Androgens are considered important in normal prostate physiology and prostate cancer (PCa) pathogenesis. However, androgen-targeted treatment preventing PCa recurrence is still lacking. This indicates additional mediators contributing to cancer development. We sought to determine the prognostic significance of estrogen receptors, ERα and -β, and the aromatase enzyme in PCa. Tissue microarrays were created from 535 PCa patients treated with radical prostatectomy. Expression of ERα, ERβ and aromatase were evaluated using immunohistochemistry. Representative tumor epithelial (TE) and tumor stromal (TS) areas were investigated separately. Survival analyses were used to evaluate the markers correlation to PCa outcome. In univariate analyses, ERα in TS was associated with delayed time to clinical failure (CF) (p = 0.042) and PCa death (p = 0.019), while ERβ was associated with reduced time to biochemical failure (BF) (p = 0.002). Aromatase in TS and TE was associated with increased time to BF and CF respectively . Multivariate analyses supported these observations, indicating an independent prognostic impact of all markers. When stratifying the analysis according to different surgical centers the results were unchanged. In conclusion, significant prognostic roles of ERα, ERβ and aromatase were discovered in the in PCa specimens of our large multicenter cohort. Prostate cancer (PCa) is continually a challenge as one of the leading causes of cancer-related death amongst men2567CYP19A1, and it’s association to PCa has received interest and is undergoing investigation1415The involvement of androgens in PCa has led to an increased interest in the involvement of other sex steroid hormones and their synthesis in PCa development. Local estrogen production happens thorough the conversion of androstenedione to estrone, and testosterone to estradiol which is catalyzed by the aromatase enzyme CYP 19). This process takes place in several tissues, including the prostate. This pr1012910The involvement of estrogens in PCa is not a novel concept2The effects of estrogens are mediated through two different receptors, ERα and ERβ1920212224252627282930In order to understand the ERs involvement in PCa we have investigated the epithelial and stromal expression of ERα, ERβ and aromatase in different tissue compartments in a large cohort of 535 prostatectomy specimens. We further analyzed their prognostic impact on patient outcome and correlation to clinicopathological variables. All three markers were detected in either tumor related stromal cells (TS), tumor epithelial cells (TE) or both and correlated to PCa outcome.Primary tumor tissue from 535 radical prostatectomy (RP) patients was included in this study. The tumor tissue was retrospectively collected from the Departments of Pathology at the University Hospital of Northern Norway (n = 248), Nordland Hospital (n = 59) and St. Olavs Hospital (n = 228) from the period 1995–2005. Patients who had (I) radiotherapy to the pelvic region prior to surgery, (II) other malignancies within 5 years prior to the PCa diagnosis, (III) inadequate paraffin-embedded tissue blocks, and (IV) lack of clinical follow-up data, (V) received hormonal therapy prior to or at the time of the prostatectomy, were excluded. All primary cancers were histologically reviewed by two pathologists (ER and LTB) and the tumors were graded according to the modified Gleason grading system32The Regional Committee for Medical and Health Research Ethics (2009/1393), the Data Protection Official for Research (NSD), and the National Data Inspection Board approved this study. All patients were made anonymous with each trial number. These numbers were initially linked to identity for only one purpose prior; to collect clinical information. Written consent from the patients was considered, but as this was a retrospective study where most of the material was more than 10 years old and most of the patients deceased, it was considered not needed. The aforementioned parties accepted this solution. All data was analyzed anonymously.Tissue Microarray (TMA) construction was chosen for high-throughput molecular pathology analysis. For each case, a pathologist (ER) identified and marked two representative areas of tumor tissue , two with tumor stromal tissue, one area with normal epithelial tissue, and one area with normal stromal tissue. From each of these areas, cores were sampled from each donor block in order to construct TMA blocks.The TMAs were assembled using a tissue-arraying instrument . We used a 0.6 mm diameter needle to harvest cores from the marked tissue areas from the corresponding formalin-fixed paraffin-embedded (FFPE) tissue blocks. The samples were inserted into an empty recipient paraffin block according to a coordinate pattern. To include all core samples, twelve tissue array blocks were constructed. Multiple 4 μm sections were cut with a Micron microtome (HM355S), affixed to glass slides, and sealed with paraffin. The detailed methodology has been reported previouslyThe following antibodies were used in this study: Rabbit polyclonal ERα antibody , mouse monoclonal ERβ antibody , and goat polyclonal aromatase (CYP-19) antibody . The TMA slide sections were deparaffinised and rehydrated and antigen retrieval was performed by microwaving (450 W) in 0.01 M citrate buffer at pH 6.0 for 20 minutes. The sections were cooled to room temperature (RT) and endogenous peroxidase activity was blocked by incubation with a solution of 0.5% hydrogen peroxide for 10 minutes. The sections were then incubated in 5% normal serum ABC kit (Vector Laboratories) for 1 h at RT to block nonspecific binding. Subsequently, the sections were incubated overnight at 4 °C with primary antibodies, however for goat polyclonal aromatase the incubation time was 45 minutes at RT. After washing, the sections were incubated with the corresponding secondary antibodies for 1 h at RT. The Vectastain ABC kit (Vector Laboratories) was used for the avidin-biotin complex method according the manufacturer’s instructions. The sections were lightly counterstained with hematoxylin, dehydrated through an ethanol series, cleared in xylene and mounted. Two different controls for our staining method were applied. Firstly, control staining of the sections with an isotype-matched control antibody without the primary antibody. Secondly, multiple organ tissue microarray as positive and negative tissue controls were used to verify the specificity of the staining in every staining procedure. The positive tissue controls comprised ovary for ERα, colon adenocarcinoma for ERβ and placenta for aromatase; Negative tissue controls were samples of normal pancreas and liver. Details regarding antibody validation are presented in The ARIOL imaging system  was used to scan and digitalize the IHC stained TMA slides. The slides were loaded in the SL 50 automated slide loader and scanned at a low resolution (1.25x) and high resolution (20x) using an Olympus BX61 microscope with an automated platform . Images of the cores were uploaded into the ARIOL Software. All samples were de-identified and scored manually by two experienced parties independent of each other: ERα and ERβ by two pathologists (ER and SFI) and aromatase by one pathologist (ER) and one MD student (TG) trained by an experienced pathologist. Consequently, all reported marker expressions are based on two separate evaluations of the tissue cores. The scoring was done semi-quantitatively and both parties were blinded to any pathological or clinical information. In case of discrepancy of more than one, the slides were re-examined. When selecting the representative pictures of IHC stained TMA cores depicted in st quartile . Marker expressions were evaluated in all the different PCa compartments: Normal epithelia (NE), normal stroma (NS), hyperplasia (H), TE and TS. Further, marker expression in the different compartments and their correlation with biochemical failure (BF), clinical failure (CF) and prostate cancer death (PCD) was analyzed.Overall, the percentage of ERα and ERβ positive cells varied between the different cores, there was however little variation in staining intensity. ERα and ERβ density was therefor given a score between 0–3, reflecting the percentage of positive cells in the examined compartment. The applied scoring system was as follows: 0: 0%, 1: ≤5%, 2: 6–50%, 3: >50%. For aromatase there was an overall high percentage of positive cells, but variation in staining intensity was observed. The degree of aromatase protein expression in cytoplasm was therefore graded according to the dominant staining intensity. The scoring was done using the following system: 0 = negative, 1 = weak, 2 = moderate, 3 = strong. For each case, mean scores were calculated. Further, the scoring values were dichotomized as high and low intensity or density of stained cells. Both median, mean and quartile cut off values were considered, but the optimal cut off was chosen based on adequate number of patients in each group and statistical trends. The cut off for ERβ in TS and aromatase in TE was defined as the median  value. For ERα and aromatase in TS the cut off was set to the value ≥1All statistical analyses were performed using the statistical package IBM SPSS, version 21 . A Wilcoxon signed rank test was used to assess differences in ERβ, ERα and aromatase expression between the different compartments: TE vs. TS. Spearman correlation coefficient was performed to examine the association between ERβ, ERα, aromatase expression and clinicopathological variables. The Kaplan-Meier method was used for the univariate survival analysis, and log-rank test was used to assess statistical significance. Univariate analyses were constructed for the following end-points: (1) Biochemical failure free survival (BFFS), (2) Clinical failure free survival (CFFS) and (3) PCa death free survival (PCDFS). BF was determined as PSA recurrence ≥0.4 ng/ml in a minimum of two different blood samples postoperativelyAn overview of the patient’s demographic, clinical and histopathological characteristics is presented in There was a good scoring agreement between the scorers. The intra-class correlation coefficient  was 0.93 (p < 0.001) for the ERα marker and 0.79 (p < 0.001) for the ERβ marker and 0.89 (p < 0.001) for the aromatase marker respectively.ERα and aromatase staining was predominantly cytoplasmic . The staThe correlation between marker expressions and clinicopathological variables was weak or non-significant (r < 0.2). However, a positive correlation was detected between ERα and ERβ in TS . As expected, in TS both ERα and ERβ displayed a correlation to aromatase . The same