density lipoprotein (LDL), serum high-density lipoprotein (HDL), cholesterol, glycosylated hemoglobin, triglycerides, serum dehydroepiandrosterone sulfate (DHEA-S), fibrinogen, CRP, and serum creatinin. Systolic and diastolic blood pressure were measured as the average of 2 readings obtained with a digital blood pressure instrument (Boso Inc.), the waist circumference was measured in a standing position at the level of the umbilicus or, in obese persons, at the maximum between the iliac crest and the lowest rib. Blood samples for assays of HDL, cholesterol, HbA1c, and DHEA-S were obtained in the morning. All samples were immediately processed and frozen at -200 C according to standard laboratory procedures. Saliva samples were collected using cotton dental rolls held in the mouth until saturated, and then stored in Salivette tubes . Participants were instructed to give 7 samples over a single working day, with measures on waking up, 30, and 60 mins later, and then within defined time intervals throughout the day and evening . Each participant registered in a diary the exact timing when the sample was taken as well as the time of waking up. Tubes were returned to the investigators in person and salivary free cortisol was analysed using a commercial chemiluminescence immunoassay . Alpha-amylase was determined by using the automatic analyser Cobas Mira and assay kits obtained from Roche .The selection of biological indicators in our study was based on the allostatic stress load concept ,32,33. TSociodemographic data and job specific questions concerning professional status and working time were recorded.Self-perceived stress reactivity was measured with the 29-item-"Stress-Reactivity Scale ". Perceived stress reactivity is assumed to be determined by a disadvantageous pattern of personality traits. It refers to the disposition of a person to respond to stressors with immediate, intense and long lasting stress response characteristics. The SRS quantifies general stress reactivity and stress reactivity in specific domains . Two scales evaluate stress reactivity before and after stressful events in general and a total score can be generated. A satisfying Chronbach's Alpha between .71 ≤ α ≥ .82 for the various subscales and of .91 for the of the SRS total score has been reported. In the present sample internal consistencies (Chronbach's Alpha) ranges between .65 ≤ α ≥ .78 for the various subscales and .92 for the total score. Furthermore, factorial validity and correlations with construct-related personality traits, bodily complaints, sleep behaviour, chronic diseases, and cortisol reactions have been described . In addiThe amount of chronic stress within the last 3 months was measured by the 12-item global scale  of the "Trier Inventory for the Assessment of Chronic Stress (TICS)" using a four-point Likert scale .Job stress was assessed with the "Effort-Reward Imbalance" (ERI) which claims that failed reciprocity in terms of high efforts spent and low rewards received in turn is likely to elicit recurrent negative emotions and sustained stress responses in exposed people. Conversely, positive emotions evoked by appropriate social rewards promote well-being, health and survival. The ERI model has been operationalised as a standardised self-report measure containing 23 Likert-scaled items in its established short version. These items define three unidimensional scales: 'effort' (6 items), 'reward' (11 items), and 'overcommitment' (6 items) with each item rated on a five-point  Likert scale ,36.To assess the current health status of the participants, the following indicators were selected.The Giessen Subjective Complaints List (GBB-24) is a questionnaire for health-related quality of life, and assesses four domains of physical and psychosomatic complaints by means of 24 items on a five-point Likert scale, which includes heart symptoms, fatigue, stomach pain as well as arm and leg pain . AdditioAnxiety and depression was assessed with the Hospital Anxiety and Depression Scale (HADS) . This sc2-tests. Significance of differences between sample means and reference values was tested with t-tests. For the calculation of subscale values a maximum 30% missing items per subscale was accepted. To assess whether and to which extent self-perceived stress reactivity can be predicted by psychosocial and biological factors and health-related behaviour, a logistic regression model was applied with high vs. low stress reactivity as dependent variable. The criterion for independent variables to be entered into the initial model was a p-value < .250 in univariate between-group comparisons  and for two groups with low (n = 88) and high (n = 86) stress reactivity based on a median split of the SRS total score. In all between-group comparisons significance of differences in means was tested with t-tests or Mann-Whitney-U-tests, significance of differences in proportions was tested by Chi74 and foThe average age of the study participants was 40.9 (7.8) years and 98.3% were male. Although participants have achieved a professional position with leadership responsibility, more than three fourths (76.9%) have a lower educational degree with less than 11 years of formal education. 