P<0.01). The sex ratio in IVF/ICSI babies was significantly higher toward males in transfers of blastocyst (54.9%) and thawed embryo (52.4%) when compared with transfers of cleavage stage embryo (51.4%) and fresh embryo (51.5%), respectively. Multiple delivery was not associated with sex ratio. However, in multivariable logistic regression analysis after controlling for related factors, only ICSI  and blastocyst transfer  were associated with sex ratio in IVF/ICSI babies. In conclusion, the live birth sex ratio in IVF/ICSI babies was influenced by the use of ICSI, which may decrease the percentage of male offspring, or the use of blastocyst transfer, which may increase the percentage of male offspring.In order to study the impact of procedures of IVF/ICSI technology on sex ratio in China, we conducted this multi-center retrospective study including 121,247 babies born to 93,895 women in China. There were 62,700 male babies and 58,477 female babies, making the sex ratio being 51.8% . In univariate logistic regression analysis, sex ratio was imbalance toward females of 50.3% when ICSI was preformed compared to 47.7% when IVF was used ( In humans, it is estimated that the primary sex ratio after conception may be as high as 170∶100 in vitro fertilization (IVF) baby was born in 1978 in England, assisted reproductive techonology (ART) has rapidly developed all over the world. Concerns about whether IVF/ICSI has any impacts on SSR have led scholars to study SSR in IVF/ICSI babies Sicne the first Similar studies have shown that large sample size, data from different countries and regions are needed to study SSR in IVF/ICSI offspring. A careful review of the literature has shown that SSR in babies following IVF/ICSI was reported in United States, Australia and New Zealand, Japan, etc All the babies born after IVF/ICSI in this retrospective study were recruited from 18 reproductive medicine centers in China . Since no previous reports has been published concerning SSR in IVF/ICSI babies in China, this study included all the offspring after IVF/ICSI from each center from January 2002 to December 2012. Those conceived by sperm donation cycles, oocyte donation cycles, or pre-implantation genetic diagnosis cycles were excluded. Institutional Review Board (IRB) approval was obtained from First Affiliated Hospital of Zhengzhou University. Written consent forms were obtained from patients in some centers. However, all the patients were undergoing routine IVF/ICSI treatment, and patient information was totally anonymous in our analysis.The data variables included in the study were live birth cycles, the babies' number and sex, insemination method (IVF or ICSI), stage of embryo transferred (cleavage stage embryo or blastocyst), type of embryo (fresh or thawed), and type of birth (singleton or multiple delivery). Cleavage stage embryo was defined as a day-2 or day-3 embryo. Blastocyst transfer was a transfer of a day-5 to day-7 embryo. Live birth was defined as any birth event in which at least one baby is born alive P value for the final univariate analysis. Multivariable binary logistic regression was used to generate the adjusted OR. A P value <0.05 was considered to be significant. SPSS 17.0  was used for analysis.For univariate analysis of categorical variables, chi-square tests were used. Simple binary logistic regression was used to generate the crude odds ratio (OR), 95% of confidence interval and In total, 18 reproductive medicine centers were included into this study. The study population included 121,247 babies born to 93,895 women . There wP<0.01). In contrast, the SSR was significantly higher towards males in blastocyst transfers compared with that in cleavage stage embryo transfers . In addition, we also found a sex ratio imbalance towards males of 52.4% in thawed ETs compared with 51.5% in fresh ETs (P<0.01). However, the SSR in singleton and multiple delivery were similar .Firstly, the sex ratio was analyzed according to different ART procedures and singleton or multiple delivery . There wP<0.01) and blastocyst transfer  were still significantly associated with SSR in IVF/ICSI babies. However, the association between fresh/thawed transfer and SSR was not significant after adjusting for other factors .Results of multivariable logistic regression analysis were showed in In order to clarify the different effects of ART procedures on live birth sex ratio, we analyzed all the data from each subgroup –3. As shRecently, IVF/ICSI has been developed rapidly, and clinical pregnancy and live birth rates are stable. It is shown that around 130,000 IVF/ICSI babies were born in the year of 2005 in America, which accounts for 1% new