correlation was observed in TE for ERα, ERβ and aromatase respectively .Variables significant for BF were pT-stage (p < 0.001), pN-stage (<0.001), preoperative PSA (p < 0.001), Gleason score (p < 0.001), tumor size (p < 0.001), perineural infiltration , positive surgical margin , apical PSM (p = 0.040), non-apical PSM (p < 0.001), and lymphovascular infiltration . For CF, significant prognostic factors were: pT-stage (p < 0.001), pN-stage (p < 0.001), Gleason score (p < 0.001), tumor size (p = 0.019), PNI (p = 0.001), PSM (p = 0.038), non-apical PSM (p < 0.001) and LVI (p < 0.001). For PCD the prognostic factors were: pT-stage (p = 0.027), pN-stage (p < 0.001), Gleason score (p < 0.001), PNI (p = 0.002), non-apical PSM (p = 0.029) and LVI (p = 0.009).Results from univariate analysis of molecular markers according to BFFS, CFFS and PCDFS are presented in When merging the expression levels of ERα and aromatase in TS, a combined high level of the two markers  was associated with increased CFFS (p = 0.029) (S1 Table 2). The same tendency was also displayed when merging ERα in TS and aromatase in TE. A combined high level  was associated with increased CFFS (p = 0.038) and PCDFS (0.003), but not BFFS (p = 0.854) (S1 Table 2). Further, when merging the stromal expression of ERβ and aromatase, a beneficial effect of a combined level low ERβ and high aromatase (low/high) in BFFS stood out compared to the high ERβ and low aromatase (high/low) combination which was associated with reduced time to BFFS (p < 0.001) (S1 Table 2). When combining ERβ in TS and aromatase in TE, no obvious trends or significant results were displayed.Results from multivariate analysis are presented in In our large cohort of 535 PCa specimens, an independent association was detected between PCa outcome and ERα, ERβ and aromatase expression. In TS, high-density of ERα was independently and significantly associated with both increased CFFS and PCDFS. In contrast, a high ERβ density level was independently and significantly associated with reduced BFFS. Further, a strong staining intensity of aromatase in both TS and TE was significantly and independently associated with increased BFFS and CFFS respectively. In addition, a correlation and an additive effect were discovered when analyzing the combined expression of ERs and aromatase. A major strength of our study is the large multicenter cohort and the long follow-up. In addition, our results were validated in two different cohorts, yielding data tending towards the end results in the total cohort. In addition, few studies have investigated these markers independently in both epithelial and stromal areas of PCa with a clinical event-free survival.et al. discovered using IHC, in vitro invasion assays and in vivo studies that ERα in TS is beneficial for PCa patientset al.et al. detected an improved overall survival for CRPC patients with stromal ERα expressionet al. who noted survival to be significantly reduced in PCa patients with low stromal ERα expressionIn accord with previous publications, ERα density level was predominantly negative in NE in the PCa patients21242930et al. reported that increased ERβ expression in hormone naïve PCa (HNPC) was associated with a worse outcomeet al. reported that non-androgenic proliferation of PCa can occur through estrogen-mediated activation of AR in complex with ERβ and proline-, glutamic acid- and leucine rich protein 1 (PELP1), an AR cofactor known for its proto-oncogenic abilitiesIn our material ERβ was expressed, to various extents, in the majority of both epithelial and stromal cells. This is in agreement with previous publications23242425262723252022Recently, several ERβ isoforms have been isolated and different functions of these isoforms have been hypothesized, including several with tumor promoting abilities2526CYP191A, has also been a topic of interest. There have been reports, however with equivocal results, indicating that different single nucleotide polymorphism (SNPs) in CYP191A influences PCa risk and survival14Our study demonstrated a wide distribution of aromatase in stromal and epithelial cells of both benign and malignant prostate tissue. Aromatase has previously been detected in both epithelial and stromal tissue, but agreement regarding its compartmental expression is however currently lacking91031By detecting aromatase expression in the PCa specimens, in addition to the strong correlation between aromatase and the ERs in TS and TE, we confirm a local production of estrogens in PCa and its stimulation of the local receptors. This indicates estrogens’ ability to directly act upon the prostate gland, not only thorough negative feedback on the hypothalamic-pituitary-gonadal axis. This is of particular interest since it is still unresolved whether locally produced or circulating hormones effect PCa moreThere are several factors that may explain some of the discrepancies regarding these hormonal biomarkers. The reproducibility of prognostic biomarker studies is always a challenge241120441213In the present study, ERs and aromatase emerged as potential prognostic biomarkers for PCa in addition to other well-established markers. This is demonstrated by the significant impacts in the multivariate analyses . In addiWe found both ERs and aromatase to be significantly and independently associated to PCa outcome. In TS, a high expression of ERα was associated with increased CFFS and PCDFS, while a high expression of ERβ was associated with reduced BFFS. In addition, high aromatase expression in both TS and TE was favorable with respect to BFFS and CFFS, respectively. For CFFS, the impact of these markers added prognostic relevance within each stage group. This knowledge may be valuable for the development of future prognostic biomarkers in PCa, but further validation is warranted before clinical application.How to cite this article: Grindstad, T. et al. Estrogen receptors α and β and aromatase as independent predictors for prostate cancer outcome. Sci. Rep.6, 33114; doi: 10.1038/srep33114 (2016).


Achieving a secure airway in rabbits is generally considered more difficult than in cats or dogs. Their relatively large tongue, small oropharyngeal cavity and glottis limit direct visualization. A rabbit-specific supraglottic airway device (SGAD) may offer benefits over blind orotracheal intubation.−1 IM) and midazolam (0.5 mg kg−1 IM), followed by induction with alfaxalone (0.3 mg kg−1 IV). Two CT scans of the head and neck were performed, following sedation and SGAD/ETT placement. The following were recorded: time to successful device insertion, smallest cross-sectional airway area, airway sealing pressure, and histological score of tracheal tissue. Data were analyzed with a Mann–Whitney test.Fifteen adult New Zealand white rabbits were randomized to SGAD or orotracheal intubation (ETT). All animals were sedated with dexmedetomidine  kg, SGAD; n = 7, 2.7 (2.4–5.0) kg]. SGAD placement was significantly faster [33 (14–38) s] than ETT [59 (29–171) s]. Cross-sectional area (CSA) was significantly reduced from baseline [12.2 (6.9–3.4) mm2] but similar between groups . In the SGAD group, the device tip migrated into the laryngeal vestibule in 6/7 rabbits, reducing the CSA. ETT airway seals were higher [15 (10–20) cmH2O], but not significant . ETT resulted in significantly more mucosal damage [histological score 3.3 (1.0–5.0)], SGAD; 0.67 (0.33–3.67).Two rabbits were excluded following failed ETT. Body masses were similar [ETT; The SGAD studied was faster to place and caused less damage than orotracheal intubation, but resulted in a similar CSA. General anesthesia and sedation in rabbits carries a relatively high risk, with an overall incidence of death of 1.39% reported, approximately 6–8 times higher than that in cats and dogs .Orotracheal intubation provides a secure airway by facilitating the flow of gas during spontaneous ventilation, allowing intermittent positive pressure ventilation (IPPV), preventing aspiration of foreign material into the respiratory tract, and limiting workplace pollution with waste anesthetic gas. Achieving atraumatic, timely orotracheal intubation is difficult without training and practice and is hindered by the oropharyngeal anatomy of rabbits, with their relatively large tongues, narrow oropharyngeal cavity, and small glottis –11. RabbAs a result of difficulties achieving orotracheal intubation, it is not commonly performed and anesthesia may be induced and maintained with a facemask; however, this commonly leads to inadequate ventilation , 12, 13.The aim of this study was to assess the performance of this rabbit-specific SGAD against blind orotracheal intubation with an ETT. Primary outcome measures included time to successful device insertion, cross-sectional airway area, histological score, and airway seal. Compared with ETT, we hypothesized that the SGAD would be faster to place and maintain a larger cross-sectional airway area with less airway damage but create a less-effective airway seal.n = 12 males, n = 3 females). Rabbits from the commercial supplier had a minimum of 1 week to acclimatize before experimentation. Rabbits were either group or individually housed (depending on location) in an environmentally controlled unit . All rabbits were provided with ad libitum pellets, hay, and water with a small amount of fruit and vegetables. Experiments were performed between 1700 and 2000 hours.In a prospective, randomised study design, adult New Zealand white rabbits were obtained as surplus stock from the Animal Resource Center of the University of Calgary and a commercial supplier  SGAD (Sarah Engbers). These investigators both completed an online training program provided by the manufacturer and practical experience was gained with two rabbits scheduled for euthanasia. Investigators could not be blinded to group assignment, with the exception of the veterinary pathologist (Cameron Knight), who performed necropsies off-site. Animals were fasted for approximately 2 h before the experiment. Physical examinations  were performed immediately before each experiment. Exclusion criteria included any indication of systemic illness resulting in assigning an American Society of Anesthesiologists physical classification status >2.