23.6% achieved a master-level degree. For the total sample an average of 1.9 hours of unpaid overtime per month has been reported. 63% of all participants did shift work. Looking at the number of sick leave days per year, our sample is comparable to the total workforce of the company: 55.7% reported no sick leave days  and 12.6% were more than 10 days absent   was 17.29 in our sample compared to 14.37 for the reference population . LookinFor the group with higher self-perceived stress reactivity a mean value of 63.21 (6.1) has been calculated. For the 50% with lower self-perceived stress reactivity the mean score was 45.95 (5.2). For demographic and professional variables, significant differences between high and low stress-reactive employees were found only for the educational background of the subjects, with more persons with master-level degree in the low stress-reactive group , especially with respect to musculoskeletal and cardiovascular complaints. Also for depression and anxiety we found poorer results for high stress-reactive persons. Looking at health behaviour the two groups showed a significant difference for intensive sports activity  and all ERI-scales table .High vs. low stress-reactive employees did not show significant differences in any specific biological stress indicators, nor in the revised global allostatic load index. The mean allostatic load value was 3.32 (SD 2.2) for the high stress-reactive group and 3.05 (SD 2.1) for the low stress-reactive group p = .484)  showed a 13-fold (95% CI 3.6-46.6) and those with the lowest educational level (school until age 15) a 6.3-fold (95% CI 2.2-17.8) higher risk for high stress reactivity. The risk of employees in the highest tertile of cardiovascular complaints was 5.1-fold higher (95% CI 2.0-13.0) compared to those with low level cardiovascular complaints. Self-assessed increased chronic stress was also associated with increased self-perceived stress reactivity. As to the effort-reward ratio, employees with moderate values in this scale are found at the highest risk for high stress reactivity, and not those with the worst imbalance.Equally examined in the model were the effects of esteem, security, and promotion , gastrointestinal complaints and musculoskeletal complaints (GBB-24), HADS anxiety and depression, intensive sports, smoking, total cholesterol, and daytime slope amylase; they did not significantly change the prediction and were thus removed by the stepwise procedure.Referring to the main hypothesis (H1), our study shows that in industrial employees holding a middle management ("sandwich") position in manufacturing, increased individual stress reactivity is indeed associated with heightened psychosocial work stress, a higher rate of psychosomatic and physical complaints, poorer psychological health, and negative health behaviour. In addition, high stress-reactive employees experience an increased effort-reward imbalance , show higher levels of depression and anxiety, and suffer more often from musculoskeletal and cardiovascular complaints. Compared to the normal population, our industrial workplace sample showed poorer results in terms of self-rated chronic stress, effort-reward ratio, anxiety, a variety of other related psychological problems, and obesity. These findings are of considerable interest since a study on work-related stress in a population of employed Swedish women showed an association between work-related stress, illness symptoms and sick-leave . FurtherIn respect to H2, our data revealed that education, cardiovascular complaints, chronic stress, and effort-reward imbalance were moderate predictors for perceived stress reactivity. Contrary to our expectations and to the few findings available of somatic correlates of stress phenomena in working place samples ,45, we d1. Unlike the only workplace study investigating the interaction of job profile and allostatic load , our parSome more general factors may also explain our cross sectional finding: lack of longitudinal data, age of the participants, and a selection bias due to voluntary participation in the study.We do recognise that there are some additional limitations of our study. First, our results cannot be generalised to other working populations. As participation was voluntary it can be assumed that all participants in the current study were in at least some positive stage of change . Based oFinally, we conclude that the concept of stress reactivity as measured by the SRS correlates with various important aspects of psychosocial impairment and maladaptive health behaviour and thus indeed may be a valid tool to identify employees potentially prone to suffer from chronic job stress.The results of this cross sectional study in a sample of middle management employees in the production line of an international plant demonstrate that in a relevant subgroup of the whole sample psychosocial stress parameters and well known health risk factors are significantly increased. Self-report data on stress reactivity allow to identify employees who are under stress according to established models and who suffer from stress-associated complaints. They may thus be at risk for severe stress sequelae. However, this cross sectional study failed to demonstrate a significant relationship between psychosocial and biological stress indicators. Longitudinal data may further elucidate the pathways of perceived stress and health status, and thus may prove if the measurement of dispositional stress reactivity may indeed serve as a predictor for adverse long term stress-related health outcomes.The authors declare that they have no competing interests.HL, PA, MH, BMM, UN and HG made substantial 1) contributions to conception and design, acquisition, analysis and interpretation of data 2) have involved in drafting the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/252/prepub


