Brachyspira hyodysenteriae, swine dysentery, is a severe production limiting disease of swine. Recently, pigs in western Canada with clinical signs indistinguishable from swine dysentery were observed. Despite the presence of spirochetes on fecal smears, recognized Brachyspira spp. including B. hyodysenteriae could not be identified. A phylogenetically distinct Brachyspira, called “B. hampsonii” strain 30446, however was isolated. The purpose of this study was to experimentally reproduce mucohaemorrhagic colitis and characterize strain 30446 shedding following inoculation.Mucohaemorrhagic diarrhea caused by Eighteen 13-week-old pigs were randomly assigned to inoculation (n = 12) or control (n = 6) groups in each of two trials. In trial 1, pigs were inoculated with a tissue homogenate collected from clinically affected field cases. In trial 2, pigs were inoculated with a pure broth culture of strain 30446. In both trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls, all of which remained healthy. In animals with mucohaemorrhagic diarrhea, significantly more spirochetes were observed on Gram stained fecal smears, and higher numbers of strain 30446 genome equivalents were detected by quantitative PCR (qPCR). Strain 30446 was cultured from colon and/or feces of all affected but no control animals at necropsy.“Brachyspira hampsonii” strain 30446 causes mucohaemorrhagic diarrhea in pigs following a 4–9 day incubation period. Fecal shedding was detectable by day 4 post inoculation, and rarely preceded the onset of mucoid or haemorrhagic diarrhea by more than 2 days. Culture and 30446-specific qPCR are reliable methods of detection of this organism in feces and tissues of diarrheic pigs. The emergence of a novel Brachyspira spp., such as “B. hampsonii”, creates diagnostic challenges including higher risk of false negative diagnostic tests. We therefore recommend diagnostic laboratories routinely use Brachyspira culture, nox-based and species-specific PCR, and DNA sequencing to diagnose Brachyspira-associated colitis in pigs. Brachyspira hyodysenteriae. The first clinical description of swine dysentery was published in 1921, although it was not until 1971 that B. hyodysenteriae (then Treponema hyodysenteriae) was recognized as the cause B. pilosicoli, which is characterized by diarrhea (non-haemorrhagic with a wet cement consistency) and poor feed conversion in chronic cases Brachyspira are varyingly associated with disease, including B. murdochii, B. intermedia, B. innocens and the provisionally named ‘B. suanatina’, although considerable strain level differences in pathogenicity (particularly among B. intermedia) are apparent Lawsonia intracellularis and Salmonella spp., and despite large numbers of spirochetes seen on Gram strained fecal smears, B. hyodysenteriae and B. pilosicoli were not detected. The apparent spirochetosis prompted further testing of samples by PCR using genus-specific primers targeting the Brachyspira NADH oxidase (nox) gene “Brachyspira hampsonii”“B. hampsonii” clade 2 “B. hampsonii” (clades 1 and 2), and the pathogenicity of strain 30446 may not be reflective of all “B. hampsonii” isolates. As the species has not been formally recognized this study will refer precisely to “B. hampsonii” strain 30446.Swine dysentery is a mucohaemorrhagic colitis causing severe production losses in pigs, resulting from infection with the intestinal spirochaete “Brachyspira hampsonii” strain 30446 in experimentally infected pigs. The results of two infection trials in grower pigs, involving inoculation with either tissue homogenate  or pure culture  are presented.The purpose of this study was to investigate the pathogenicity of Both trials were designed and conducted in accordance with the Canadian Council for Animal Care and approved by the University of Saskatchewan Committee on Animal Care and Supply (Protocol #20110038).“Brachyspira hampsonii” strain 30446 infected material was obtained from clinically affected 13-week-old pigs from a porcine reproductive and respiratory syndrome (PRRS) negative farm. Following necropsy, the colonic and caecal mucosa were removed from the underlying sub-mucosa and muscularis by scraping with the edge of a glass microscope slide, and then frozen at −80°C within four hours of collection. To confirm the absence of pathogens other than strain 30446, sections of small and large intestine were processed routinely for histopathology, bacterial culture, and PCR.Brachyspira was cultured by streaking out approximately 10 µg of feces or intestinal contents onto BJ and CVS agar plates Brachyspira spp., partial 16S rRNA, cpn60, nox, adh, alp, est, gdh, glpK, pgm and thi were amplified and sequenced using previously published primers (DNA was extracted from samples using either the QIAmp DNA stool mini kit (feces or colon contents) or DNEasy blood and tissue kit   and 2 µl of extract used as template in PCR reactions. DNA was extracted in triplicate in terminal colon tissues and colonic contents. To differentiate strain 30446 from other  primers .“B. hampsonii” strain 30446, B. hyodysenteriae and B. pilosicoli, SYBR green qPCR assays were developed targeting either nox (strain 30446), or cpn60 (B. hyodysenteriae and B. pilosicoli) (B. hyodysenteriae and 111 bp for B. pilosicoli. Quantitative PCR reactions were conducted on a Bio-Rad MyiQ thermocycler with iQ SYBR green supermix (Bio-Rad Laboratories (Canada) Ltd., Mississauga, Ontario) according to the manufacturer’s instructions. Quantification was accomplished by use of a serially diluted standard curve of plasmids containing target sequences. All reactions were run in duplicate and each run included both extraction negatives and no template controls. For samples that resulted in a Ct value higher than the lowest standard, but with dissociation curves consistent with the expected product, a result of detected but not quantifiable (DNQ) was reported. The detection limits of the assays were defined by the linear portion of the standard curve for each assay, which were 101–107 copies per reaction (103–109 copies per gram of feces or tissue) for B. hyodysenteriae and B. pilosicoli, and 102–107 copies per reaction (104–109 copies per gram of feces or tissue) for “B. hampsonii”.To specifically detect osicoli) . ProductBrachyspira spp. The pigs were conveniently selected, of average body weight compared to their cohorts and all appeared healthy. Farm selection was based on the screening of four and seven week old pigs from three farms for B. hyodysenteriae, B. pilosicoli and strain 30446 prior to trial 1. Animals were randomly assigned to control  and inoculated  groups on arrival, and held for a 10-day acclimation period prior to inoculation. They were fed a commercially prepared, non-medicated, pelleted starter diet ad libitum, and housed in separate rooms in 4′×6′ pens each containing 3 pigs. During the acclimation period all pigs were tested for strain 30446, B. hyodysenteriae and B. pilosicoli in feces by qPCR ten, seven and five days and immediately prior to first inoculation. Fecal DNA extracts for pre-trial screening were tested in duplicate from DNA extraction through PCR .Eighteen five-week old, Landrace male weanling piglets were purchased from a PRRS negative high health commercial farm in Saskatchewan, Canada, with no history of swine dysentery or previous laboratory diagnosis of Brachyspira species by a novel nox sequence , thi , glpk  and est . The concentration of “B. hampsonii” strain 30446 in the inoculum was determined by qPCR and three daily inoculum doses of 3.42×108, 1.80×108 and 6.37×107 genome equivalents were given. These doses were intermediate to recent trials with B. murdochii where 106 colony forming units were used and a ‘B. suanatina’ sp. nov. trial where 30 ml of a 108 to109 cells/mL inoculum was used The inoculum was prepared by mixing approximately one part mucosal scraping and three parts 0.1 M pH 7.0 phosphate buffered saline (PBS) in a sterile blender. Strain 30446 was identified in the inocula and differentiated from other sequence  and usinPigs were inoculated on three consecutive days  as previously described “B. hampsonii” strain 30446. For statistical analysis, the qPCR results were categorized: 0 = not detected; 1 = DNQ; and 2 = quantifiable.The pigs were observed and scored twice daily for responsiveness, skin colour, appetite and body condition, respiratory effort and fecal consistency. Fecal consistency was scored daily as: 0 = formed, normal; 1 = soft, wet cement consistency; 2 = runny or watery; 3 = mucoid diarrhea; or 4 = bloody diarrhea. Gram stained fecal smears were made from rectal swabs collected daily from each pig. An investigator blinded to the slide ID, evaluated and scored all slides as: 0 = negative; 1 = less than 1 spirochete/high power field (hpf); 2 = between 2 and 10 spirochetes/hpf; 3 = between 11 and 49 spirochetes/hpf; 4 = greater than 50 spirochetes/hpf. Fecal samples collected from each pig on days 3, 7, 10 and 14 post inoculation were also tested by qPCR for Salmonella culture on brilliant green agar following enrichment in selenite broth (pooled colon and caecum), Lawsonia intracellularis PCR (ileum) B. hyodysenteriae, B. pilosicoli and “B. hampsonii” strain 30446 in triplicate as done in pre-trial screening. To detect viable strain 30446, colonic tissue was cultured for Brachyspira and if isolated, was speciated by sequencing nox PCR amplicons.After the intensity of mucohaemorrhagic diarrhea peaked (INOC), or at the end of the study (CTRL) on day 16, pigs were euthanized by cranial captive bolt and exsanguinated. A complete necropsy was performed with special attention to the stomach, duodenum, jejunum, ileum, spiral colon, caecum and rectum. Samples for histological examination, Brachyspira-like organisms associated with the lesions.The pathologist (YH) responsible for analysis of the samples was blinded to the identity of the slides. The presence or absence of superficial necrosis and inflammation were scored in Haematoxylin-Eosin stained sections of colon, caecum and rectum. Scoring for inflammation was based on the severity of neutrophil infiltration in the mucosa and fibrinous exudate on the surface. Necrosis of the mucosa was assessed by visualization of apoptotic cells and degenerated nuclei. Additionally, Warthin-Faulkner stained colon sections were examined for the presence of P-values ≤0.05 were considered significant.Statistical analysis was performed using SPSS version 18.0 . The presence or absence of mucohaemorrhagic diarrhea in INOC versus CTRL groups was compared using the Fisher’s exact test. The presence or absence of histologic and gross lesions in INOC pigs with or without mucohaemorrhagic diarrhea, and CTRL pigs was compared using the Fisher’s exact test. Spirochete slide score from colonic swabs and strain 30446 DNA concentration  in INOC pigs with and without mucohaemorrhagic diarrhea, and in CTRL pigs were compared using the Kruskal-Wallis test followed by post-hoc Mann-Whitney test if significant. Two-tailed B. hyodysenteriae, B. pilosicoli and “B. hampsonii” strain 30446. Randomly assigned CTRL (n = 6) and INOC (n = 12) pigs were held for an 8 day acclimation prior to inoculation. Groups were housed in separate rooms containing 2 pigs (INOC) or 3 pigs (CTRL) per pen. During the acclimation period, feces were collected eight, five and two days, and immediately prior to first inoculation and tested for Brachyspira spp. by PCR and culture as described above.The methodology for trial 2 was similar to that of trial 1; only major differences will be noted below. Pigs were sourced from the same farm following pre-screening of three and six week old pigs for “B. hampsonii” strain 30446 was cultivated in vitro on JBS broth from one of the pig colons used in trial 1 (isolate 6953). Approximately 2 cm2 of solid media with haemolytic zones were used to inoculate JBS broth (brain heart infusion with 5% (v/v) fetal calf serum, 5% (v/v) sheep’s blood, and 1% (w/v) glucose). Broth cultures were incubated in glass vials with magnetic stir bars anaerobically at 39°C for 24 hours with constant stirring.