born babies in the whole country As early as 2000, scholars from French and Australia reported the SSR in IVF/ICSI babies The current study also analyzed SSRs from different regions of China. According to the location of the 18 centers, all IVF/ICSI babies were divided into 6 regions: Northern China, Central China, Southern China, Eastern China, Northwestern China, and Southwestern China. The SSRs were different among these 6 regions. Thus, here we advocate that it is necessary to include multiple centers from different regions in such studies in the future.ICSI, which has been mainly used to treat male factor infertility, is now widely used all over the world. The use of ICSI in the United States has increased from 11.0% of ART cycles to 57.5% of cycles between 1995 and 2004 However, since the SSR in IVF/ICSI could be affected by other factors, we explored the association between SSR and insemination methods in subgroups . Our resCurrently, there is no conclusion why the use of ICSI reduces SSR. ICSI is mainly used in patients with male factor infertility, of whom the spermatogenic function is usually compromised. It may be possible that the percentage of Y-bearing sperm is also reduced in those patients Compared with cleavage stage embryo transfer, blastocyst transfer significantly increases implantation rate, and reduces multiple pregnancy rate It is worthwhile to note that we also explored the relationship between stage of embryo transferred and SSR in subgroups . CompareIn the present study, we also explored the effect of frozen-thawed embryo transfer (FET) on SSR. To date, very few studies are reporting the difference of SSR in fresh embryo transfer and FET, and the results are controversial In most of the previous studies, only singleton or single embryo transfer cycles were included into analysis, because monozygotic twins in multiple pregnancies may also have effect on SSR To the best of our knowledge, this is the first study reporting SSR in babies born after IVF/ICSI in China. Even though with large sample size from 18 centers, the present study indeed has several limitations. Since there is no standard ART data reporting system in China, currently it is impossible to collect as much information, such as mothers' basic characteristics, as we want. Even though basic characteristics of 5,790 patients with singleton birth were obtained from only one center , and theTaken together, the present study including more than 120 thousands IVF/ICSI babies from 18 reproductive medicine centers in China showed that the overall SSR in IVF/ICSI is 51.8% (107∶100), which is similar with that in natural conception. Compared with the use of IVF, the use of ICSI reduces SSR in cleavage stage embryo transfer cycles, but not in blastocyst transfer cycles. However, blastocyst transfer increases SSR compared with cleavage stage embryo transfers, and this effect is independent of other factors. Neither FET nor multiple delivery has effect on SSR.Table S1Basic characteristics of 5,790 mothers with singleton birth in one center.(DOC)Click here for additional data file.File S1121,247 IVF/ICSI babies from 18 centers in China. IVF-ET  =  in vitro fertilization and embryo transfer; ICSI-ET  =  intracytoplasmic sperm injection and embryo transfer; Cleavage stage  =  Day 2 or day 3 embryo transfer; Blastocyst  =  Day 5 or day 6 embryo transfer.(DOC)Click here for additional data file.


Nebivolol, third-generation β-blocker, may activate β3-adrenergic receptor (AR), which has been emerged as a novel and potential therapeutic targets for cardiovascular diseases. However, it is not known whether nebivolol administration plays a cardioprotective effect against myocardial infarction (MI) injury. Therefore, the present study was designed to clarify the effects of nebivolol on MI injury and to elucidate the underlying mechanism. MI model was constructed by left anterior descending (LAD) artery ligation. Nebivolol, β3-AR antagonist (SR59230A), Nitro-L-arginine methylester (L-NAME) or vehicle was administered for 4 weeks after MI operation. Cardiac function was monitored by echocardiography. Moreover, the fibrosis and the apoptosis of myocardium were assessed by Masson's trichrome stain and TUNEL assay respectively 4 weeks after MI. Nebivolol administration reduced scar area by 68% compared with MI group (p<0.05). Meanwhile, nebivolol also decreased the myocardial apoptosis and improved the heart function after MI (p<0.05 vs. MI). These effects were associated with increased β3-AR expression. Moreover, nebivolol treatment significantly increased the phosphorylation of endothelial NOS (eNOS) and the expression of neuronal NOS (nNOS). Conversely, the