−1, Dex-domitor, Zoetis, Kirkland, QC, Canada) and midazolam  delivered intramuscularly as a single injection in the epaxial musculature. Ten minutes later, animals were placed in sternal recumbency on the CT scan table, provided oxygen by facemask (1.5 L min−1) and cannulas  placed in the auricular vein (IV) and artery. A baseline CT scan  was performed under sedation following instrumentation, followed by a second scan after ETT or SGAD placement. Scans were performed by a board-certified radiologist (Nicolas Rousset), with the scan margins ranging from the rostral margin of the nose to the thoracic inlet.Animals were sedated with dexmedetomidine . The SGAD size was chosen based on body mass as per the manufacturer’s recommendations. Where an animal fell between sizes, the larger SGAD was selected. An insertion attempt began 30 s after completing alfaxalone injection. Each animal in the SGAD group was positioned in sternal recumbency with its tongue exteriorized and the lubricated SGAD  inserted into the oropharynx until either resistance was encountered, or the incisors were within 1–2 cm of the device’s fixation tabs. Acceptable placement was confirmed with a mainstream capnograph attached to the SGAD  and visualization of six rectangular waveforms. Criteria for rescue intubation with ETT for the SGAD group were defined as: lingual cyanosis that did not resolve with repositioning the SGAD or tongue, or three unsuccessful insertion attempts (no capnograph trace observed).Following the baseline CT scan, invasive systemic arterial blood pressure (BP), ECG, and saturation of arterial hemoglobin with oxygen (pulse oximetry probe on the prepuce or vulva) monitoring began . General anesthesia was induced with alfaxalone  were collected and recorded every 30 s for 5 min, and at 15 min post-insertion. Additionally, the highest systemic systolic arterial BP observed during device insertion was recorded (from video-recording of the physiologic monitor). At 10 min, the second CT scan was performed. At 15 min, the concentration of isoflurane measured from a sampling line placed approximately 5 cm from the mouth was recorded, and the airway seal was tested by closing the adjustable pressure limiting valve on the anesthetic breathing system and squeezing the reservoir bag. Peak inspiratory pressures were increased in 5 cmH2O increments, between 5 and 20 cmH2O, until a leak was heard (or 20 cmH2O achieved). This leak test was done by the same investigators (Sarah Engbers and Daniel S. J. Pang) for all animals. Occurrences of lingual cyanosis or airway obstruction (audible wheeze during respiratory cycle or irregular capnograph trace) were recorded. In the last nine animals , an arterial blood sample was collected from the intra-arterial cannula 30 min after device placement and analyzed for blood gases and electrolytes . General anesthesia was maintained for 60 min (from time of induction) with isoflurane (1% vaporizer setting) carried in oxygen (1 L min−1) through a non-rebreathing system . All animals were euthanized with an overdose of IV sodium pentobarbital while anesthetized at the end of the experiment.Baseline systemic arterial BPs were recorded immediately before induction of general anesthesia. Beginning immediately after the insertion of the airway device, physiologic data .All measurements were taken three times by a board-certified veterinary radiologist (Nicolas Rousset) and averaged for analysis. The midline sagittal view of each SGAD rabbit post-device placement was used to measure the following distances: 1) distance between the caudal edge of the basihyoid bone (BH) to the rostral edge of the thyroid cartilage (TC)  according to a standardized method . The ton2 data, followed by a Bonferroni post hoc test if main effects were significant. A p value < 0.05 was considered significant. Data are presented as mean ± SD and median (range). 95% confidence intervals (95% CI) for mean or median differences are provided as available.Commercial software was used for statistical analyses . Data were tested for normal distribution with a D’Agostino and Pearson normality test. Non-parametric analyses were used if data were not normally distributed or sample sizes were too small for the normality test. Mann–Whitney tests were used to make the following comparisons between groups: body mass, anatomical distances, cross-sectional airway areas, device insertion time, airway seal, percentage changes in systolic BP during device insertion, arterial blood sample parameters, and histological scores. A Wilcoxon matched pairs test was used to compare changes in systolic arterial BP over time. Two-way ANOVA was applied to systemic arterial BPs, heart rate, respiratory rate, and expired COn = 6; SGAD, n = 7. Rabbits ranged in age from 3 to 12 months. There were no significant differences in body mass between groups: ETT; 2.6 (2.3–4.5) kg, SGAD; 2.7 (2.4–5.0) kg . All rabbits appeared healthy on physical examination and were assigned an American Society of Anesthesiologists physical classification status of 1. The endotracheal tubes used in the ETT group ranged from 2 to 3.5 mm ID: 2 mm, n = 2; 2.5 mm, n = 1; 3 mm, n = 1; 3.5 mm, n = 2. Three sizes of SGAD were used: R3, n = 4; R4, n = 1; and R5, n = 2.Two rabbits were excluded from the ETT group, one due to failure to achieve orotracheal intubation and the other due to prolonged intubation resulting from fecal matter in the oropharynx . CT scanp = 0.16, 95% CI −6.7 to 1.3): SGAD, 16.2 (13.2–20.3) mm; ETT, 14.5 (12.7–15.8) mm  so that baseline CSA was pooled. The CSA of the narrowest point decreased significantly between the baseline CT scan  and placement of the SGAD  and ETT  at an average of 5.3 mm (range 2.9–7.9) caudal to the rostral edge of the TC, so that the smallest CSA was formed by the SGAD dorsally and the rabbit’s airway ventrally . In the ETT group, a uniform CSA was created by the endotracheal tube.In most cases (6/7 rabbits), placement of the SGAD resulted in the tip of the device deviating ventrally to be positioned within the laryngeal vestibule . The median airway seal was higher in the ETT group [15 (10–20) cmH2O], but there was no statistical difference compared to the SGAD group  than ETT  tp = 0.03, 95% CI 0–3.3, Figure The ETT group was found to have significantly more damage to tracheal mucosa and submucosa [3.3 (1.0–5.0)] than the SGAD group  showed a wider range of percentage change in peak systolic BP than SGAD [1.0 (−3.9 to 5.8)%] though the median difference was not significant , respiration rate (p = 0.22), and expired CO2 (p = 0.74), though heart and respiratory rates tended to be higher in the SGAD group . One rabbit in each group had a single SpO2 reading <90% .There was a systematic, albeit non-significant, difference in systemic arterial BP between groups, with higher BPs observed in the SGAD group . Hypercapnea was present in both treatment groups: SGAD; 65.1 (54.0–75.1) mmHg, ETT; 51.8 (41.2–67.8) mmHg.No significant differences in the following variables were observed: pH, PaOThe findings of this study show that, in comparison with orotracheal intubation using a blind technique, the rabbit-specific SGAD studied is rapid to place and causes minimal trauma to the upper airway. As with orotracheal intubation, the SGAD can be used to successfully maintain general anesthesia in rabbits.General anesthesia and sedation in rabbits carries an unacceptably high mortality rate compared with dogs and cats. A study of 8,209 rabbits identified respiratory and cardiovascular or respiratory causes  as the most frequent causes  .Providing a secure airway and the ability to support ventilation pose a challenge to anesthetists inexperienced with rabbits and when blind orotracheal intubation is attempted. Achieving orotracheal intubation is difficult without training and practice , 22. Com2 and PaO2.With regular use, it is possible that the time to place the SGAD may be reduced further, and recorded times in this study were increased because they included observing a waveform on the capnograph . Securin2O , a small sample chamber (dead space volume of 1 mL) was used and the rise time of the capnograph is reported as <60 ms (for 10–90% step change). Optimization of SGAD placement by confirming clear breath sounds with a stethoscope placed on the larynx has also been suggested  in humans, with associated morbidity and mortality from arrhythmias, intracranial hemorrhage, and elevated intracranial pressure –28. The In conclusion, these results show the rabbit-specific SGAD studied to be consistently faster to place and to cause less trauma to laryngeal and tracheal tissue than a blind orotracheal intubation technique. The SGAD was capable of maintaining general anesthesia, though the presence of hypercapnea underlines the importance of the option to provide IPPV.Institutional ethics approval was provided by the Veterinary Science Animal Care Committee of the University of Calgary, which operates in accordance with the Canadian Council on Animal Care (VSACC AC14-0229).Study design: SE, AL, MJ, NR, CK, and DP. Data collection: all authors. Data analysis and interpretation: SE, AL, NR, CK, and DP. Manuscript drafting and final approval: all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ewes were euthanized, and tissue samples were collected from the gravid uterus and placentomes. Based on BCS (2.0 ± 0.02), glucose (2.4 ± 0.33), and BHBA (0.97 ± 0.06) concentrations, ewes (n = 10) were grouped as healthy (n = 5) and subclinical SCPT (n = 5) ewes. The mRNA expressions were determined by quantitative PCR method, and prediction of miRNA partners and target genes for the predicted miRNA were identified using miRDB (http://mirdb.org/miRDB/). Top ranked target genes were used to identify associated biological functions and pathways in response to SPCT using PANTHER. The angiogenesis genes VEGF and PlGF, and AdipoQ, AdipoR2, PPARG, LEP, IGF1, IGF2, IL1b, and TNFα mRNA expressions were lower in abundances, whereas hypoxia genes eNOS, HIF1a, and HIF 2a, and sFlt1 and KDR mRNA expressions were greater in abundances in uterus and placenta of SCPT ewes compared to healthy ewes (P < 0.05). The predicted miRNA and associated target genes contributed to several biological processes, including apoptosis, biological adhesion, biological regulation, cellular component biogenesis, cellular process, developmental process, immune system process, localization, metabolic process, multicellular organismal process, reproduction, and response to stimulus. The target genes were involved in several pathways including angiogenesis, cytoskeletal regulation, hypoxia response via HIF activation, interleukin signaling, ubiquitin proteasome, and VEGF signaling pathway. In conclusion, genes associated with blood vessel remodeling were lower in abundances and that the genes associated with hypoxic conditions were greater in abundances in the uteroplacental compartment of SCPT ewes. It is obvious that the factors that influence placental vascular development and angiogenesis as noted in this study set the course for hemodynamic changes and hence have