“B. hampsonii” strain 30446 was administered by intra-gastric tube for three consecutive days at doses of 2.78×106, 5.04×108 and 4.50×108 genome equivalents, followed by 50 ml PBS. CTRL pigs were mock inoculated with an equivalent volume of sterile JBS broth followed by 50 ml PBS. One INOC pig (#683) did not receive a complete dose of inoculum on D1, and the CTRL group inadvertently did not have feed removed prior to the second inoculation. Daily observations and fecal consistency scoring was performed as described above. The Gram stained smears made from rectal swabs were scored: 0 = less than 1 spirochete/hpf; 1 = between 2 and 10 spirochetes/hpf; 2 = between 11 and 49 spirochetes/hpf; 3 = greater than 50 spirochetes/hpf. Daily rectal swabs were cultured for Brachyspira and results were scored: 0 = negative; 1 = less than 10 colonies/1° streak; 2 = less than 10 colonies/2° streak; 3 = less than 10 colonies/3° streak; or 4 = less than 10 colonies/4° streak. Fecal samples collected daily from each pig were tested by qPCR for “B. hampsonii” strain 30446. For statistical analysis, the qPCR results were categorized: 0 = not detected; 1 = DNQ; and 2 = quantifiable.Brachyspira cultured from feces collected on the day of euthanasia and from terminal colon tissues was verified by nox PCR and speciated by sequencing.Necropsy examinations were performed after the intensity of mucohaemorrhagic diarrhea peaked (INOC), on day 14 for non-diarrheic INOC, or day 15 for CTRL. Histopathologic and microbiologic assessments of tissues were performed as described above. Statistical analysis was identical to that described for trial 1 above. In addition, daily fecal culture results in INOC pigs with and without mucohaemorrhagic diarrhea, and in CTRL pigs were compared using the Kruskal-Wallis test followed by post-hoc Mann-Whitney test if significant.B. hyodysenteriae and B. pilosicoli by qPCR. DNQ levels of “B. hampsonii” strain 30446 were found in 1/20 pigs prior to trial 1 and 3/20 pigs prior to trial 2. As DNQ levels of strain 30446 were detected at all three potential source farms tested, the farm with the lowest prevalence was used to supply the pigs for both trials. The pre-trial screening was conducted on different pigs from those used in each experiment.All pigs tested at the source farm during the pre-trial screening were negative for 5 genome equivalents/g) and day 10 PI . Of the 12 INOC pigs, nine developed mucohaemorrhagic diarrhea between days 4 and 10 PI (P<0.001).During the acclimation period, 3/12 INOC pigs had DNQ levels of strain 30446 DNA present in feces at single time points . The stund 10 PI . Of the P = 0.011) or CTRL (P<0.001). Similarly, in terminal colon, strain 30446 concentrations were significantly higher in pigs with mucohaemorrhagic diarrhea (1.84×107–1.32×108) than either INOC without mucohaemorrhagic diarrhea  or CTRL . Neither B. hyodysenteriae nor B. pilosicoli were detected in terminal colon tissue or contents from any pig.In pigs that developed mucohaemorrhagic diarrhea, the number of spirochetes seen on Gram stained slides increased concurrently with elevated fecal consistency scores . In one Lawsonia intracellularis, B. hyodysenteriae, B. pilosicoli, Salmonella spp., and PRRS virus were not detected in terminal samples from any pig. A single INOC pig (#57) was weakly positive for PCV2, and all other animals were negative. Culture of colonic tissue revealed “B. hampsonii” strain 30446 in nine, and B. intermedia  in one (#54) INOC pigs. Two INOC pigs (#61 and #63) were culture negative. The nox sequence of an isolate similar to B. intermedia  was also isolated by culture from one CTRL pig (#56).Gross pathological findings were consistent with clinical signs. Lesions were significantly more common in pigs with mucohaemorrhagic diarrhea than in INOC pigs without mucohaemorrhagic diarrhea . In affeHistologically, necrosis of the superficial colonic mucosa, which was covered by a bacteria-rich, mucoid exudate that extended into the superficial crypts, was observed. When examined using Warthin-Faulkner silver stains, numerous long, thin spirochetes were seen in the crypts and among the mixed bacteria on the surface . There w“B. hampsonii” strain 30446 was detected at DNQ levels in various INOC (8 of 12) and CTRL (4 of 6) pigs during the acclimation period, but generally as single events (4 copies/g) on the day of inoculation.e events . One INO4 to 2.67×105 genome copies/g) were detected by qPCR in the feces of three of these pigs , and positive culture results were obtained for #683, #688 and #695. Mucohaemorrhagic diarrhea was significantly more common in INOC (8 of 12) than CTRL (0 of 6) (P = 0.013).The study continued for 14 days following the first inoculation. All CTRL animals remained healthy throughout the study. Although low numbers of spirochetes were seen by direct microscopic examination in feces of CTRL pigs, no sample was positive by qPCR or culture during following inoculation. Of the 12 INOC pigs, eight developed mucohaemorrhagic diarrhea between 5 and 9 days PI , 5, 6. O). Gram stain slide scores of 3+ were seen before or concurrently with diarrhea among pigs that developed mucohaemorrhagic diarrhea, whereas scores above 2+ were not observed in pigs without mucohaemorrhagic diarrhea. Similarly, 2+ or 3+ cultures were only observed in pigs that developed mucohaemorrhagic diarrhea  and more frequent isolation of 30446 by culture . A significantly higher number of genome equivalents of strain 30446 DNA was detected by qPCR in the colonic tissue of pigs with mucohaemorrhagic diarrhea than CTRL (P<0.001). Although the same association was seen between pigs with mucohaemorrhagic diarrhea (9.65×103–1.85×107 genome copies/g) and INOC without mucohaemorrhagic diarrhea (DNQ - 1.03×104 genome copies/g), the non-parametric method of analysis was insensitive to this difference (P = 0.157). Sequencing of nox PCR amplicons from terminal fecal and colon tissue cultures revealed isolates with 99–100% sequence similarity to strain 30446 in all pigs with mucohaemorrhagic diarrhea and one INOC pig without mucohaemorrhagic diarrhea (#684). An isolate with sequence similarity to B. intermedia (97% identity over 825 bp of the nox gene to B. intermedia ATCC 51140T) was grown from one other INOC pig without mucohaemorrhagic diarrhea (#695). All CTRL pigs were culture negative. Importantly, nox sequence of the Brachyspira species isolated from the terminal fecal samples from the INOC group matched the nox sequences of the isolates from terminal colon culture.While fecal consistency score, spirochete slide score, culture results and qPCR counts varied daily, when elevated, they tended to move together as a group . Gram sdiarrhea . CompareLawsonia intracellularis, B. hyodysenteriae, B. pilosicoli, Salmonella spp. and PRRS were not detected in terminal samples from any pig. Pig #695 tested positive for PCV2 in ileum and lymph node by immunohistochemistry, while all others tested negative.Gross pathological findings were consistent with the clinical signs. Lesions were significantly more common in pigs with mucohaemorrhagic diarrhea than in INOC pigs without mucohaemorrhagic diarrhea or CTRL . In affeThe distribution of histologic lesions was significantly associated with clinical signs and was similar to that observed in trial 1 . There wnox PCR from terminal colon tissue from all INOC pigs. Sequencing revealed 11 amplicons with 99–100% sequence identity to strain 30446  and one amplicon with 97% sequence identity to B. intermedia ATCC 51140T . When tested by qPCR, strain 30446 specific amplicons were generated for all 12 INOC pigs and no CTRL pigs. When sequenced, 10 of these amplicons were 99–100% similar to strain 30446, while sequencing failed for the other two. The colon contents from one CTRL pig (#685) were positive for B. pilosicoli by qPCR (1.65×104 copies/g of feces) and its identity was confirmed by sequencing. Neither B. hyodysenteriae nor B. pilosicoli were detected in any other pigs.Amplicons were generated from B. hyodysenteriae“B. hampsonii” strain 30446 poses diagnostic challenges for producers, veterinarians and diagnosticians “B. hampsonii” strain 30446 necessitates a reliance on laboratory tests to make a specific etiological diagnosis. Unfortunately there is no standardized method of identification. Although there have been no attempts to standardize testing amongst laboratories to date, the use of direct examination, culture followed by genus specific PCR and sequencing, and species-specific PCR are recommended. A number of Brachyspira species specific PCR assays have been published Brachyspira species, the presence or absence of spirochetes is useful for interpreting molecular test results.Swine dysentery, which by definition is caused by “Brachyspira hampsonii” strain 30446 induces a mucohaemorrhagic diarrhea and colitis in pigs that is indistinguishable from swine dysentery. Trial 1 preceded our ability to grow “B. hampsonii” strain 30446 in broth culture, but the development of JBS broth in October 2011 made it possible to use a pure culture inoculum in trial 2. This pure broth culture was prepared from the tissue inoculum used in trial 1. The results of trial 2 demonstrate that strain 30446 causes mucohaemorrhagic diarrhea indistinguishable from the field cases where it was first observed, establishing this organism as a pathogen of swine.This research confirms that B. murdochii8 genome equivalents in trial 1 and 3.19×108 genome equivalents in trial 2 were used, intermediate to those used in previous studies. Disease developed between four and ten days PI in trial 1 and five to nine days PI in trial 2 indicating that a dose sufficient to cause disease was used. Interestingly, the incubation period observed in these trials was consistent with an infection trial conducted as part of the initial description of swine dysentery in 1921 The design of these trials was based on a successful, previously published trial utilizing “B. hampsonii” strain 30446 colonization (reported as DNQ) in 3/18 pigs in trial 1, and 12/18 pigs in trial 2 based on qPCR. In addition, 1 pig in trial 2 (#684) had 6.80×104 genome equivalents/g of “B. hampsonii” strain 30446 in feces on D0  only received two of three inoculum doses, and one (#684) was the pig with 10L. intracellularis, however the ileum of all pigs were negative by PCR for L. intracellularis and in no pigs were lesions typical of ileitis seen histologically. Whether or not these mild ileal lesions are a feature of strain 30446 associated disease or an incidental finding is unknown.Pathological findings in affected pigs, characterized by mucoid or mucohaemorrhagic colitis and muco-fibrinous typhlitis, were consistent with previous reports describing swine dysentery Brachyspira have been reported to cause disease in pigs nox sequence, strain 30446 clusters separately from the other known species with good bootstrap support . Furthermore, three of seven primer sets from a previously published MLST scheme for Brachyspira sp, failed to yield a product, while novel sequences were generated for the other four.A number of atypical or novel, phylogenetically distinct, strongly β-haemolytic  support . The parBrachyspira strains including “B. hampsonii” strain 30446 isolated from pigs with signs of swine dysentery in Iowa between 2008 and 2011. These experiments were the first to demonstrate the causal relationship between strain 30446 and mucohaemorrhagic typhlocolitis in pigs. The authors found that strongly β-haemolytic strains of Brachyspira spp., including strain 30446, produced disease and colonic lesions typical of those associated with B. hyodysenteriae. In the porcine experiment Lawsonia intracellularis and Salmonella spp., were negative. Collectively, these data provide substantive evidence of causality and provide the first report characterizing fecal shedding using a Canadian “B. hampsonii” 30446 isolate. While strain 30446 used for the present research clearly falls within “B. hampsonii” clade 2, the relationship between this strain and other members of the species, particularly clade 1, is unclear. Hence, additional research is necessary to more fully understand the clinical relevance of this novel and diverse Brachyspira species in swine.Two recent reports describe infection experiments using murine “Brachyspira hampsonii” strain 30446 and mucohaemorrhagic diarrhea in swine. The emergence of “Brachyspira hampsonii” strain 30446 therefore poses potential diagnostic challenges since the specificity of some currently used PCR assays may not detect this organism, and Brachyspira culture is not widely used in diagnostic laboratories in many countries. To date, cases have been diagnosed in pigs from Alberta, Saskatchewan, Iowa, Illinois, Minnesota, Missouri and North Carolina “B. hampsonii” strain 30446 are entirely unknown. Research is ongoing in our lab to address these and other important questions.In summary, our results confirm the causal relationship between