MicroRNAs (miRNAs) are small ~22-nt regulatory RNAs that can silence target genes, by blocking their protein production or degrading the mRNAs. Pig is an important animal in the agriculture industry because of its utility in the meat production. Besides, pig has tremendous biomedical importance as a model organism because of its closer proximity to humans than the mouse model. Several hundreds of miRNAs have been identified from mammals, humans, mice and rats, but little is known about the miRNA component in the pig genome. Here, we adopted an experimental approach to identify conserved and unique miRNAs and characterize their expression patterns in diverse tissues of pig.By sequencing a small RNA library generated using pooled RNA from the pig heart, liver and thymus; we identified a total of 120 conserved miRNA homologs in pig. Expression analysis of conserved miRNAs in 14 different tissue types revealed heart-specific expression of miR-499 and miR-208 and liver-specific expression of miR-122. Additionally, miR-1 and miR-133 in the heart, miR-181a and miR-142-3p in the thymus, miR-194 in the liver, and miR-143 in the stomach showed the highest levels of expression. miR-22, miR-26b, miR-29c and miR-30c showed ubiquitous expression in diverse tissues. The expression patterns of pig-specific miRNAs also varied among the tissues examined.Identification of 120 miRNAs and determination of the spatial expression patterns of a sub-set of these in the pig is a valuable resource for molecular biologists, breeders, and biomedical investigators interested in post-transcriptional gene regulation in pig and in related mammals, including humans. Only recently, the discovery of microRNAs  has greatly expanded our knowledge of the cellular mechanisms that regulate gene expression at the post-transcriptional level in eukaryotes [Spatio-temporal regulation of gene expression is vital for normal growth and development in living organisms and for optimal response to environmental stimuli and endogenous cues. This regulation is achieved by multiple mechanisms operating at transcriptional, post-transcriptional and post-translational levels ,2. Transkaryotes ,4. Recenkaryotes  and/or akaryotes ,7.Biochemical studies in animals support a compartmentalized, two-step maturation of miRNAs derived from their precursor transcripts originating from the miRNA genes located in the intergenic regions. Interestingly, many miRNAs in mammals are derived from the introns of the protein coding genes. The miRNAs are loaded into a silencing complex, called RNA-induced silencing complex (RISC) and can guide the complex to the target mRNAs. In most cases, animal miRNAs recognize their target transcripts by base pairing with the 7- to 8-nt complementary region usually located in the 3'UTRs on the target mRNAs and thus repressing their expression , althougCaenorhabditis elegans [Since the initial discovery that lin-4 acts as a regulatory RNA in  elegans , interes elegans . Many mi elegans . As a re elegans ,19 and s elegans ,20,21 ha elegans ,23. Howe elegans .Sus scrofa), is one of the most widely eaten meats in the world [Pork, derived from pigs   in our sequences Table  and the In animals and plants, miRNAs exist as multigene families (two or more closely related sequences and vary by 1 or 2 nt only). With use of small RNA blot analysis or probe-based miRNA micro array, discriminating individual members within a miRNA family is difficult. The expression information of a member of a miRNA family is useful for understanding its functions and for practical use for gene manipulations . The cloSeveral recent studies found a number of lineage-specific miRNAs ,19 or spThe expression levels of individual miRNAs can be determined by small RNA blot analysis ,39. The miR-1 is one of the highly conserved miRNAs and found to be abundantly and specifically expressed in the heart and other muscular tissues ,42. Our Because of their location within the introns of myosin genes and their specific expression in myogenic cells, miR-208 and miR-499 were referred to as MyomiRs . miR-208We observed specific and abundant expression of miR-122 in the liver Figure . PreviouOne of the key features of a functional immune system is its ability to distinguish antigens of foreign origin from those derived endogenously and subsequently to mount an immune response against such antigens. With respect to T cells, this goal is achieved through antigen recognition by T-cell receptors and a highly ordered developmental process in the thymus . Many mimiR-143 expression