a major impact on the rate of transplacental nutrient exchange, fetal growth, and health of the dam.The objective was to elucidate gene expression differences in uterus, caruncle, and cotyledon of ewes with subclinical pregnancy toxemia (SCPT) and healthy ewes, and to identify associated biological functions and pathways involved in pregnancy toxemia. On Day 136 (±1 day) post-breeding, ewes ( Pregnancy toxemia is a metabolic disorder of pregnant ewes, caused by an abnormal metabolism of carbohydrates and fats, which occurs during the final stage of pregnancy. The disease occurs more frequently in lean [body condition score (BCS) <2 in the 5-point scale] or obese BCS ≥4) animals, as well as in animals carrying two or more fetuses –4. animals,In ewes, glucose is the principal carbon source for placental and fetal oxidative metabolism and tissue formation , 6. A toPregnancy requires an expansion of maternal blood volume, an increase of cardiac output, and a redistribution of blood to the uterus to meet the needs of the growing fetus. Normal maternal vascular adaptation during pregnancy includes enhanced vasodilation, with the greatest effect seen in the uterus. A range of pathophysiological factors including maternal stress due to poor nutrition, hyperthermia, or metabolic diseases such as pregnancy toxemia and eclampsia may affect important metabolic, transport, and hemodynamic functions of placentas. Excessive accumulation of free radicals affects placental development and function, and may subsequently impact both the fetus and dam –14.in utero .n = 6), (2) 1,000 mg of γ-tocopherol (n = 7), or (3) no treatment (n = 5) received a placebo. Animals were supplemented orally, once daily, from approximately 100 to 136 (±1) days post-breeding. On Day 136 (±1) post-breeding, all ewes received BCS , and blood samples were collected by jugular venipuncture for plasma glucose and BHBA (in tubes anticoagulated with heparin 10 U/mL) and serum 8-isoprostane (in tubes without heparin) determination. All ewes were euthanized (Day 136 ± 1), and tissue samples were collected immediately from the gravid uterus (full thickness) and placentomes (caruncle and cotyledon). Placentome and uterine samples were collected close to the umbilicus for consistency. Cotyledons were separated from caruncles by applying strong pressure. Tissue samples were placed in RNAlater  in 5-mL Nalgene® cryogenic vials  and snap frozen immediately and stored at −70°C for subsequent evaluation of mRNA expression. This study was approved by Virginia Tech Institutional Animal Care and Use Committee . Tissue Use Protocol was approved by IACUC at Washington State University (ASAF #03922-001).Blood and tissue samples were collected during a previously reported clinical trial to determine the effect of daily tocopherol supplementation during late stage of pregnancy. Briefly, 18 pregnant ewes  with similar breeding dates were selected and were maintained under normal pasture conditions. One week prior to the trial, the selected ewes were moved to the research facility. The ewes had access to 35 sq. ft/ewe paddock lots. In addition, they were fed free choice hay. The ewes were randomly assigned to receive (1) 500 mg of α-tocopherol  as described in the previous reports .Blood samples (with heparin) were centrifuged at 3,500 ×  reports , 19, andg for 10 min, and serum was separated and stored at −20°C until analyzed. Isoprostane in serum samples were estimated by direct ELISA as described previously , and serum concentrations of isoprostane were calculated from standard curves.Blood samples (without heparin) were centrifuged at 1,200 × eviously . BrieflyTotal RNA was extracted from uterus, caruncle, and cotyledon tissues with RNeasy Mini Kit  according to the manufacturer’s protocol. RNA concentration was measured using a NanoDrop spectrophotometer . Sample absorbance ratio of 260/280 wavelength was observed to ensure the purity of RNA and they were 1.96–2.00. DNase treatment was performed using deoxyribonuclease 1 . Briefly, the 1 μg of RNA sample was added with 1 μl of 10× DNase I reaction buffer, 1 μl of 10 DNase I enzyme, and DEPC-treated water. The mix was incubated for 15 min at room temperature. After the reaction, the enzyme was inactivated by adding 1 μl of 25 mM EDTA and heating to 65°C for 10 min. Then, the RNA samples were stored at −20°C until complementary DNA (cDNA) preparation.The mRNA was reverse transcribed to cDNA. The cDNA samples were prepared using the iScript cDNA Synthesis kit . A 500-ηg sample of RNA was reverse transcribed in 20-μL reaction at the incubating conditions of 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min; 25 ηg/μL RNA equivalent cDNA was obtained. Qiagen Tag PCR master mix , a pre-mixed solution, was used to amplify the fragment of the genes of interest. Final concentration of the primers was 0.3 μM. Initial denaturation was set at 94°C for 3 min. Followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C were programed. A final extension step at 72°C for 10 min was included in thermocycling conditions. Primers  were designed either using the NCBI website or primer express version 3.0 . Consideration was given to the set of primers (forward and reverse primers) to ensure separation of at least an intron and melting temperatures, and CG content were set at optimal, or close to optimal level. Amplicon was run on a 2% agarose gel and stained with ethidium bromide for visualization to ensure a single amplicon for a set of primers.SYBR green chemistry was applied to observe relative mRNA expression. Fast SYBR green master mix (2×)  was used to prepare the reaction mix. The final concentration of each primer was 0.3 μM. A 20-μL aliquot of three technical replicates was used for each sample. A 1.6-μL volume of 25 ng/μL RNA equivalent cDNA was present in the total volume of the three triplicates. StepOne Plus instrument  was used for the real-time PCR runs. Precycling stage was maintained at 95°C for 20 s. Forty cycles of amplification was carried out with the conditions of 95°C for 3 s and 60°C for 30 s (fast ramp speed conditions for the fast mixture). A continuous dissociation step was added to look for additional amplification products.Carboxy-X-rhodamine (ROX) dye was set up for the passive internal reference. The baseline was automatically adjusted to obtain threshold cycles of each sample. Threshold cycles were normalized to an endogenous control, β-actin. A standard curve was obtained using one in five dilutions for each set of primer in order to check the amplification efficiency. Correlation coefficient for the dilution curve was ≥0.9900.The placentome samples collected for histological evaluation were fixed in 10% neutral buffered formalin, sectioned at 5 μm, and stained with hematoxylin and eosin. They were evaluated on a Nikon E400 Eclipse microscope, and photomicrographs (100×) were taken with a Nikon camera with a 3 chip. Images were processed with Nikon Act 1 software. Image processing and morphometry analysis were performed using ImageJ 1.42q  to evaluate the fractal dimension and lacunarity as described previously . A fracthttp://mirdb.org/miRDB/) for the predicted miRNAs, and top ranked predicted genes were run using PANTHER classification system  were grouped as healthy (n = 5) and SCPT (n = 5) ewes  ewes . In the  5) ewes , 22, 28,−ΔΔCt values to ascertain statistical significance of any differences in genes expressions between healthy and SCPT ewes . Mean ± SEM differences in BCSs and plasma glucose and BHBA concentrations between healthy and SCPT ewes were calculated using ANOVA. Mean ± SEM differences in alpha tocopherol and gamma tocopherol concentrations in placenta, uterus, and serum of healthy SCPT ewes were calculated using ANOVA. The differences in fractal dimension and lacunarity in uteroplacental unit between healthy and SCPT ewes were tested by ANOVA. The PCR data were subjected to ANOVA using 2CPT ewes .n = 5) and for ewes with SPCT (n = 5) are presented in Table P < 0.05). The serum isoprostane concentrations were different between healthy and SCPT ewes, 244.6 ± 14.2 and 292.9 ± 9.1, respectively (P < 0.05).Mean ± SEM BCS and plasma glucose, and BHBA concentrations for healthy ewes . The sFlt1, KDR, eNOS, HIF1a, and HIF2a mRNA expressions were greater in abundance in cotyledon, caruncle, and uterus of SCPT ewes compared to healthy ewes (P < 0.05). The HIF2b mRNA expression was not different between healthy and SCPT ewes (P > 0.05) in cotyledon, caruncle, and uterus. The IL-8 mRNA expression was lower in abundance in cotyledon and in uterus (P < 0.05) but not in caruncle (P > 0.1) of SCPT ewes compared to healthy ewes. The AdipoR1 mRNA expression was lower in abundance in both units of the placenta (P < 0.05) but not in the uterus (P > 0.1) of SCPT ewes compared to healthy ewes. The IL6 mRNA expression was lower in abundance in the uterus of SCPT ewes compared to healthy ewes (P < 0.05) and the expression was not different in both units of placenta of SCPT and healthy ewes (P > 0.1).The P < 0.05).The healthy ewes had increased fractal dimension and decreased lacunarity in their placental vascular network compared to SCPT ewes  associated to vascular remodeling were lower in abundances in SGPT ewes; the genes  associated to hypoxic condition were greater in abundances in SGPT ewes; morphometry analysis of angiogenic parameters of uteroplacental unit displayed reduced vascularization suggestive of hypoxic conditions in SCPT ewes compared to healthy ewes.The goal of this study was to elucidate the differences in gene expressions of the uteroplacental compartment and morphometry of placental vascular network between healthy and SCPT ewes. We used BCS and biochemical parameters to distinguish SCPT ewes from healthy ewes. The SCPT ewes had lowered BCS and blood glucose concentration and increased BHBA concentration without any clinical signs of the disease . The finIn this study, the BCS and concentrations of glucose were lower and BHBA was greater in SCPT ewes compared to healthy ewes. Susceptible, thin ewes develop ketosis because a chronically inadequate ration is offered or because other diseases such as lameness or dental diseases limit intake and, with increasingly insufficient energy to meet increasing fetal demands, the ewe or doe mobilizes more body fat, with resultant ketone body production and hepatic lipidosis. The ewes included in the study were observed to have normal appetite. Ewes with a poor BCS or that are overconditioned and carrying more than one fetus are most at risk of developing pregnancy toxemia, although the condition can occur even in ideally conditioned ewes on an adequate ration. In