Angiogenesis quantification, through vessel counting or area estimation in the most vascular part of the tumour, has been found to be of prognostic value across a range of carcinomas, breast cancer included. We have applied computer image analysis to quantify vascular properties pertaining to size, shape and spatial distributions in photographed fields of CD34 stained sections. Aided by a pilot (98 cases), seven parameters were selected and validated on a separate set from 293 breast cancer patients.Two new prognostic markers were identified through continuous Cox regression with endpoints Breast Cancer Specific Survival and Distant Disease Free Survival: The average size of the vessels as measured by their perimeter , and the average complexity of the vessel shapes measured by their solidity (p = 0.004 and 0.004). The Hazard ratios for the corresponding median-dichotomized markers were 2.28 (p = 0.005) and 1.89 (p = 0.016) for the mean perimeter and 1.80 (p = 0.041) and 1.55 (p = 0.095) for the shape complexity. The markers were associated with poor histologic type, high grade, necrosis, HR negativity, inflammation, and p53 expression (vessel size only).Both markers were found to strongly influence the prognostic properties of vascular invasion (VI) and disseminated tumour cells in the bone marrow. The latter being prognostic only in cases with large vessels (p = 0.004 and 0.043) or low complexity (p = 0.018 and 0.024), but not in the small or complex vessel groups (p>0.47). VI was significant in all groups, but showed greater hazard ratios for small and low complexity vessels (6.54–11.2) versus large and high complexity vessels (2.64–3.06).i.e. the size and shape of vessels in the studied hot spots.We find that not only the overall amount of produced vasculature in angiogenic hot-spots is of prognostic significance, but also the morphological appearance of the generated vessels,  Written consent was obtained from all patients enrolled in the study.CD34-immunostained sections from 394 patients out of the 920 enrolled in the Oslo Breast Cancer Micrometastasis Project from 1995 to 1998 were examined. The current study is based on a subset of the material previously reported on regarding the prognostic significance and clinico-pathological associations of DTC and tumour vascularity The material has a known significant impact from angiogenesis quantification using CC, but not MVD Paraffin-embedded blocks with representative tumour tissue were sectioned 4 µm thick, as previously described 2; pixel pitch 0.34 µm). The image subjectively judged to be most vascular of the three was selected for each case. Cases with CD34-positive fibroblastic cells or spindle cells in the tumour stroma The image acquisition and field selection were performed as previously described Briefly, stains were identified from dynamically calculated intensity thresholds that identified all dark pixels, and pre-set colour hue thresholds which rejected any pixel associated with haematoxylin (i.e. blue). While determining vessel profiles from the distributions of CD34+ pixels, only gaps in the stains wider than 1.0 µm were considered to be lumens, and only stained objects at least 3.5 µm wide, lumens included, were considered to be microvessels. Width was defined as the diameter of the largest inscribable circle,  and Breast Cancer-Specific Survival (BCSS). Times were measured from date of surgery to event (systemic relapse and breast cancer death), or censured at last follow-up or unrelated death.2-values are reported outside the context of hypothesis testing.The data material was randomly split into two parts, pilot (25%) and validation (75%). The pilot cases  were used to aid parameter selection, while validation cases  were used to determine prognostic significance. Seven markers were validated (see parameter selection) using a common null hypothesis. The Bonferroni correction was applied with significance level α = 0.05 resulting in the test p≤β = α/7 = 0.0071. Marker validity was studied using Cox proportional hazard regression analysis (Cox regression) with statistically standardized continuous variables; hazard ratios (HR) correspond to an increase of one standard deviation. Markers that were significant  for both DDFS and BCSS were considered prognostic. χvs low-risk group. Associations with clinico-pathologic parameters were tested using the Pearsons chi-square or Linear-by-linear association exact test. Multivariate Cox regression was performed with parameters restricted to about 1 variable per 10 events.Prognostic parameters were further evaluated using median cut-off categorization. All further tests were done at the 0.05 level. Kaplan Meier analysis applied the logrank test; corresponding HRs were obtained using Cox regression for high-risk Statistical calculations were carried out using IBM SPSS Statistics (version 19); IBM, Endicott, NY, US.cf. supporting information 2-values, each parameter category was sorted according to survival performance. Based on this and findings in the literature Eighty-four pilot parameters were derived  of the field to that of the highest scoring subfield corresponding to four times higher magnification; MVluminal, the fraction of vessel profiles with at least one discernible lumen. MVscale is the average slope of the vascular content vs. field size curve in a double-logarithmic plot. The vessel profiles were first transformed to minimum connected strokes (skeleton) by removing their thickness (Σ. Square subfields were then randomly distributed across the image and the average vascular content (sum of stroke-lengths) of the fields computed using fifty different field sizes ranging from 3px to the image height. Low values are associated with uniform vessel distributions and small, simple vessels. MVSμ is the mean vessel solidity; i.e. the vascular area of the vessel divided by the area of its convex hull, i.e. the region with the shortest possible perimeter still containing the entire vessel .Seven parameters were tested in the validation material . MVPμ anMVPμ and MVSμ were found to be strongly associated with higher histologic grade, hormone receptor negativity, histologic type, presence of necrosis, moderate/marked inflammation, and p53 expression . No assoWhile the loss of information incurred by grouping makes it optimal to use continuous data whenever possible Multivariate survival analysis was performed separately for MVPμ and MSVμ. The models included clinico-pathological parameters that were significant in univariate survival analyses: histologic grade, LN-status, T-status, VI and hormone receptor status. In addition, systemic therapy was included. Neither marker came out significant in the multivariate model .Both MVPμ and MVSμ were found to strongly affect the prognostic properties of DTC and VI . In the 2-values 6.3–7.7 vs. 9.1 for MVPμ; BCSS: 12.3–12.9 vs. 14.2; all p<0.012). Thus, all would be prognostic, but MVPμ performed somewhat stronger (trend). However, total perimeter length in the field (MVPΣ) was not associated to survival at all (χ2≤1.0); it had an eight times lower χ2-value than the total vascular area (MVAΣ).MVPμ was the highest scoring of several strongly related vessel size measurements in the pilot. The others were the mean of the vascular area (MVAμ), endothelial area and vessel length (skeleton size). This result was repeated in the validation material , while it was r2 = 0.16 to MVAμ. Despite being only moderately correlated, MVSμ and MVPμ were not independently significant in the bi-variate Cox regression model. MVSμ was, however, found to significantly add prognostic information to MVAμ for DDFS (p = 0.031) and a trend for BCSS (p = 0.071). Both MVPμ and MVSμ significantly added independent prognostic information to MVAΣ—a marker closely related to Chalkley counts—for both end-points . MVAΣ was positively correlated with MVPμ (r2 = 0.35) and negatively with MVSμ (r2 = 0.17).The Pearson's correlation of MVSμ to MVPμ was found to be rIn the present study, the mean vessel size and the mean vessel shape complexity, as measured by MVPμ and MVSμ respectively, were identified as strong prognosticators in invasive breast carcinoma patients. We observed a significantly reduced survival in patients with large vessels compared to those who had smaller vessels, especially in the continuous model . The finIn this study, we used an automatic system to explore different vascular parameters that might be useful in evaluating tumour angiogenesis in a clinical setting. The size of the parameter-space far exceeded the statistical resolving power of the dataset; only a limited number of parameters could be analyzed. The literature concerning advanced quantifiers is limited and inconclusive The discovery of new independent vascular markers highlights a methodological challenge. The quantified fields are hot-spots selected in accordance with Weidner et.al. 's criteria Hot-spot vascular density assessment is interpreted as a measurement of the biologically most active part of the tumour. Thus, to some extent, it reflects the tumours growth potential, as angiogenesis is a limiting factor for both proliferation and metastasis These characteristics are reflected, not only in a strong association with necrosis, but also in how the vascular morphology affects DTC's prognostic ability . DTC+ paMVPμ and MVSμ were not found to be associated with physical tumour extension characteristics; T-status, LN-status, VI, and DTC status were insignificant. Strong associations were, however, observed for tumour grade, ER, necrosis, inflammation, and histologic type . Thus, tIn conclusion, we have identified two new prognostic vascular markers, MVPμ and MVSμ, pertaining to features at the individual vessel level. Not only is the overall amount of neo-vascularisation of prognostic value in breast cancer, but also morphological characteristics of the produced vessels. These new parameters add into our further understanding the role of angiogenesis in tumour progression, dissemination and metastasis. We find that the measurement of average vessel size and vessel shape in the hot-spot provides new prognostic information.Pilot S1Description of pilot parameters.(DOC)Click here for additional data file.