varied substantially among the 14 tissues examined Figure . The expSome other miRNAs were detected in almost all tissues that we analyzed Figure . let-7, Post-transcriptional gene regulation guided by miRNAs has emerged as one of the major gene regulatory mechanisms in higher eukaryotes. In the present study, we have identified a total of 120 conserved miRNA homologs in pig. Conserved miRNAs are likely play an important role in regulating basic cellular and developmental pathways from lower to higher organisms, whereas the non-conserved miRNAs are thought to be important in lineage-specific or species-specific pathways and functions .32P-labelled probe for detection of miR-1 and miR-133. In our view, small RNA blot analysis provides a more reliable measure of abundance. Therefore, the above discrepancy could be largely due to biased cloning efficiencies of these two miRNAs. In Arabidopsis, a similar discrepancy between the cloning frequency and small RNA blot results was observed for miR398 previously [Often, sequencing-based miRNA expression profiling has been used as a tool to measure the relative abundance of miRNAs ,60. Our eviously . What caThe expression of miRNAs is tightly regulated both in time and space. Tissue-specific miRNAs or a high level of expression of miRNAs only in certain tissues implies that these miRNAs play critical roles in the tissues where they are expressed. The observation that miR-22, miR-26b, miR-126, miR-29c and miR-30c are ubiquitously expressed in 14 different tissues of pig is interesting. Similarly, let-7, miR-98, miR-16 and miR-130a are abundantly expressed in 13 of the 14 tissues (except in pancreas) Figure . These rThe miR-17-92 cluster (polycistronic miRNA gene) encodes six miRNAs  located in the third intron of a ~7-kb primary transcript known as C13orf25 . AlthougSeveral miRNAs  have distinct tissue-specific expression patterns. Most animal miRNAs were thought to regulate several dozens of genes involved in diverse pathways and processes . AdditioThe use of three specialized tissues for library generation is a major limitation for the discovery of miRNAs in pig. Generating more small RNA libraries from diverse tissues should identify additional conserved and porcine-specific miRNAs. While our manuscript was in preparation, Kim et al.,  reportedIn summary, the present study has identified a total of 120 miRNAs in the pig. The expression for 22 conserved and three candidate miRNAs was determined in diverse tissues of the pig. The findings from this study will be a highly valuable resource for future transcriptomic and proteomic studies, as well as for annotating the pig genome in the near future.Tissues from 10 month-old crossbreed were collected and stored at -80°C by the DeSilva laboratory as part of the ongoing porcine gene expression project. Three tissues  used for the generation of small RNA library were obtained from one animal. The experiments were approved by the Institutional Animal Care and Use committees. Total RNA from different tissues was isolated with use of Trizol reagent  following the manufacturer's instructions. The RNA pellet was dissolved in de-ionized formamide for effective denaturation of the RNA. Small RNAs of the desired size range (15–30 nt) were gel-purified by resolving in denaturing 15% polyacrylamide gel. These small RNAs were sequentially ligated with the 3' and 5' adapters as described in our previous reports -33. Reve) and known non-coding RNAs  with NCBI BLASTN. In total, 304 small RNAs mapped to ncRNAs and 83 small RNAs mapped to RepBase were removed from further analysis. Then the small RNAs were mapped to the reported miRNAs in the miRBase (version 11), obtained from miRBase [Our computational methods for analyzing 454 small RNA library was reported previously . In brie miRBase . Small RSmall RNA blot analysis was performed to determine the expression patterns of 22 conserved miRNAs. Total RNA (15–30 μg) was resolved on denaturing 15% polyacrylamide gel, along with labeled RNA markers. RNA was electrophoretically transferred to Hybond-N+ (Amersham) membranes, and the membranes were UV cross-linked and baked for 1 h at 80°C. DNA oligonucleotides complementary to small RNA sequences were end-labeled with γ-32P-ATP with T4 polynucleotide kinase (Invitrogen) used as a probe. Blots were pre-hybridized for at least 1 h and hybridized overnight with PerfectHYB Plus buffer (Sigma) at 38°C. Blots were then washed three times at 50°C and autoradiographed.Note: While our manuscript is under review, two other papers have reported the identification of new miRNAs in pig using bioinformatics approach  and expeRS designed the research, coordinated the project and wrote the paper. AMR performed the expression analysis and GJ constructed the small RNA library. YZ and WZ performed the computational analysis and wrote the corresponding part. SM, GW and BR sequenced the small RNA library. UD provided the pig tissues used in this study. All authors read and approved the final manuscript.Processed procine small RNA sequences obtained using the 454 pyrosequencing. Excel spreadsheet displaying Processed procine small RNA sequences obtained using the 454 pyrosequencing.Click here for file