this study, three animals had twins and no differences between ewes carrying singletons and twins were detected.VEGF, PlGF, sFlt-1, KDR, and HIFs , placental vascularization , and hypoxic condition (regulation of nitric oxide synthase and hypoxia).Remarkably, the genes investigated and predicted miRNAs and targeted genes were related to several biological functions. The targeted genes contributed to several biological processes, such as apoptotic process, biological adhesion, biological regulation, cellular component organization or biogenesis, cellular process, developmental process, immune system process, localization, metabolic process, multicellular organismal process, reproduction, and response to stimulus. The target genes were involved in several pathways including angiogenesis, cytoskeletal regulation, hypoxia response Pregnancy is a dominant physiological state during which an alteration in metabolism may be expected because of a greater demand for nutrients by developing fetus. In addition to alterations in nutrient partitioning, the placentogenesis are tightly associated with diverse pathophysiological changes in the feto-maternal compartment. The findings of the study indicated that expressions of the genes associated with vascular remodeling were lower in abundances and that the genes associated with hypoxic condition were greater in abundances in the uteroplacental compartment in SCPT ewes. In addition, morphometry analysis of angiogenic parameters of uteroplacental unit displayed reduced vascularization suggestive of hypoxic conditions in SCPT ewes compared to healthy ewes. It is obvious that the factors that influence placental vascular development and angiogenesis as noted in this study set the course for hemodynamic changes and hence have a major impact on the rate of transplacental nutrient exchange, fetal growth, and health of the dam.Acquisition of funding; conception, design, and collection of data; morphometry analysis; data analysis and interpretation; and drafting of the manuscript, tables, and figures. The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Using C57Bl6 mice, we administered 4 million hAECs intraperitoneally 24 hours after bleomycin challenge. Outcomes were measured at days 3, 5, and 7. hAEC administration resulted in significant changes to T‐cell, macrophage, dendritic cell, and monocyte/macrophage infiltration and phenotypes. Endogenous levels of lipoxygenases, LXA4, and the lipoxin receptor FPR2 were elevated in hAEC‐treated animals. Furthermore, we showed that the effects of hAECs on macrophage phagocytic activity and T‐cell suppression are LXA4 dependent, whereas the inhibition of neutrophil‐derived myleoperoxidase by hAECs is independent of LXA4. This study provides the first evidence that lipid‐based mediators contribute to the immunomodulatory effects of hAECs and further supports the growing body of evidence that LXA4 is proresolutionary in lung injury. This discovery of LXA4‐dependent communication between hAECs, macrophages, T cells, and neutrophils is important to the understanding of hAEC biodynamics and would be expected to inform future clinical applications. Stem Cells Translational Medicine2017;6:1085–1095Human amnion epithelial cells (hAECs) have been shown to possess potent immunomodulatory properties across a number of disease models. Recently, we reported that hAECs influence macrophage polarization and activity, and that this step was dependent on regulatory T cells. In this study, we aimed to assess the effects of hAEC‐derived proresolution lipoxin‐A This molecule works by encouraging interactions between a variety of immune cells in the lungs to effect repair. Following hAEC administration, fewer proinflammatory immune cells were attracted to the lungs and their activity was suppressed to achieve resolution of lung injury. The findings from this study will help inform the design of safe and efficacious hAEC therapy for lung disease.In this study, stem‐like cells derived from the human placenta, called human amnion epithelial cells (hAECs), were shown to prevent lung injury by producing a lipid‐based molecule called lipoxin A+/CD28+/CD25+/Foxp3+ regulatory T‐regulatory cells and higher numbers of Th1 effector CD4+ T cells in the lung ; We evaluated the effect of hAECs and hAEC‐derived LXA4 on T‐cell phenotype, function, and behavior. In comparison with the control treatment , hAECs significantly decreased IL‐2 and increased IL‐17A levels in vitro ‐β1 from day 14 onward [Polarity of Th1 and Th2 cells is a stochastic process usually directed by proinflammatory  and anti‐inflammatory  cytokines, which provides positive feedback signaling for downstream inflammatory response. Bronchoepithelial cells express IL‐4 receptors, which, upon activation by IL‐4, results in the production of cytokines  and increased migration of human airway epithelial cells , 32. IL‐4 onward . However4 onward . In our Conversely, we found significant reduction in Th1 drivers IL‐6 and IL‐2 by day 7, suggesting a repression of inflammatory responses. This is supported by an earlier report that showed blocking IL‐6 at days 8, 9, and 10 after bleomycin instillation can ameliorate lung fibrosis . Surpris4 is a lipid mediator capable of anti‐inflammatory signaling by regulating neutrophil infiltration and proresolving signaling through macrophage polarization and nonphlogistic uptake of apoptotic PMNs. Their innate ability to perpetuate and limit inflammation is of particular importance in our study to define probable immune regulatory roles of hAECs. As our first step in identifying the importance of LXA4 in hAEC‐mediated repair, we evaluated endogenous levels of LXA4 and gene expression of its precursor enzymes ALOX‐5, ‐12, and ‐15 in vivo. Here, hAECs significantly increased levels of LXA4 in BALF at day 7 after bleomycin challenge. Gene expression of the enzymes ALOX‐5, ‐12, and ‐15 responsible for the biosynthesis of lipoxin was significantly increased in hAEC‐treated animals at day 7 (ALOX‐5) and day 5 (ALOX‐12 and ‐15), suggesting that hAEC treatment had an effect on endogenous LXA4 and lipoxygenase synthesis in bleomycin‐induced lung injury. However, changes to lipoxygenase expression varied between days 5 and 7 and may be a consequence of the behavior of lipoxygenase‐expressing immune cells [Lipoxin Ane cells . Treatmeepi‐lipoxin A4 in vitro [In our study, we saw no change in neutrophil numbers across all days between injured and treated animals, suggesting that, in our study at least, hAECs did not alter neutrophil infiltration to the site of injury and may, instead, mediate activity of neutrophils to resolve injury. Neutrophils are cells recruited as the first line of inflammatory defense, hence behavioral changes to these cells will inevitably influence downstream inflammatory processes. Neutrophil release of MPOs contributes to alveolar damage and, subsequently, fibrosis in patients with IPF, and their activity can be regulated by aspirin‐triggered 15‐in vitro , 42, 43.in vitro . In thisin vitro , 45. Takhi monocytes, which secrete inflammatory cytokines TNF‐α and IL‐1β; and Ly6Clo monocytes, which patrol the endothelium. The percentage of CD11b+Ly6Chi monocytes was significantly higher in hAEC‐treated animals at days 3 and 5 of this study. This was unexpected because there was less inflammation and honeycomb injury in those mice. Although denoted as inflammatory monocytes, CD11b+Ly6Chi cells have the ability to differentiate into mature alternatively activated macrophages [hi inflammatory monocytes during the progressive fibrotic phase led to increased numbers of M2 macrophages and exacerbation of fibrosis [2+ motility [Next, we assessed the changes to pulmonary monocyte subpopulations where two distinct subtypes can be found: Ly6Crophages . Gibbonsfibrosis , 48, 49.fibrosis , an outcfibrosis , 50. Colmotility , 52. Thimotility  and is cmotility , FPR2‐an−/−fpr2 have defective phagocytic ability for apoptotic PMN [Our observations suggest that hAECs exert their effects, at least in part, through LXA4, which is a potent stimulus for macrophage phagocytosis , 56. Macotic PMN , 60, fur4 per mouse at days 7 and 10 significantly reduced inflammatory cell infiltration and reversed established fibrosis [LXA4 is a potent regulator of inflammation and resolution; however, other specialized lipid mediators released by hAECs remain to be investigated , 62, 63.fibrosis . Furtherfibrosis , 65, 66,fibrosis .Dendritic cells (DCs) are antigen‐presenting cells known to influence T‐cell proliferation and plasticity. In this study, we showed that hAEC treatment significantly reduced DC infiltration (day 5) to the site of injury and this might subsequently have led to changes in T‐cell numbers that we saw on day 7. This study identified possible mechanisms of hAEC‐mediated regulation of T‐cell behavior and phenotype. Our coculture migration and proliferation assays found that T‐cell migration toward CCL17 was unaffected by the hAEC‐conditioned medium but was able to suppress T‐cell proliferation. To elucidate the consequence of hAEC‐derived LXA4 on T‐cell proliferation, we found that addition of NDGA mitigated hAEC suppression of T‐cell proliferation. This may be due to the inhibition of extracellular signal‐regulated kinase signaling, which is known to regulate T‐cell proliferation .The bleomycin mouse model of pulmonary fibrosis has come under recent scrutiny . IPF is Our current study provides evidence that hAECs can alter neutrophil, macrophage, and T‐cell migration, phenotype, and behavior in resolving bleomycin‐induced lung injury, in part through LXA4. In the process of lung repair, hAECs promote resolution by upregulating macrophage phagocytosis, partly through LXA. It was highlighted at the 2012 National Institutes of Health, National Heart, Lung, and Blood Institute workshop that understanding the mechanism of action of stem cells is paramount for the advancement of cell‐based therapies . Acumen J.L.T.: study design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; Y.Z.T., R.M., S.T.C., and S.N.L.: collection and/or assembly of data, data analysis and interpretation, final approval of manuscript; J.C.M.: administrative support; final approval of manuscript; E.M.W.: study design, data analysis and interpretation, manuscript writing, final approval of manuscript; R.L.: study design, data analysis and interpretation, manuscript writing.S.T.C. has compensated employment and intellectual property rights. E.M.W. holds a patent for use of amnion cells as a lung repair therapy. The other authors indicated no potential conflicts of interest.Supporting InformationClick here for additional data file.