The development of pancreatic cancer is a process in which genes interact with environmental factors. We performed this study to determine the effects of the ABO blood group, obesity, diabetes mellitus, metabolic syndrome (MetS), smoking, alcohol consumption and hepatitis B viral (HBV) infection on patient survival.A total of 488 patients with pancreatic cancer were evaluated.p = 0.001) and more predominantly male (p = 0.020) than those never exposed to HBV. Patients with MetS had later disease staging (p = 0.000) and a lower degree of pathological differentiation (p = 0.008) than those without MetS. In a univariate analysis, the ABO blood group, smoking and alcohol consumption were not associated with overall survival. HBsAg–positivity and elevated fasting plasma glucose were significantly associated with unfavorable survival though not in the multivariate analysis. The presence of MetS , age ≥65, an elevated CA19–9 baseline level, TNM staging, the type of surgery, the degree of differentiation and chemotherapy were independently associated with overall survival.Patients who presented as chronic carriers of HBV infection were younger at disease onset (We report, for the first time, that patients with chronic HBV infection may represent a special subtype of pancreatic cancer, who have a younger age of disease onset and male dominancy. Patients with MetS had later disease staging and a poorer histological grade. Patients with MetS demonstrated significantly poorer survival. Pancreatic cancer is the fifth most common cancer and the fourth leading cause of cancer-related mortality for men and women in Western societies It has been increasingly recognized that the development of cancer is a process in which genes interact with environmental factors. Several risk factors related to pancreatic cancer have been previously explored, including genetics Therefore, we performed this study including patient clinicopathological characteristics and survival dates to determine the prognostic value of several risk factors related to pancreatic cancer.Clinical and laboratory examinations were performed after obtaining informed written consent from all patients and approval from the independent Institute Research Ethics Committee at the Cancer Center of Sun Yat-sen University.Between January 1, 1998, and December 30, 2010, 645 patients with histologically confirmed pancreatic adenocarcinoma treated at the Sun Yat-sen University Cancer Center in Guangzhou, China, were retrospectively analyzed. Patient follow-up was completed until October 1, 2011. At the end of follow-up, survival data were unavailable for 157 patients due to incorrect contact information. A total of 488 patients with complete survival dates were included in this study. A 3-dimensional, contrast-enhanced computed tomography (CT) scan or magnetic resonance imaging (MRI) scan was used for clinical staging. In patients who were considered unresectable, pathological assessment was based on a cytological diagnosis through fine-needle aspiration. A biopsy specimen was used for pathological diagnosis in patients who had undergone exploration only. In patients who had undergone surgical resection, the pathological diagnosis depended on the resected specimen.Serum samples were collected to test the ABO blood groups and the presence of HBV infection. The ABO blood types  were tested using mouse-derived monoclonal antibodies . An enzyme-linked immunosorbent assay was used to detect hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), hepatitis B e antigen (HBeAg), hepatitis B e antibody (anti-HBe), and hepatitis B core antibody (anti-HBc) . Data on patients’ age, sex, baseline blood pressure, height, weight, fasting plasma glucose, triglyceride and HDL levels, cigarette smoking, alcohol intake, personal history of diabetes mellitus and hypertension were collected by clinical staff.2 and categorized into four groups .Metabolic syndrome was defined as a clustering of three or more of the following five risk factors: (1) fasting plasma glucose ≥5.6 mmol/l (100 mg/dl) or currently taking medication for diabetes mellitus; (2) blood pressure ≥130/≥85 mmHg or currently taking medication for hypertension; (3) triglycerides ≥1.7 mmol/l (150 mg/dl); (4) HDL-cholesterol: for men: <1.03 mmol/l (40 mg/dl) and for women: <1.29 mmol/l (50 mg/dl); (5) obesity: for men: waist circumference >102 cm and for women: waist circumference >88 cm, which was suggested by the National Cholesterol Education Program Adult Treatment Panel (NCEP-ATP) III guidelines 2 test and Student’s t-test were used to compare the variables. The statistical analyses were performed using the SPSS statistical package . Overall survival (OS) was calculated from the date of diagnosis to the date of patient death from cancer or the last date of follow-up. A Cox regression was used for univariate analysis. Variables that were significantly prognostic in the univariate analysis were selected for use in the final multivariable Cox proportional hazards regression analysis using the forward stepwise method. OS curves were compared using the two-sided log-rank test and Kaplan-Meier survival analyses. P<0.05 was defined as statistically significant.Descriptive statistics of the clinicopathological data of patients with pancreatic cancer were calculated with mean, standard deviation (SD) and frequencies, depending on the type of data. To test whether the clinicopathological characteristics differed for those with HBV infection and MetS, Pearson’s χThe associations among clinicopathological characteristics, potential risk factors and OS of patients with pancreatic cancer are shown in p trend = 0.000). According to the univariate analysis, factors associated with OS included the presence of MetS (p = 0.000) (p = 0.034), age ≥65 (p = 0.002), pretherapeutic weight loss ≥5 percent (p = 0.001), elevated fasting plasma glucose (p trend = 0.006), elevated baseline CA19–9 levels (p = 0.000), higher TNM staging (AJCC) (p = 0.000), type of surgery (p = 0.000), degree of differentiation (p = 0.000) and chemotherapy (p = 0.019). In addition, there was a trend toward chronic carriers of HBV infection  having a negative effect on prognosis compared with patients who were never exposed to HBV  (p = 0.059). Patients with obesity, elevated levels of triglycerides and low levels of HDL were also shown have a likely shorter survival duration . However, ABO blood type, gender, smoking, alcohol intake, history of chronic pancreatitis, family history of pancreatic and other cancers, and tumor location were not associated with overall survival  , HBsAg-psurvival .p trend = 0.188). When MetS was included in the multivariate model, it remained significant and independently associated with cancer mortality . Otherwise, age ≥65 (p = 0.032), elevated baseline CA19–9 levels (p = 0.046), higher TNM staging (AJCC) (p = 0.010), type of surgery (p = 0.039), degree of differentiation (p = 0.006) and chemotherapy (p = 0.015) were also independently associated with OS. However, HBsAg-positivity was not an independent prognostic factor (p = 0.711)  .p = 0.001) (p = 0.020). HBV infection was not significantly associated with the other clinicopathological characteristics of patients with pancreatic cancer. Patients who were older than 65 years old more frequently presented with MetS compared with patients who were younger than 65 years old (p = 0.038). Sixty-six (90.41%) patients who presented with MetS were at stage III or IV compared with 279 (67.23%) patients who were not (p = 0.000) (p = 0.008)  . A total= 0.000) . Patient= 0.008)  Table 3p = 0.001To the best of our knowledge, this is the first study to determine the prognostic effects of several risk factors and survival in patients with pancreatic cancer. Although previous studies have provided evidence in support of the association between the ABO blood type, smoking, alcohol consumption, obesity, diabetes, HBV infection and increased risk of pancreatic cancer, our study did not find an effect of smoking, alcohol drinking, or the ABO blood group on the prognosis of patients with pancreatic cancer. However, patients with HBsAg-positivity and elevated fasting plasma glucose levels were associated with unfavorable survival; however, these were not independent prognostic factors. The presence of MetS was better than hyperglycemia, and MetS was independently associated with OS.Recently, several epidemiological observations have found a relationship between ABO blood group genotypes and pancreatic cancer risk Some epidemiological observations have found an association between HBV infection and the risk of pancreatic cancer development Diabetes has been consistently linked to the risk of pancreatic cancer. However, studies investigating the prognostic value of diabetes mellitus and pancreatic cancer have shown inconsistent results Recently, several clinical studies have found a reduced incidence of tumors treated with the antidiabetic agent metformin There are some limitations to this study. First, only 18 patients (3.69%) were categorized as obese in our study. Due to the lack of a direct measure of central adiposity in this study, BMI was utilized as a proxy variable for waist circumference Overall, our study did not provide evidence of the impact of the ABO blood group, elevated fasting plasma glucose, obesity, smoking, alcohol consumption or HBV infection on prognosis in patients with pancreatic cancer. However, we report for the first time that patients with chronic HBV infection may represent a special subtype, who present with earlier disease onset and male dominancy. Patients with MetS had later disease staging and a poorer histologically differentiated grade than those without MetS. Patients with MetS demonstrated significantly poorer survival. Additional large-scale studies are needed to extend and confirm our results.


Dyslipidemia is an extremely prevalent but preventable risk factor for cardiovascular disease. However, many dyslipidemia patients remain undetected in resource limited settings. The study was performed to develop and evaluate a simple and effective prediction approach without biochemical parameters to identify those at high risk of dyslipidemia in rural adult population.Demographic, dietary and lifestyle, and anthropometric data were collected by a cross-sectional survey from 8,914 participants living in rural areas aged 35–78 years. There were 6,686 participants randomly selected into a training group for constructing the artificial neural network (ANN) and logistic regression (LR) prediction models. The remaining 2,228 participants were assigned to a validation group for performance comparisons of ANN and LR models. The predictors of dyslipidemia risk were identified from the training group using multivariate logistic regression analysis. Predictive performance was evaluated by receiver operating characteristic (ROC) curve.P<0.001).Some risk factors were significantly associated with dyslipidemia, including age, gender, educational level, smoking, high-fat diet, vegetable and fruit intake, family history, physical activity, and central obesity. For the ANN model, the sensitivity, specificity, positive and negative likelihood ratio, positive and negative predictive values were 90.41%, 76.66%, 3.87, 0.13, 76.33%, and 90.58%, respectively, while LR model were only 57.37%, 70.91%, 1.97, 0.60, 62.09%, and 66.73%, respectively. The area under the ROC cure (AUC) value of the ANN model was 0.86±0.01, showing more accurate overall performance than traditional LR model (AUC = 0.68±0.01, The ANN model is a simple and effective prediction approach to identify those at high risk of dyslipidemia, and it can be used to screen undiagnosed dyslipidemia patients in rural adult population. Further work is planned to confirm these results by incorporating multi-center and longer follow-up data. Dyslipidemia is a widely recognized risk factor for cardiovascular diseases, a leading cause of death in both developed and developing countries Estimating an individual’s risk across a range of presumed risk factors is fundamental to prevent dyslipidemia Logistic regression (LR) is often used to identify significant factors that correlate with disease, and has commonly been used to develop models for clinical diagnosis and treatment This study was a cross-sectional survey, and subjects were selected randomly from eligible candidates in the residential registration record from the rural areas in Henan Province, China. The eligibility of the candidate was defined as those who were stable residents for at least 10 years in the study areas aged 35–78 years, and were free from the following conditions: 1) severe psychological disorders, physical disabilities, cancer, Alzheimer’s disease, or dementia, within 6 months; or 2) currently diagnosed with tuberculosis, acquired immune deficiency syndrome (AIDS), and other infectious diseases. Ultimately, a total of 8,914 residents who met the criteria enrolled in the study. Informed consent was obtained from all participants. The procedure of the study was approved by the Zhengzhou University Medical Ethics Committee, and written informed consent was obtained for all participants.A home interview was conducted by physicians or public health workers from the local Centers for Disease Control and Prevention and the community hospital. All investigators and staff successfully completed a training program that oriented them both to the aims of the study and to the specific tools and methodologies used. At the training sessions, interviewers were given detailed instructions on administration of the study questionnaire.Education level was classified into five categories: no education, primary school, middle school, high school, college and above. Marital status was categorized as unmarried, married/cohabitation, and divorced/widowed. Occupation was categorized as farmers, laborers, professionals and employers/managers. Individual annual income was calculated by total household income divided by the number of family members. Positive family history was defined as the participant’s parents or siblings having a history of dyslipidemia at or before the baseline examination.Three-day dietary intake data were collected from each subject using a 24-hour diet recall and a 2-day diet record. The daily intake of energy, nutrients, food and food groups for each subject were calculated using China Food Composition Table Current smoking status included smoker and non-smoker. Participants who currently smoked and had smoked at least 100 cigarettes during their lifetime were classified as current smokers if they answered affirmatively to the following questions: “Do you smoke cigarettes now?” and “Have you smoked at least 100 cigarettes during your lifetime?” Physical activity level was classified as low, moderate, or high according to the International Physical Activity Questionnaire (IPAQ) scoring protocol Body weight and height were measured twice in light indoor clothing without shoes to the nearest 0.1 kg and 0.1 cm, respectively. Waist circumference (WC) was measured twice at the mid-point between the lowest rib and the iliac crest to the nearest 0.1 cm, after inhalation and exhalation. Central obesity based on WC  was defined according to WHO criteria for the Asia-Pacific region population An overnight fasting blood specimen was collected in a vacuum tube containing EDTA for measurement of lipid profile. Blood specimens were centrifuged at 4°C and 3,000 rpm for 10 minutes, and the plasma was transferred and stored at −20°C for biochemical analyses. Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured enzymatically on an automatic biochemical analyzer  with reagents purchased from Wako Pure Chemical Industries .According to the China Adult Dyslipidemia Prevention Guide (2007 Edition) criteria 2 = 2,228) were assigned to the validation group for performance comparisons of ANN and LR models. The proportion of dyslipidemia between the training group and validation group was similar to the surveyed population data, and there was no statistically significant difference by χ2 test for gender and age between the training and validation datasets  were randomly selected to provide the training group for constructing ANN and LR prediction models. The remaining 25% of participants (Ndatasets .P) = 0β+1 χβ1+2 χ2β+…+ i χiβP = 1/(1+eP)-logit used for calculating the probability of dyslipidemia in this study, where 1 =  dyslipidemia and 0 =  non-dyslipidemia. A stepwise algorithm was used to construct the multivariate LR model. At each step, independent variables not yet included in the equation were tested for possible inclusion. The variable with the strongest significant contribution to improving the model was included. Variables already included in the logistic regression equation were tested for exclusion on the basis of a likelihood ratio test. The analysis ended when no further variables for inclusion or exclusion were available The LR model generates the coefficients for the following formula used in logit transformation for predicting the probability of an event among patients with certain characteristics of interest: Logit  curve analysis.P-values were two-sided, and P-values less than 0.05 were considered statistically significant.The LR model and ROC curve analysis were constructed using SAS 9.1 . The ANN model was performed with MATLAB 7.1 . All reported P>0.05), confirming the reliability of random subject selection.1N = 6,686). With the construction of a multivariable model . Factors significantly associated with dyslipidemia were age (odds ratio OR = 1.042), higher-educational level (OR = 1.362), smoking (OR = 1.165), more high-fat diet (OR = 1.403), positive family history of dyslipidemia (OR = 1.876), and central obesity  (OR = 2.327). There was also an inverse relationship for male gender (OR = 0.758), more vegetable and fruit intake (OR = 0.844), and more physical activity (OR = 0.924).A − 0.2774 XG +0.092 XEL +0.1529 XS +0.3385 XHFD - 0.1701 XVFI +0.6290 XFH - 0.0786 XPA +0.8444 XWC. According to the predictors of dyslipidemia risk from the LR analysis, the ANN model was built using the training group data. The predictors used as the model input were XA, XG, XEL, XS, XHFD, XVFI, XFH, XPA, and XWC. The probability of whether an individual had dyslipidemia was the output variable. The analysis structure of the neural network included three layers: input, hidden and output layers. The logit probability of having dyslipidemia was described by the following LR model: −2.7155+0.0410 X2N = 2,228). The ANN model detected 911 dyslipidemia patients from the 1011 actual dyslipidemia patients, whereas the LR model only detected 582 dyslipidemia subjects. CI: 0.85–0.88) than that of the LR model  (P<0.001).The ANN and LR models could successfully distinguish an individual’s risk of having dyslipidemia. We compared the individual’s predicted risk from the two models with the actual status using the validation group Click here for additional data file.


Echocardiography is the most convenient method used to evaluate right ventricular function, and several echocardiographic parameters were studied in previous studies. But the value of these parameters to assess the right ventricular function in patients with pulmonary arterial hypertension (PAH) has not been well defined.Patients with PAH were observed prospectively. Right heart catheterization, echocardiography and cardiac magnetic resonance (CMR) were performed within 1 week interval. The correlations between echocardiographic parameters and right ventricular ejection fraction (RVEF) derived from CMR as well as hemodynamics were analyzed.r = 0.440, P = 0.015; tricuspid annular systolic excursion velocity [S’], r = 0.444, P = 0.016; isovolumic acceleration [IVA], r = 0.600, P = 0.001; right ventricular fraction area change [RVFAC], r = 0.416, P = 0.022; ratio of right ventricular transverse diameter to left ventricular transverse diameter [RVETD/LVETD], r = −0.649, P<0.001; RVMPI, r = −0.027, P = 0.888). After adjusted for mean right atrial pressure, mean pulmonary arterial pressure and pulmonary vascular resistance (PVR), only IVA and RVETD/LVETD could independently predict RVEF. Four echocardiographic parameters displayed significant correlations with PVR .Thirty patients were enrolled including 24 with idiopathic PAH, 5 with PAH associated with connective tissue diseases and 1 with hereditary PAH. All echocardiographic parameters except right ventricular myocardial performance index (RVMPI) correlated significantly with RVEF (tricuspid annual plane systolic excursion [TAPSE], The echocardiographic parameters IVA and RVETD/LVETD can reflect RVEF independently regardless of hemodynamics in patients with PAH. In addition, TAPSE, S’, RVFAC and RVETD/LVETD can also reflect PVR in PAH patients. Pulmonary arterial hypertension (PAH), caused by vascular remodeling of the small pulmonary arteries, is characterized by mean pulmonary arterial pressure (mPAP) ≥25 mmHg and pulmonary capillary wedge pressure (PCWP) ≤15 mmHg at rest assessed by right heart catheterization (RHC). Pulmonary vascular remodeling and the RV dysfunction both play important roles in the pathophysiological process of PAH. Comprehensive evaluation of the condition of pulmonary hypertension (PH) patients requires the consideration of both the pulmonary circulation and RV function. RVEF that can reflect intrinsic RV contractility has been demonstrated to be an independent predictor of survival. Echocardiography is the most common and convenient method used to evaluate RV function and hemodynamics of pulmonary circulation, and several echocardiographic parameters have been studied in previous studies, including RV fractional area changes (RVFAC), RV myocardial performance index (RVMPI), Thus, in the present study, we attempted to analyze the correlations between the echocardiographic parameters and RVEF as well as hemodynamics, in order to systematically elucidate the role of these parameters in assessing the severity of PAH.PAH patients who were referred to Fuwai hospital between October 2010 and April 2012 for treatment were evaluated for this study. Inclusion criteria were mPAP≥25 mmHg and PCWP≤15 mmHg by RHC at rest. We excluded (1) patients with congenital heart diseases; (2) patients with unstable conditions; (3) patients with contraindication of CMR; (4) patients who refused to participate. Finally, thirty patients were recruited, and all of them underwent RHC, echocardiography and CMR within 1 week. The written informed consent was acquired from all patients enrolled. This study complied with the Declaration of Helsinki and was approved by the Institutional Review Board of Fuwai Hospital.Exercise capacity was evaluated by the WHO functional class (WHO-FC) and 6-minute walk distance (6MWD) according to the guidelines of the American Thoracic Society. Echocardiography was performed by the Philips iE33 system, and images were analyzed offline after the procedure. TAPSE was acquired by M-mode image, and the cursor was placed through the lateral tricuspid annulus, the displacement of which from the end-diastole to the end-systole was measured. The S’ known as tricuspid annular systolic excursion velocity was measured by Tissue Doppler image (TDI) in the apical 4-chamber view. The RV isovolumic acceleration was calculated as the peak isovolumic myocardial velocity divided by the time to peak velocity measured by TDI at the lateral tricuspid annulus. RVFAC was calculated as follows: RVFAC = (RV diastolic area-RV systolic area)/RV diastolic area×100%. RV diastolic area and RV systolic area were obtained from the two-dimensional apical 4-chamber view. The RVMPI is the ratio of the isovolumic time and the ejecting time, which was measured in the same pulsed TDI. The isovolumic time was calculated by subtracting the ejecting time from the tricuspid closure time. The ratio of RV transverse diameter to LV transverse diameter was measured at the base in the end-diastole using the apical 4-chamber view.2; matrix, 150×256 pixels; pixel size, 1.9×1.3; slice thickness, 6 mm; slice distance, 4 mm). The endocardium and epicardium outline of RV and LV were drawn manually using Argus software  by an experienced radiologist to obtain volumes of RV and LV.CMR was performed on a Siemens 1.5 T Sonata system , and phase offset errors was corrected by simultaneous ECG recording. Short-axis cine images including the whole LV and RV from apex to base were acquired by the gradient-echo pulse sequence (True-FISP by Siemens) .Descriptive data for continuous variables with Gaussian distribution were presented as means ± standard deviation. Continuous variables were tested for adherence to a normal distribution with the Kolmogorov-Smirnov method. Pearson’s