The extreme sparsity of IgE+ cells in vivo has confined their study almost entirely to mouse models.IgE‐expressing  IgE+germinal centre (GC)‐like B cells, (ii) IgE+PC‐like ‘plasmablasts’ and (iii) IgE+PCs. The same phenotypic stages were also observed for IgG1+ cells. Total tonsil B cells give rise to IgE+PCs by direct and sequential switching, whereas the isolated GC B‐cell fraction, the main source of IgE+PCs, generates IgE+PCs by sequential switching. PC differentiation of IgE+ cells is accompanied by the down‐regulation of surface expression of the short form of membrane IgE (SmIgE), which is homologous to mouse mIgE, and the up‐regulation of the long form of mIgE (LmIgE), which is associated with an enhanced B‐cell survival and expressed in humans, but not in mice.We have identified three phenotypic stages of IgE+PCs from tonsil GC B cells occurs mainly via sequential switching from IgG. The LmIgE/SmIgE ratio may be implicated in survival of IgE+ B cells during PC differentiation and allergic disease.Generation of IgE BCRB‐cell receptorCSRClass switch recombinationeGCEarly germinal centreEMPDExtramembrane proximal domainFACSFlow cytometry or fluorescence‐activated cell sortingGCGerminal centreLmIgELong‐form membrane IgESmIgEShort‐form membrane IgEPBPlasma blastPCPlasma cellsSHMSomatic hypermutations+ B cells in vivo has hindered the attempts to investigate their development, particularly in the human system, while reliance on the results from mouse models often fails to predict the outcome of proposed therapies IgE antibodies mediate the activation of IgE effector cells and antigen‐presenting cells by allergen and hence are central to allergic disease In vivo, CSR occurs in lymphoid tissues and at sites of inflammations , or allergen in the case of IgE It is well established that T‐cell helper type 2 (Th2) cytokines, IL‐4 and/or IL‐13, in association with CD40 cross‐linking on B cells, promote class switch recombination (CSR) to IgE, which may be direct, from IgM to IgE, or sequential, via IgG + B cells is dramatically different from that of IgG1+ B cells, which express the most abundant and most thoroughly investigated isotype + cells exhibited a PC phenotype and were excluded from the GCs + B cells determined their ultimate fate + GC cells with an impaired B‐cell receptor (BCR) signalling, due to the low expression of the BCR, leading to cell death + PCs with elevated BCR expression and was associated with the secretion of high‐affinity IgE antibodies + B cells are needed for the generation of a memory IgE response Recent studies in the mouse revealed that the fate of IgE+ B cells express two forms, one short and one long form, of mIgE, mIgES and mIgELL containing a longer extra‐membrane proximal domain (EMPD) region, an additional 52‐amino acid residue between the C‐terminal Ig domain, Cε4 and the transmembrane M1 domain The relevance of results in the mouse to human allergy has been questioned ex vivoex vivo tonsil human B‐cell culture system, we have now investigated the ontogeny of human IgE+ PCs. We point out many similarities, but also important differences from studies in the mouse models that may illuminate the mechanisms in allergy.We have previously characterized the capacity of various tonsil B‐cell subsets to undergo CSR to IgE + as determined by flow cytometry analysis.With informed written consent and ethical approval from Guy's Research Ethics Committee, we obtained human tonsils from donors undergoing routine tonsillectomies. Mononuclear cells were separated according to the density on a Ficoll gradient , and B cells were isolated using 2‐aminoethylisothiouronium bromide‐treated sheep red blood cells . B cells were >95% CD196 freshly isolated tonsil B cells were stimulated with IL‐4  and anti‐CD40 antibody  for up to 12 days.To induce CSR to IgE, B cells were cultured as previously described + cells was performed as previously described Surface and intracellular staining of IgEanova, with Bonferroni correction, unless otherwise stated. A value of P < 0.05 was considered significant .Statistical analysis was performed using the one‐way + cell populations  and IgG1hi (IgG1hiCD138− and IgG1hiCD138+) cells , a marker of proliferation, and their cell cycle progression declines, with the majority of the IgE+ and IgG1+ PCs being at the quiescent G0 stage of the cell cycle . In sum, these observations, consistent with those on the cell surface markers, indicate that IgElo (IgEloCD138−) cells have a phenotype with GC B cell‐like characteristics, whereas the IgEhi cells represent a later stage of differentiation into a PC‐like (IgEhiCD138−) ‘plasmablast’ phenotype, and only a small proportion appear to be fully differentiated PCs (IgEhiCD138+).Bcl‐6 and Pax‐5, important for maintaining the B‐cell identity and the GC phenotype lls Fig. B. The oplls Fig. B. There + cells + cells , memory (CD27+CD38−CD77−), early GC (CD27−CD38+CD77+) and GC (CD27+CD38+CD77+) B cells + PCs.This observation led – because GC B cells are the main source of IgE+ PCs. To confirm this, we cultured tonsil naïve, memory, eGC and GC B cells with IL‐4 and anti‐CD40, with unfractionated total B cells as controls, and determined the IgEhi/IgElo cell ratio as a measure of the rate of differentiation of the newly switched human IgE+ cells. We found that this ratio was lowest in naïve (0.19 ± 0.14) and highest in GC B‐cell cultures (2.24 ± 1.1), whereas the memory (0.95 ± 0.59), eGC (1.18 ± 0.64) and total B‐cell cultures (0.67 ± 0.42) had IgEhi/IgElo cell ratios intermediate to that in the naïve and GC B‐cell cultures .The above results suggest that the GC environment may favour the generation of IgE+ cells, derived from these cells, towards the PC lineage. We therefore examined the PC differentiation of IgE+ cells in each of the sorted B‐cell cultures . The data demonstrate that IgE+ B cells have a much higher frequency of PC differentiation than IgG1+ cells.The difference in the yields of IgG1PCs Fig. C,D. Howe+ B cell is required for the generation of high‐affinity IgE antibodies + GC cells and sequential CSR from IgG to IgE leads to IgE+ PCs + cells in our total B‐cell cultures, we examined switch circle transcripts (SCTs) by a nested PCR. Analysis of SCTs shows that both IgM to IgE (Iε‐Cμ) and IgG to IgE (Iε‐Cγ) SCTs were present in IgEloCD138−, IgEhiCD138− and IgEhiCD138+ cells  IgE+ GC‐like (IgEloCD138−), (ii) IgE+ PC‐like “plasmablasts” (IgEhiCD138−) and (iii) IgE+ PCs (IgEhiCD138+). The same developmental sequence was seen to prevail in the human tonsil IgG1+ cells. In contrast, mouse in vivo and ex vivo studies show the existence of only two distinct IgE+ (IgEloCD138− and IgEhiCD138+) and IgG1+ (IgG1loCD138− and IgG1hiCD138+) cell populations The developmental pathway of human IgEe IgEloCD8−, (ii) + cells appeared to differentiate more swiftly into PCs than IgG1+ cells + cells in vivo as well as ex vivo + cells, compared with their IgG1+ counterparts, differentiate into PCs. The mechanisms contributing to the apparent propensity of IgE+ cells to differentiate into the PC lineage remain unclear.In the mouse, nascent IgE+ and IgG1+ cells is similar and therefore does not account for their differences in PC differentiation.The transgenic Blimp‐1‐deficient mouse B cells undergo CSR to IgE, but fail to differentiate into PCs + cells. We have previously shown that CD23 plays an important role in IgE synthesis + cells towards the PC lineage could also result from the activity of CD23.Both mIgG1 and mIgE have cytoplasmic tails that contribute to their enhanced signalling capacity hi cells are mainly IgE+ PCs hi cells in total tonsil B cells become CD138+ PCs. It is possible that in these cultures, IgE+ PCs are generated, but fail to survive. However, we have demonstrated that increased IL‐4 concentration improved the yields of IgE+ PCs by maintaining the GC B cells. This implies that GC B cells are important for the generation of IgE+ PCs and that IL‐4 is essential not only for CSR to IgE but also for the maintenance of GC B cells, which have very high rates of cell death An important way in which the mouse and human systems diverge is that whereas mouse IgE+ cells that differentiated into IgE+ PCs at the end of the GC B‐cell cultures demonstrates the importance of GCs in the IgE+ PC generation. Yet, despite the different levels of Blimp‐1 and Xbp‐1 expression, eGC and memory B‐cell cultures display similar IgE+ cell propensity to PC differentiation, implying that other factors are important, for example the response to cytokines that affect the balance between cell division and cell death. In contrast, the majority of IgE+ switched cells in naïve B‐cell cultures, which undergo similar levels of CSR to IgE In vivo memory B cells are generated in GCs following SHM and affinity maturation, resulting in cells with high‐affinity BCRs The high proportion of IgE+ cells + cells + GC cells, attributed to their low level of mIgE expression, and impaired BCR signalling + GC cells fail to undergo the canonical B‐cell differentiation programme that potentiates IgG1 memory immune responses + cells was determined by their switching pathway; direct switching from IgM+ cells generated only a transient population