correlation coefficients were calculated to analysis the association of interesting parameters. Multivariate linear regression analysis was carried out to explain the role of echocardiographic parameters in predicting RVEF. Statistical significance was defined as 2. The subtypes of PAH were, idiopathic PAH 24 patients (80%); PAH associated with connective tissue diseases, 5 patients (16.7%); hereditary PAH, 1 patient (3.3%). The mPAP was 58±16 mmHg and PVR was 1168.5±509.4 dyn·s·cm−5. The cardiac index measured by thermodilution method was 2.39±0.98 L/min/m2. The data of echocardiography and CMR are presented in Demographic, clinical and hemodynamic data of the thirty patients enrolled are shown in r = 0.440, P = 0.015; S’, r = 0.444, P = 0.016; RVFAC, r = 0.416, P = 0.022; IVA, r = 0.600, P = 0.001; RVETD/LVETD, r = −0.649, P<0.001). However, RVMPI had no significant correlation with RVEF .Correlations between echocardiographic parameters and CMR-derived RVEF were shown in r = −0,615, P<0.001; S’, r = −0.557, P = 0.002; RVFAC, r = −0.454, P = 0.012; RVETD/LVETD, r = 0.543, P = 0.002). Moreover, TAPSE , IVA  and RVETD/LVETD  had significant relationships with mRAP. TAPSE, RVFAC and RVMPI showed significant correlations with mPAP.Correlations between echocardiographic parameters and hemodynamic parameters are presented in P = 0.043 and P = 0.015)  . The modSeveral echocardiographic parameters have been reported to assess the RV function and hemodynamics of pulmonary circulation. However, the value of these parameters remains controversial. In the present study, we used echocardiography and CMR to assess RV function and structure in PAH patients. We systemically analyzed the correlations between the echocardiograpic parameters and RV function as well as the hemodynamics, which may provide reference for comprehensively assessing the severity of PAH patients.Despite several studies have validated the value of some echocardiographic parameters in assessing RV function, Among the six echocardiographic parameters, TAPSE, S’, RVFAC, IVA and RVETD/LVETD correlated significantly with RVEF. However, after adjusting for the hemodynamics including mRAP, mPAP and PVR, multivariate regression analysis indicated that IVA and RVETD/LVETD were the only two independent predictors of RVEF. Unaffected by pre−/after-load, et al reported that TAPSE and S’ (r = 0.86 and r = 0.63) significantly correlated with RVEF derived from CMR, et al, PVR of the patients were lower than that of the population in our study and the RV function of were relatively better (with RVEF of 38%±11% vs. 27%±12%). The differences may be due to the heterogeneity of patients included. With regard to the dilated RV in PH patients, circumferential contractility may also contribute a lot to RVEF. A CMR study by Kind et al reported that transverse wall movements provided important information of RV function in PH patients, in which TAPSE measured by CMR also had relatively weak relationship with RVEF (r = 0.458). Both TAPSE and S’ can reflect the longititudinal systolic function of the RV. TAPSE from M-mode echocardiograph has been reported to significantly correlate with RVEF derived from radionuclide angiography. Although the RVFAC is a common parameter for assessing RV systolic function, its two-dimensional measuring view can not completely reflect RVEF which is a three-dimensional parameter. Moreover, the hypertrophied RV trabeculae hinder us to distinguish the endocardium clearly in PH patients, therefore, accurate assessment of RVFAC can’t be assured. So the correlation coefficient in this study is moderate as well as the previous study, The RVMPI has been reported to assess the combined systolic and diastolic function, With regards to the hemodynamic data, TAPSE, S’, RVFAC and RVETD/LVETD correlated with PVR significantly, which may provide some information for PH diagnosis and severity evaluation. In line with the previous study, RVMPI showed significant correlation with mPAP, but not PVR. Firstly, echocardiography, CMR and RHC were not performed simultaneously, which may affect accurate comparisons of multiple methods. However, we excluded the patients with unstable conditions during the study period to reduce the bias. Secondly, the significance of imaging data may vary in different stages of PAH. The number of patients in the present study was small, which did not allow us to evaluate the data in patients with different stages.The echocardiographic parameters IVA and RVETD/LVETD can reflect RVEF independently regardless of hemodynamics in patients with PAH. In addition, TAPSE, S’, RVFAC and RVETD/LVETD can also reflect PVR in PAH patients.


P(Z) <10−5, P(Q) <10−4; dominant model: OR = 1.21, 95%CI = 1.14–1.27, P(Z) <10−5, P(Q) <10−4; recessive model: OR = 1.18, 95%CI = 1.12–1.24, P(Z) <10−5, P(Q) = 0.04]. In addition, our data revealed that rs2046210 conferred greater risk in estrogen receptor (ER)-negative tumors  than in ER-positive ones . When stratified by ethnicity, significant associations were found in Caucasian and Asian populations, but not detected among Africans. There was evidence of heterogeneity (P<0.05), however, the heterogeneity largely disappeared after stratification by ethnicity. The present meta-analysis demonstrated that the rs2046210 polymorphism may be associated with increased BC susceptibility, but this association varies in different ethnicities.Genome-wide association studies (GWAS) have identified several genetic susceptibility loci for breast cancer (BC). One of them, conducted among Chinese women, found an association of rs2046210 at 6q25.1 with the risk of BC recently. Since then, numerous association studies have been carried out to investigate the relationship between this polymorphism and BC risk in various populations. However, these have yielded contradictory results. We therefore performed a meta-analysis to clarify this inconsistency. Overall, a total of 235003 subjects based on 13 studies were included in our study. Significantly increased BC risk was detected in the pooled analysis [allele contrast: OR = 1.13, 95%CI = 1.10–1.17,  BRCA1 and BRCA2, have been identified as risk factors for BC, but only account for less than 5% of overall BC cases ESR1 gene, which encodes estrogen receptor α (ERα) and may be involved in the development of BC. Therefore, numerous studies have been carried out to investigate this susceptibility subsequently. However, a proportion of them have produced conflicting results, which may be caused by the restriction of sample size or ethnic diversity. And individual studies may have insufficient power to achieve a reliable conclusion.Breast cancer (BC) is the most common cancer among women worldwide To clarify this inconsistency, we therefore performed the first comprehensive meta-analysis to determine the overall effect of the rs2046210 polymorphism on the risk of BC.Eligible literatures in English language published before January 10th 2013 were identified by a search of PUBMED, EMBASE and ISI Web of Science using combination of following keywords: breast cancer, 6q25.1, rs2046210, single nucleotide polymorphism (SNP or polymorphism). All the references cited in these studies and previous review articles were reviewed to identify additional relevant publications.P-value; (4) the studies described the genotyping methods, equipment and protocol used or provide corresponding reference to them; (5) the diagnosis of BC patients was confirmed histologically or pathologically. The major reasons for exclusion of studies were (1) used overlapping samples; (2) were case-only studies or review articles.For inclusion, eligible studies had to meet all of the following criteria: (1) the studies were published in peer-reviewed journals and were original papers having independent data from other studies; (2) the studies investigated the association between rs2046210 and BC risk using either case–control or cohort design; (3) the studies have sufficient data to calculate the odds ratio (OR) with its 95% confidence interval (CI) and For each study, the following data were extracted independently by two participants: first author’s surname, year of publication, diagnosis criteria, age, ethnicity, study site, study strategy, Hardy-Weinberg equilibrium (HWE) status, genotyping method, source of control, estrogen receptor (ER) status, total number of cases and controls, and genotype frequency among cases and controls. The results were compared and disagreements were discussed and resolved by consensus.2) test was used to test for deviation from Hardy-Weinberg equilibrium (HWE). Odds ratio (OR) with its 95% confidence interval (CI) was used to assess the strength of association between rs2046210 and BC risk. We used allele contrast (A vs. G), dominant (AA+AG vs. GG) and recessive (AA vs. GG+AG) genetic models to examine the relationship between risk allele or genotypes and BC susceptibility. Cochran’s Chi square-based Q statistic test was performed to assess possible heterogeneity across studies. If heterogeneity existed (P<0.05), the random effects model was adopted to calculate the overall OR value Z test.The chi-square .Subgroup analyses were performed according to ethnicity , ER-positive/negative status, sample size (<5000 and ≥5000), and source of controls. In addition, sensitivity analysis was performed to assess the stability of the results by removing each study in turn from the total and re-analyzing the remainder. Funnel plot and Egger’s regression test were used to assess publication bias. All The literature search yielded 127 references, and the study selection process is shown in P(Z) <10−5, P(Q) <10−4; dominant model: OR = 1.21, 95%CI = 1.14–1.27, P(Z) <10−5, P(Q) <10−4; recessive model: OR = 1.18, 95%CI = 1.12–1.24, P(Z) <10−5, P(Q) = 0.04] as shown in P(Z) <10−5, P(Q) = 0.03; dominant model: OR = 1.12, 95%CI = 1.10–1.14, P(Z) <10−5, P(Q) = 0.10; recessive model: OR = 1.14, 95%CI = 1.11–1.18, P(Z) <10−5, P(Q) = 0.99] and Asians . However, there was no evidence indicating significant association among African population in any genetic model (Main results of our study investigating the effect of the rs2046210 polymorphism on BC risk are listed in ic model .P(Z) <10−5, P(Q) <10−4] than ER-positive tumors  .Each study included in the meta-analysis was removed from the total each time to reflect the influence of individual data set to the pooled ORs. According to the result of sensitivity analysis, the pooled ORs were not qualitatively altered after the removal of any data set, even for two studies by Cai et al. The rs2046210 polymorphism was first identified as a risk variant for BC by Zheng et al In the stratified analysis by ethnicity, significant associations were found among Caucasians and Asians in all genetic models, while no association was detected in African population. In fact, the frequency of the risk-associated A-allele was substantially higher in African population (e.g. 69% in HapMap YRI) than in Caucasian (e.g. 29% in HapMap CEU) or Asian populations (e.g. 38% in HapMap CHB). It has been reported that the A-allele may consist of different haplotypes with different risk associations in African population. The study by Ruiz-Narváez et al. Meta-analysis is often dominated by a few large studies, which may remarkably reduce the evidence from smaller studies. However, in the stratified analysis according to sample size, significantly increased BC risk was found in both large and small studies for all genetic models. In the subgroup analysis based on control source, we also found significant associations in all the subgroups , and there was no obvious difference between these subgroups. In addition, our data indicated that rs2046210 was more closely related to ER-negative BC, which is consistent with previous reports in Chinese women ESR1 gene (29 kb upstream of the first untranslated region and 180 kb upstream of the first coding exon) ESR1 encodes estrogen receptor α (ERα), which regulates signal transduction of estrogen, a sex hormone that plays a central role in the etiology of BC. Elevated estrogen levels have been reported to be associated with increased BC risk ESR1 and ESR2, have been the focus of numerous epidemiologic studies on BC risk ESR1, the rs2046210 polymorphism or SNPs in LD with it may modulate the expression of ESR1 and alter its downstream signaling, and then is likely to affect BC susceptibility. Interestingly, it is found in a recent GWAS study that the 6q25.1 locus is associated with bone mineral density, which is also a phenotype affected by estrogen This polymorphism is located upstream of the Despite our efforts to investigate the relationship between rs2046210 and BC risk, some limitations should be acknowledged. First, the subgroup meta-analysis examining the interaction between the ER status and the effect of rs2046210 on BC susceptibility was performed based on limited studies with such information available. Therefore, selection bias may have occurred and the corresponding result may be over inflated. In this context, more reliable result can be expected if relevant statistical data are available for a pooled analysis. Second, results in this meta-analysis were based on unadjusted estimates. If all the individual raw data were provided, a more precise analysis could be conducted which would allow for the adjustment by other covariates including age, drinking status, cigarette consumption and other lifestyles. Third, there was substantial between-study heterogeneity found in the overall pooled analysis. However, the heterogeneity was effectively decreased after stratification by ethnicity. In spite of these limitations, no publication bias was observed, and the large number of study subjects still guarantees the statistical power of our analyses.To conclude, this meta-analysis showed that the rs2046210 polymorphism was significantly associated with increased rick of BC, particularly in Caucasian and Asian populations. Further studies with more accurate phenotype and genotype data, detailed individual information, larger sample size of different ethnic populations and standard statistical methods will be needed to validate our results. Moreover, the interactions between rs2046210 and other SNP loci which are in LD with it should also be evaluated to further elucidate the underlying relationship between this polymorphism and the risk of BC.Figure S1Flow diagram of the literature selection process.(DOCX)Click here for additional data file.Figure S2Funnel plot of the association between rs2046210 and breast cancer risk .(DOCX)Click here for additional data file.Figure S3Funnel plot of the association between rs2046210 and breast cancer risk (dominant model).(DOCX)Click here for additional data file.Figure S4Funnel plot of the association between rs2046210 and breast cancer risk (recessive model).(DOCX)Click here for additional data file.PRISMA Checklist S1(DOC)Click here for additional data file.


Background. Skin biopsy is an established method for allying the dermatologist in overcoming the diagnostic dilemmas which occur during consultations. However neither do all skin biopsies produce a conclusive diagnosis nor the dermatologists routinely perform this procedure to every patient they consult. The aim of this study was to investigate the favourable clinical diagnoses set by dermatologists when performing skin biopsy, the diagnoses reached by the dermatopathologists after microscopic examination, and the relationship between them and finally to comment on the instances that skin biopsy fails to fulfill the diagnostic task. Methods. Six thousand eight hundred and sixteen biopsy specimens were reviewed and descriptive statistics were performed. Results. The mean age of the patients was 54.58 ± 0.26 years, the most common site of biopsy was the head and neck (38.3%), the most frequently proposed clinical diagnoses included malignancies (19.28%), and the most prevalent pathological diagnosis was epitheliomas (21.9%). After microscopic examination, a specific histological diagnosis was proposed in 83.29% of the cases and a consensus between clinical and histological diagnoses was observed in 68% of them. Conclusions. Although there are cases that skin biopsy exhibits diagnostic inefficiency, it remains a valuable aid for the dermatology clinical practice. The management of skin diseases requires a pertinent diagnosis, which in many occasions constitutes an intricate process. Skin biopsy is an established diagnostic procedure which connects clinical diagnostic methodology with the invisible to the unaided eye microscopic field of skin pathology. Taking under consideration the potentials and limitations of optical microscopy and the indications of performing an invasive technique, dermatologists often rely on skin biopsy for enhancing their diagnostic abilities. The aim of this study was to investigate the favourable clinical diagnoses set by dermatologists when performing skin biopsy, the diagnoses reached by the dermatopathologists after microscopic examination, as well as the relationship between them, and finally to comment on the instances that skin biopsy fails to fulfill the diagnostic task.Six thousand eight hundred and sixteen (6816) biopsies were reviewed which were included in 5941 histopathology report forms and were processed in the “Andreas Sygros” hospital during the years 2004–2006. Furthermore, a topographic anatomy coding system was developed along with an ad hoc coding system for skin diseases in order to meet the requirements of the study (data not shown). Each of the 5941 patients underwent at least one and at most seven skin biopsies at any session. The frequencies of the various sites of biopsy, the percentages of all clinical diagnoses proposed by the dermatologists, and the percentages of the histological diagnoses set by the dermatopathologists were calculated and statistical significance was evaluated.Data were analyzed using PASW Statistics version 18 . Descriptive statistics were applied including frequencies and percentages, as well as the chi-square test, both for one-way and contingency tables. The level of significance was set at less than 0.05.n = 2862) were males and 51.8% (n = 3075) females, with n = 4 missing data. The mean age was 54.58 ± 0.26 and the median 57 years. The mean age for males was 56.54 ± 0.37 (median 60 years) and for females 52.79 ± 0.36 (median 54 years).Out of 5941 patients that underwent skin biopsy, 48.2% (n = 2515), the anterior and lateral tegument 14.3% (n = 941), the posterior tegument 12.3% (n = 810), the pelvis 6.9% (n = 454), the upper extremities 11.1% (n = 729), and the lower extremities 17.1% (n = 1124). Out of 6573 valid biopsy sites (n = 243 missing) the most common specific locations were the back 8.8% (n = 579), the scalp 5.9% (n = 387), the nose 3.3% (n = 218), and the abdomen 3.3% (n = 217). After performing the chi-square test, the differences in the frequencies were found statistically significant .The site of each biopsy was studied regarding both anatomic regions and specific locations. Regarding anatomic regions, the respective frequencies were found to be the head and neck 38.3% (n = 158 cases (2.4%). A classification of 14 categories of all clinical diagnoses is presented in n = 2158 (19.28%), “papulosquamous dermatoses” n = 1358 (12.13%), and “nevi” n = 1176 (10.51%) including melanocytic nevi, congenital nevi, Spitz nevi, blue nevi, dysplastic nevi, junctional-compound-intradermal nevi, nonmelanocytic nevi, and epidermal nevi, all expressed forms of “dermatitis” n = 941 (8.4%) including dermatitis, contact dermatitis, acute or chronic dermatitis, dyshidrotic eczema, nummular eczema, atopic dermatitis, and seborrheic dermatitis; also, “connective tissue diseases” n = 803 (7.17%), noninfectious granulomas and “granulomatous diseases” n = 389 (3.48%), “immunobullous diseases” n = 376 (3.36%), “cutaneous infections” n = 351 (3.14%), “benign tumors” n = 344 (3.07%), “drug eruptions” n = 314 (2.8%), “vasculitides” n = 230 (2.06%), acne and “acneiform eruptions” n = 119 (1.06%), hemangiomas and “vascular malformations” n = 118 (1.05%), and miscellaneous dermatoses n = 2517 (22.49%). After applying the chi-square test, the differences in the frequencies were found statistically significant .In order to study the clinical diagnoses that were proposed by the dermatologists, 6733 out of 6816 biopsies (98.78%) were evaluated. There were 11194 valid specific diagnoses, divided in 367 different terms of skin diseases, after excluding clinical descriptions and intangible expressions , producing a ratio of 1.66 proposed diagnoses per skin biopsy. No diagnosis at all was given in n = 1037), “melanocytic nevus” 7.9% (n = 880), “dermatitis” 6.1% (n = 685), “plaque psoriasis” 4.6% (n = 515), and “squamous cell carcinoma” 3.9% (n = 436).Out of the 367 different clinical expressions, the most common specific diagnoses were “basal cell carcinoma” 9.3% (n = 1224 (21.9%) comprising basal cell carcinoma, squamous cell carcinoma, basosquamous carcinoma, collision tumors with any epithelioma as a component , keratoacanthoma, fibroepithelioma, and lymphoepithelioma-like carcinoma of the skin, “melanocytic nevi” n = 965 (17.2%), “benign tumors and cysts” n = 700 (12.5%), “dermatitis” n = 484 (8.6%), “papulosquamous dermatoses” n = 425 (7.6%), “premalignant skin lesions” n = 253 (4.5%), “connective tissue diseases” n = 244 (4.4%), “conditions of the skin appendages and acneiform eruptions” n = 205 (3.7%), “cutaneous vasculitides” n = 135 (2.4%), “drug eruptions and urticaria” n = 106 (1.9%), “immunobullous diseases” n = 101 (1.8%), “noninfectious granulomas” and granulomatous diseases n = 99 (1.7%), “malignant melanomas” n = 84 (1.5%), “other malignancies” besides melanomas and epitheliomas n = 70 (1.3%), “cutaneous infections” n = 61 (1.1%), and miscellaneous dermatoses n = 441 (7.9%). Performing the chi-square test, the differences in the frequencies were found statistically significant . Also, applying the test for contingency tables, the site of biopsy and histological diagnosis were found dependent  with the most important associations being between epitheliomas followed by conditions of the skin appendages in the head and neck region, cutaneous vasculitides on the lower extremities, melanocytic nevi on the posterior tegument, and dermatitis on the anterolateral tegument.The study of the histological diagnoses that were produced by the dermatopathologists included 6720 skin biopsies  and their distinctive pathology that were previously diagnosed clinically by the dermatologists. Five thousand five hundred and ninety-seven (5597) specific histological diagnoses were suggested (83.29%), divided in 259 different terms of skin diseases. A classification of 16 categories of all histological diagnoses is presented in n = 903 (16.1%), “basal cell carcinoma” n = 858 (15.3%), and “squamous cell carcinoma” n = 304 (5.4%).Among the 259 different expressions used by the dermatopathologists, the most common were “junctional, compound, and intradermal nevi” n = 5597 instances (83.3%), no specific histological diagnosis in n = 754 (11.2%), whereas two or more were proposed in n = 369 (5.5%) of the cases. Useful data orientating the dermatologist in establishing a final clinical diagnosis (cases 1–4 and 7–10 in n = 6533 (97.2%) of all biopsies. Histological and clinical diagnoses were found substantially consistent  of instances. The dermatologists did not provide any specific clinical diagnosis (cases 1 and 6) in n = 232 (3.5%) of instances. Moreover, the lack of a specific clinical diagnosis combined with the absence of usable histological data (case 6 only) occurred in n = 25 (0.4%) of all cases. With the chi-square test the differences in the frequencies between the cases were found statistically significant . There was also a dependence of the relationship between clinical and histological diagnosis with the site of biopsy , By interpreting the adjusted residuals, it was found that the correlation lied mostly between case 3  specimens were considered as destructed or inappropriate for microscopic examination and n = 39 (5.2%) were found of inadequate quantity, where in n = 27 (3.6%) the site of biopsy was regarded as not representative or adjacent to the examined lesion, in n = 24 (3.2%) the pathological features were altered due to previous treatment, in n = 23 (3.1%) optical microscopy with standard staining was considered as inappropriate for a specific diagnosis, and in n = 16 (2.1%) the examined lesion was identified as either not fully developed or resolved. The remaining 534 (70.8%) cases were documented as not pathognomonic and without exhibiting distinctive features.In There are many occasions in which a clinician is challenged by a strenuous diagnostic problem. Skin biopsy constitutes a simple and inexpensive procedure performed in the dermatology setting which facilitates clinical decisions regarding diagnosis and treatment. Also, various studies consider histological confirmation as the standard for the correct diagnosis in dermatology as compared to the clinical evaluation, and the results produced in