of IgE+ GC cells, whereas switching from IgG1+ cells generated IgE+ PCs, accompanied by the up‐regulation of mIgE A novel observation in the mouse was the inheritance of IgE memory from IgGboth direct and sequential CSR can give rise to IgE+ GC B cells and IgE+ PCs in our tonsil B‐cell cultures. This is supported by studies on a chimeric mouse model containing the human M1′ sequence inserted into a murine ε gene reporter construct + GC B cells were longer‐lived than in other mouse models and able to differentiate into both IgE+ memory B cells and IgE+ PCs. Memory IgE+ B cells and IgE+ PCs can also be detected in the peripheral blood of humans In contrast to the results in the mouse, we observe that + PCs generated from the human GC‐derived B‐cell cultures. The major implication of sequential CSR is the probability of affinity maturation of the IgG+ B‐cell precursors, by analogy to observations in the mouse Furthermore, we have also presented evidence of only sequential CSR in IgEL and mIgES expression during PC differentiation. Earlier studies have shown that mIgEL and mIgES are the predominant isoforms in the IL‐4‐ and anti‐CD40‐stimulated and unstimulated peripheral blood lymphocytes, respectively L expression is important for IgE homeostasis in the human system. Expression of this isoform is unique to humans and could well be one reason why allergic disease occurs naturally in humans, but apparently not in mice L is a validated antibody target for immunotherapy of allergic disease, now under development in several laboratories with a view to therapeutic application Our experiments also reveal the modulation of the surface mIgEex vivo human system, we have investigated the ontogeny of IgE+ B cells and IgE+ PCs. We believe that our results have a direct relevance to the discovery of novel targets for the treatment of allergy.In summary, using a tonsil F.R. designed and performed experiments, analysed data and wrote the paper. H.B., N. U., P.S.H. and Y‐C.C. performed experiments and analysed data. J‐B.C. and T.W.C contributed vital new reagents and critically reviewed the manuscript. J.M.M. and B.J.S. provided guidance and critically reviewed the manuscript. D.J.F provided guidance, analysed data and critically reviewed the manuscript. H.J.G. designed experiments, analysed data and wrote the manuscript. All authors reviewed the final manuscript.The authors declare that they have no conflict of interests.Data S1 Supplemental MethodsClick here for additional data file.


SCs), has provided enticing alternative seed cells for stem cell‐based therapy. MenSCs are enriched in the self‐regenerative tissue, endometrium, which shed along the periodic menstrual blood and thus their acquisition involves no physical invasiveness. However, the impact of the storage duration of menstrual blood prior to stem cell isolation, the age of the donor, the number of passages on the self‐renewing of MenSCs, the paracrine production of biological factors in MenSCs and expression of adhesion molecules on MenSCs remain elusive. In this study, we confirmed that MenSCs reside in shedding endometrium, and documented that up to 3 days of storage at 4°C has little impact on MenSCs, while the age of the donor and the number of passages are negatively associated with proliferation capacity of MenSCs. Moreover, we found that MenSCs were actually immune‐privileged and projected no risk of tumour formation. Also, we documented a lung‐ and liver‐dominated, spleen‐ and kidney‐involved organic distribution profile of MenSC 3 days after intravenous transfer into mice. At last, we suggested that MenSCs may have potentially therapeutic effects on diseases through paracrine effect and immunomodulation.Successful isolation of human endometrial stem cells from menstrual blood, namely menstrual blood‐derived endometrial stem cells (Men During the past decade, the laboratory data and clinical trials have established the knowledge that adult stem cell (ASC)‐based therapy could improve a myriad of diseases due to their intrinsic potential of regeneration and immunoregulation As a newly identified type of ASC, human menstrual blood‐derived endometrial stem cells (MenSCs) were first reported in 2007 and show advantages with regard to non‐invasive collection procedure, high proliferation rate, multipotency, low immunogenicity and risk of karyotypic abnormalities, comparing to the other types of stem cell The aim of this study was to firstly confirm the localization of MenSCs; subsequently address the influence of storage duration of menstrual blood preceding stem cell isolation, the age of the female donors and the number of passages on the quality of MenSCs; thirdly investigate the immunogenicity, tumorigenicity and organic distribution of MenSCs upon adoptive transfer; and finally determine the potential paracrine effect of MenSCs and the expression of adhesion molecules on MenSCs.All the MenSCs used in this study were harvested with the informed consent of the donors. PC12 cells were kept in our laboratory. This study was approved by Ethics Committee of the Xinxiang Medical University, and experimental procedures for menstrual blood samples and MenSC were carried out in accordance with the approved guidelines. We received informed consents from all of the donors, and all the donors provided consent for the use of their menstrual blood samples in scientific research.Six‐ to eight‐week‐old BALB/c mice (20–25 g) and nude mice‐BALB/c were purchased from Vital River Laboratories . Handling of mice and experimental procedures were carried out in accordance with the guidelines of Animal Care Committee of Xinxiang Medical University. The mice were bred and housed in a specific pathogen‐free condition on a 12 hrs light–dark cycle.2 plastic cell culture flasks at 37°C with 5% humidified CO2. After 2 days of incubation, the non‐adherent cells were washed away leaving behind the adherent cells that were growing as fibroblastic cells in clusters and the medium was replaced every 3 days. When the cells reaching 80–90% confluence (P0), the cells were detached by 0.25% trypsin/1 mM EDTA and subcultured to new flasks by the ratio of 1:3.The menstrual blood samples were collected from healthy female donors by menstrual cup during the first few days of menses, and the samples were mixed with equal volume of phosphate‐buffered saline (PBS) containing 0.25 mg/ml amphotericin B, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM ethylenediaminetetraacetic acid (EDTA). Shortly thereafter, the part of the supernatant of the samples was used to check the contamination of bacteria, and menstrual blood samples were delivered into laboratory and subject to standard Ficoll procedures within 72 hrs. After centrifugation, the karyocytes and the deciduous endometrium suspending in buffy coat were transferred into new tube, then washed in PBS for twice, generally suspended in growth medium  and seeded into 25 cmIn addition, for confirming the localization of MenSCs, the procedures including natural sedimentation and stainless steel mesh filtration were used. After density gradient centrifugation, the karyocytes and the visible deciduous endometrium suspending in buffy coat were firstly separated by 2 min. standing; subsequently, the cell suspensions without visible sediments were filtered through a 100‐μm stainless steel mesh; finally, these three kinds of samples  were seeded into 6‐well plate and cultured as described above.n = 6 for each group), the cells were suspended in growth medium and respectively seeded at the density of 1.25 × 104 cells/ml, 2.5 × 104 cells/ml and 5 × 104 cells/ml into 96‐well plates. After incubation at 37°C with 5% humidified CO2 for 1, 3, 5, 7 and 9 days, respectively, proliferative response was determined by MTT assay, and the absorbances were analysed at 490 nm.To assess the proliferative capacity of different passages of MenSCs  among 20 ± 2‐year, 30 ± 2‐year and 40 ± 2‐year groups (6 cells) was washed by PBS for twice and incubated with monoclonal antibodies at 4°C in the dark for 30 min. After washing with PBS, the samples were analysed by Cytomics FC 500 MPL cytometer .MenSCs harvested from different passages were used for immunophenotyping analysis. Mouse anti‐human monoclonal antibodies: FITC‐conjugated CD29, CD73, CD90, HLA‐ABC, HLA‐DR, CD34 and CD45 and PE‐conjugated CD105, and rat anti‐human monoclonal antibodies: FITC‐conjugated CD44  were used. As a control, isotype PE and FITC‐conjugated IgG were used. The cell suspension , and the induction medium was replaced every 3 days except for neurogenic differentiation. Control cells were cultured in growth medium.Adipogenic, osteogenic, chondrogenic, neurogenic and cardiogenic differentiation was performed and identified as reported. Briefly, for differentiation assays, P3 MenSCs were suspended in growth medium and seeded at the density of 2 × 10Adipogenic differentiation medium: growth medium + 1 μmol/l dexamethasone + 10 μg/ml recombinant human insulin + 200 μM indomethacin + 0.5 mM IBMX, for 14 days.Osteogenic differentiation medium: growth medium + 0.1 μmol/l dexamethasone + 0.05 mmol/l ascorbic acid + 10 mM β‐glycerophosphate, for 21 days.Chondrogenic