such manner are used in determining the epidemiological characteristics and patterns of skin diseases , 2. TherA few studies have been conducted in order to assess the diagnostic accuracy of skin diseases by physicians by comparing the clinical to the histological diagnosis. One of these studies measured the diagnostic yield of nondermatologists between 34% to 45% and that of dermatologists being 71% and 75% for inflammatory dermatoses or neoplasms and cysts, respectively . Anothern = 25 (0.4%) of all skin biopsies were lacking both clinical diagnosis and usable histological data. Also there were n = 232 (3.5%) without specific clinical diagnosis and n = 158 (2.4%) without any clinical description or diagnosis. Although these cases were infrequent, they would probably cause therapeutic problems. Hence, a closer cooperation between the dermatologist and the dermatopathologist is advisable.The data presented herein supports the empirically acquired knowledge of every dermatologist that although skin biopsy is performed for the diagnosis of a wide range of dermatoses, it is used predominantly for the determination of malignancies, mainly melanomas and epitheliomas, and also for inflammatory dermatoses such as dermatitis and psoriasis. Nevertheless, despite the high diagnostic usefulness of skin biopsy (97.2% in this study) with a diagnostic accuracy of 83.3%, there have been 11.2% of all instances lacking histological diagnosis. The possible reasons for this discrepancy have not been quantitatively assessed in the literature. Technically speaking, this could be attributed to several factors such as inadequate and inappropriate specimens, as previously analyzed. However, a number of Despite the fact that a plethora of modern techniques have been developed and utilized in the diagnosis of skin disease, dermatologists still rely vastly on biopsy for diagnostic purposes. As discussed in this study, there is a wide range of diseases that allow dermatologists to select skin biopsy in order to confirm their suspected diagnosis, and the histological perspective proves to be both helpful and reliable in the majority of cases. However, there are also limitations in this method and there are cases that the performance of a biopsy does not produce diagnostic results. As a consequence proper diagnosis is delayed and all imminent therapeutic decisions rely heavily upon the dermatologist's comprehension of the situation. Therefore an optimal use of the process is suggested with comprehensive descriptions and relevant diagnoses by the dermatologist along with a closer cooperation with the dermatopathologist performing clinicopathological correlation whenever possible.


We found that IO−3 added to the root solutions was converted to I− in the presence of roots. Iodate reduction occurred over the course of several hours. Furthermore, the iodate reduction activity of “Nipponbare” (iodine-sensitive) and “Gohyakumangoku” (iodine-tolerant) roots increased after adding IO−3 or I−. The roots of barley and soybean also showed iodate reduction activity and the activity responded to iodine treatment either with IO−3 and I−. This study suggests that plant roots biologically reduce iodate to iodide and indicates that the iodate reduction activity of roots responds to external iodine conditions.Although iodine is not an essential nutrient for higher plants, their roots take up and transport the element. However, the exact mechanisms involved in iodine uptake and metabolism in higher plants have yet to be elucidated. In this study, we compared two cultivars differing in iodine tolerance (“Nipponbare” and “Gohyakumangoku”) to increasing levels of I Iodine, an essential element for humans, is an important component of thyroid hormones. About 15.8% of the world's population suffers from goiter, which is primarily caused by iodine deficiency. Moreover, an additional one-third of the world's population has been estimated as being at risk of iodine deficiency  in buffer solutions caused by rice root to illuminate the detailed mechanism of iodine uptake by higher plants. Furthermore, we examined the role of rice roots in regard to iodine reduction. From these data, we concluded that iodate reduction is a primary physiological response of rice roots to the presence of iodine.In the present study, we investigated any changes in the chemical species of inorganic iodine  for 17 days. Composition of the culture media (pH 5.5) was 1.5 mmol L−1 NH4NO3, 1.5 mmol L−1 K2SO4, 0.25 mmol L−1 Ca(NO3)2, 0.5 mmol L−1 NH4H2PO4, 1 mmol L−1 MgSO4 and adequate levels of micronutrients. The media was renewed three times a week. At the end of the iodine treatment, shoot length was measured. For the measurement of iodine concentration in shoots of soil-cultured rice, rice seedlings were subjected to iodine treatment (IO−3 was mixed with the soil at 1 mmol kg soil−1) in the green house under natural light condition. After the IO−3 treatment, shoots were harvested for the determination of iodine concentration.For the test of tolerance to iodine excess, three cultivars of rice were used: For the measurement of iodate reduction activity using rice roots without iodine treatment, rice seedlings (cv. “Koshihikari”) were water-cultured for 27 days in a growth chamber  with culture solution (pH 5.5) mentioned above. The media were renewed once a week.− treatment was performed at 0, 0.025, 0.25, 2.5, 25 μmol L−1, and IO−3 treatment was at 0, 0.25, 2.5, 25, 50, 100 μmol L−1. Iodine containing media were renewed twice a week.For the measurement of iodate reduction activity using rice roots with iodine treatment, water-cultured 21 days-old seedlings (cv. “Nipponbare” and “Gohyakumangoku”) were subjected to iodine treatment for 1 week in a temperature-controlled green house . IHordeum vulgare L. cv. “Mikamogolden”) were subjected to iodine treatment in a temperature-controlled  green house under natural light condition. Water-cultured 15 days-old soybean seedlings (Glycine max cv. “Tachinagaha”) were subjected to iodine treatment in the temperature-controlled  green house under natural light condition. Twenty-one days-old rice seedlings (cv. “Gohyakumangoku”) were subjected to iodine treatment in the temperature-controlled green house . I− treatment was performed at 0, 5, 10, 20, 50 μmol L−1, and IO−3 treatment was at 0, 50, 100, 200, 500 μmol L−1. Iodine containing media were renewed twice a week after the start of iodine treatment.For the comparison of iodate reduction activity with barley and soybean roots, water-cultured 10 days-old barley seedlings  containing 0.16 μmol L−1 (20 μg L−1 as I) of I− or IO−3. After 24 h of incubation in the dark at 20°C, the buffer solutions were filtered (0.2 μm) and frozen at −80°C immediately. pH of assay buffer was determined to prevent loss of produced I− by automatical change 2 I−→I2 at lower pH. The concentrations of I− and IO−3 in the buffer solutions were determined by ion chromatography and inductively coupled plasma-mass spectrometry system (IC-ICP-MS)  containing IO−3 0.1 mmol L−1, followed by 6 h' incubation at 25°C in the dark. After the incubation, the buffer solution was filtered (0.2 μm) and frozen immediately at −80°C until the measurement. Iodate reduction activity was evaluated by the amount of I− reduced from IO−3 by 1 g (FW) of excised roots per hour. I− concentration in the buffer was determined by 4,4′-methylenebis-chloramine T reaction  and inductively coupled plasma-mass spectrometry  system (IC-ICP-MS) .About 0.1 g of plant samples (shoots and roots) dried at 80°C were digested by 25% tetramethyl ammonium hydroxide  in a 6 ml PFA vial (Savillex Co.) overnight at 80°C  and iodate (IO−3). Our results clearly demonstrate the degree of tolerance to excess iodine (I− and IO−3) among these cultivars: “Nipponbare” < “Koshihikari” < “Gohyakumangoku”  was higher than that of “Nipponbare” (sensitive) in both the IO Figures , 6B, A1Bs Figure . Howevers Figure  also reps Figure  or waters Figure . Therefo−3 or I− treatment. In “Gohyakumangoku,” iodate reduction activity decreased at concentrations of iodine that resulted in visible iodine-toxicity symptoms .The induction of iodate reduction by I−, more toxic form of iodine. Lower concentration of iodine in roots of “Gohyakumangoku” might be related to lower iodate reduction activity in I− treatment. Further detailed studies are needed to fully clarify these responses.The decrease in iodate reduction under high iodine conditions seems to correlate with iodine tolerance. Because lower iodate reduction activity will be helpful under excess iodine condition since high iodate reduction activity can contribute to uptake of I−3 treatment was suppressed by removing iodine from culture media (data not shown). We also demonstrated that barley and soybean roots converted IO−3 to I−, and that iodate reduction activity of barley and soybean roots increased with both IO−3 and I− treatment . Under excess iodine condition, it is considered that I− is mainly absorbed by roots due to high iodate reduction activity of iodate reductase. The existence of iodate reductase induced by iodine would be suggested by the induction of iodate reduction activity of crude proteins extracted from rice roots treated with iodine (data not shown). Iodide oxidase might also exist and be related to the iodate reduction in I− treatment. The uptake of iodine could be regulated by transporters in plasma membrane. Transport of iodine from roots to shoots could be also regulated by transporters. In barley treated with higher I−, iodine concentration in shoots reversed that of roots. In IO−3 treatment, iodine concentration in shoots was higher than that of roots only in barley. These results suggest the existence of iodine transporters. The major chemical form of soluble iodine in soil solutions is I− under flooded conditions (Muramatsu et al., −3 under non-flooded conditions (Yuita, − and IO−3 treatment. This might suggest the difference in the mechanism of uptake and transport of iodine between paddy-rice and upland crops.This is the first report demonstrating that plant roots have an ability to reduce IOs Yuita, . SoybeanThe 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.