differentiation medium: growth medium + 0.1 μmol/l dexamethasone + 0.2 mmol/l ascorbic acid + 1% insulin‐transferrin‐selenic acid + 10 ng/ml TGF‐β3, for 21 days.Neurogenic differentiation medium: growth medium + 1 mM β‐mercaptoethanol, for 1 day; growth medium + 35 ng/ml all‐trans‐retinoic acid, for 3 days; growth medium + 5 ng/ml platelet‐derived growth factor + 10 ng/ml basic fibroblast growth factor + 14 μM fsk + 126 ng/ml GGF‐2, for 14 days.Cardiogenic differentiation medium: growth medium + 20 μM 5‐azacytidine .At the end of the induction period, cells were washed and fixed. Adipogenic differentiation was confirmed by oil red O staining; osteogenic differentiation was confirmed by alizarin red staining; chondrogenic differentiation was confirmed by alcian blue stainin neurogenic differentiation was confirmed by immunofluorescence assessment of GFAP ; and cardiogenic differentiation was confirmed by immunofluorescence assessment of cTnT .During a 5‐day culture, P0 MenSCs were fixed with 4% PFA for 20 min. and permeabilized with 0.05% Triton X‐100 for 10 min.; non‐specific binding sites were blocked using 5% goat serum for 30 min. Anti‐SOX2, anti‐SSEA4, anti‐c‐Myc and anti‐Nanog (Abcam) were separately added and incubated at 4°C overnight. Alexa Fluor 488‐conjugated goat antimouse or rabbit secondary antibodies  were incubated with the cells at 37°C for 1 hr. Cell nuclei were stained with DAPI. Finally, the cells were observed and imaged under fluorescence inverted microscope .6 cells/ml in PBS. For transplantation, 1 × 106 DiI‐labelled cells (0.2 ml) were injected into BALB/c mice from tail vein (n = 3); the mice received 10 μg DiI in 0.2 ml PBS were taken as controls, and then, all the mice were killed after 72 hrs. The liver, lung, spleen and kidney were fixed in 4% formaldehyde solution overnight and then dehydrated in 18% sucrose solution overnight. Subsequently, the specimen was embedded in OCT compound , frozen in liquid nitrogen and stored at −80°C. Finally, the samples were adjacently sectioned with 20 μm thickness on the poly‐L‐lysine coated slides with a cryotome (Leica 1850) and imaged under a fluorescence microscope (Leica DFC425C).To track the transplanted cells after i.v. injection, P3 MenSCs were labelled by the fluorescent lipophilic tracer DiI . For labelling, the cells were cultured in growth medium supplemented with DiI at a final concentration of 2.5 μg/ml before harvest. After incubation for 12 hrs, the cells were detached and centrifuged at 1000 r.p.m. for 5 min., washed twice with PBS and then adjusted to the concentration of 5 × 10in vivo immune response to MenSCs, male BALB/c mice were randomly divided into three groups (n = 6): control group received 0.2 ml PBS by intraperitoneal injection, experiment group 1 received 1 × 106 P3 MenSC in 0.2 ml PBS by intraperitoneal injection, and experiment group 2 received 1 × 106 P3 MenSCs in 0.2 ml PBS by intravenous injection from tail vein. For examination, the blood samples were separately collected after 3 days and 7 days, and sent to Xinxiang Assegai Medical Laboratory Center  within 8 hrs. Routine blood tests were performed by the ADVIA2120 haematology analyser ; the activity of associated enzymes  was determined by velocity method; the content of urea and creatinine (CR) was quantified by dehydrogenase and oxidase methods.To examine the n = 5): MenSCs‐treated group (1 × 106 P3 MenSCs in 0.2 ml PBS were injected subcutaneously into the right axilla of each nude mouse) and PC12 cells‐treated group (1 × 106 PC12 cells in 0.2 ml PBS were injected subcutaneously into the right axilla of each nude mouse). The tumour formation was recorded at the time‐point of 1, 2, 3 and 12 weeks, respectively.For determining tumorigenicity potential of MenSCs, nude mice were randomly divided into two groups (n = 5). Adhesion molecule arrays (GSH‐CAM‐1) were used to measure the expression levels of 17 adhesion molecules on P3 MenSCs. Positive signals were captured on glass chips using a laser scanner , and the observed fluorescence intensities were normalized to the intensities of the internal positive controls.Angiogenesis and inflammation arrays  were used according to the manufacturer's instructions to measure the expression levels of 11 angiogenesis‐associated biological factors and 11 cytokines in the conditional medium of MenSCs . After being cultured for another 48 hrs, the conditional medium was collected and ten times concentrated by ultrafiltration.Preparation of the conditional medium of MenSCs: two million P3 MenSCs were seeded into 75 cmP < 0.05 was considered to be statistically significant.Results were presented as the mean ± S.D., and Student's paired two‐tailed test was used to determine statistical significance. Different from the conventional isolation of PBMC from peripheral blood, after density gradient centrifugation of menstrual blood, besides karyocytes, the flocculent membranes (deciduous endometrium) were also clearly observed in the buffy coat layer. For determining the localization of MenSCs, the procedures including natural sedimentation and stainless steel mesh filtration were used. As shown in Figure in vitro  culture period, MenSCs formed colonies with radioactive or helix growth pattern outgrew the non‐adherent mononuclear cells, which were readily removable through refreshing the culture medium Fig. G–I. Aftetro Fig. A–C. Substro Fig. P. Severatro Fig. L–O. To fn = 6). MenSCs were isolated, and their proliferation capacities were evaluated when seeded at various densities with cells that were maintained at various passages . During early passage (P3), the proliferation rates of MenSCs from differently aged donors were only slightly different, in an age‐coupled fashion: the older the donor, the slower the proliferation, especially at low density  during MenSCs transfer. No major alteration was observed. Thus, MenSCs transfer has little adverse effects on the immune system of recipients ; moreover, the further biochemical indicators tests also demonstrated no adverse effect on the function of key organs after MenSCs transplantation. These findings not only provided support for that MenSCs which are immune‐privileged, but also suggested the safety of allogeneic MenSCs transplantation in further clinical application in vitro secreted higher levels of angiogenic factors ; meanwhile, the pro‐inflammatory cytokines  were expressed at higher levels than anti‐inflammatory cytokines , which could recruit endothelial cells and associated leucocytes to sites of injured tissues. Although the above results confirmed the angiogenesis‐promoting and immunomodulatory potential of MenSCs through paracrine effect in vitro, the therapeutic effect in vivo still need to be testified in animal models because of the huge difference in microenvironment.It is well known that paracrine functions performed by ASCs play therapeutic roles in ASC‐based therapy. Also, the transplantation of synthetic cell‐mimicking microparticle (CMMP), in which the secreted proteins isolated from cardiac stem cells or bone marrow‐derived mesenchymal stem cells were packaged by associated stem cell membrane, could improve cardiomyocyte functions without immune activation Furthermore, adhesion molecules analysis demonstrated the higher expression of ALCAM and ICAM‐1 on MenSCs. ALCAM plays a role in the binding of T and B cells to activated leucocytes, as well as homophilic adhesion (ALCAM‐ALCAM) was shown to play important roles in tight cell‐to‐cell interaction and regulation of stem cell differentiation. Simultaneously, ICAM‐1 also plays a role in the binding of leucocytes through binding to LFA‐1 (lymphocyte function‐associated antigen 1), and recently, it is reported that low‐level expression of ICAM‐1 on ASCs has a critical impact on ASC‐mediated immunosuppression in vitro, practical delivery methods, the dosing of the cells and adjuvant strategies will have to be performed before MenSCs are used routinely in the wider field of regenerative medicine.In conclusion, our findings not only confirmed the biological characteristics of the MenSCs, such as localization, typical ASC's morphology, high proliferative capacity, classical ASC's surface markers and multilineage differentiation potential, but also suggested the safety of MenSCs‐based therapies. Furthermore, protein arrays suggested that MenSCs may have potentially therapeutic effects on diseases through paracrine effect and immunomodulation. These complete characterizations of MenSCs have improved our understanding of their biological features and provide alternative, readily available therapeutic approaches for human diseases. Then, the further pre‐clinical studies regarding the standardization of the MenSCs isolation and proliferation, storage J. L. and Y. L. were the principal investigators and take primary responsibility for the paper. J. L., Y. L. and P. S. conceived and designed the experiments. Y. L., R. N., F. Y., Y. S., S. L. and Y. Y. performed the experiments. J. L., Y. L. and P. S. analysed the data and wrote the paper and prepared figures. All authors reviewed this manuscript.The authors declare that there is no conflict of interests regarding the publication of this paper.