cardiac protective effects of nebivolol were abolished by SR and L-NAME. These results indicate that nebivolol protects against MI injury. Furthermore, the cardioprotective effects of nebivolol may be mediated by β3-AR-eNOS/nNOS pathway. Acute myocardial infarction (AMI), inducing permanent loss of cardiomyocyte mass and pathological left ventriclar remodeling, is one of the leading causes of death worldwide Nebivolol is the third-generation β-blocker approved by the Food and Drug Administration (FDA) for the treatment of hypertension. Given its greatest selectivity for cardiac β1-adrenergic receptors without intrinsic sympathomimetic activity, nebivolol reduces systemic vascular resistance and improving diastolic function Although previous studied indicated that nebivolol could reduce cardiac remodeling and preserve cardiac function through β3-AR pathway, it is not known whether nebivolol administration plays a cardioprotective effect against MI injury. Therefore, we designed the present study to explore the potential role of nebivolol during MI and to elucidate the underline mechanism of their protective effects.One hundred and fifty adult C57BL6/J mice  were housed in a temperature-controlled animal facility with a 12-h light/dark cycle. All procedures were approved by the Second Artillery General Hospital of Chinese People's Liberation Army Committee on Animal Care.  and were in compliance with Guidelines for the Care and Use of Laboratory Animals, as published by the National Academy Press. Mice were euthanized by cervical dislocation after anesthesia with 5% isoflurane.Mice were randomly allocated into 5 groups with n = 30 each: (1) sham group (Sham); (2) MI group (MI); (3) MI + Nebivolol group (Nebivolol); (4) MI + Nebivolol+SR59230A group (Nebivolol+SR); (5) MI + Nebivolol+ L-NAME group (Nebivolol+L-NAME). After MI model, all of the above drugs or vehicles were administrated for 4 weeks respectively. Blood pressure was monitored with a tailcuff system see .Myocardial infarction (MI) model was constructed by left anterior descending (LAD) artery ligation as previous described Mice of MI+ Nebivolol group were administrated with Nebivolol at 2 µg/kg/hour via osmotic mini-pumps  one day after MI operation. Mice treated with β3-AR antagonist were administrated with SR59230A at 0.1 mg/kg/hour via another osmotic mini-pump. Moreover, mice from Nebivolol+L-NAME group were treated with Nitro-L-arginine methylester (L-NAME) by intraperitoneal injection at 25 mg/kg for 4weeks after LAD ligation.Mice were euthanized by cervical dislocation after anesthesia with 5% isoflurane for histological assay at 4 weeks after MI Echocardiography was performed to assess the cardiac function after MI in a blinded manner Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed to evaluate myocardial apoptosis 4 weeks after cell transplantation, hearts were excised from peri-infarcted areas in LV for Western blotting assay following standard protocol Ser1177, p-eNOS Thr495, p-eNOS Ser1141∶500, Cell Signaling Technology); iNOS ; nNOS  and β-actin .The following primary antibodies were used: β1-adrenergic receptor ; β2-adrenergic receptor ; β3-adrenergic receptor ; eNOS ; Phospho-eNOS at Ser1177,Thr495, Ser114 . Statistical comparisons for different groups were performed using one-way ANOVA followed by Student's paired, two-tailed t test for two groups' comparison. P values <0.05 were considered statistically significant.To study the effects of nebivolol on the extent of fibrosis after MI, we performed Masson's trichrome staining. As illustrated in Furthermore, echocardiogram studies were performed to evaluate the cardiac function after MI in all groups. Serial echocardiographic analysis indicating that nebivolol administration manifested a trend towards improvement of cardiac performance after MI. Compared with sham group, MI increased the LVEDd and LVESd which were abolished by nebivolol treatment . FurtherWe next performed TUNEL staining to confirm the protective effect of nebivolol on the apoptosis of cardiomyocytes induced by MI. Representative immunofluorescence results in Western blotting assay was performed to investigate the cardiac expressions of β-adrenoreceptor, including β1/β2-AR and β3-AR. Representative bloting results  indicatePrevious studies suggested that β3-AR stimulation results in NO production via NOS which includes three isoforms . Therefore, we further performed Western blot assay to evaluate the effect of nebivolol on the expressions of NOS isoforms after MI. Representative bloting results and semiquantitative analyses  demonstrSer1177significantly decreased, whereas the expression of p-eNOSThr495 increased in MI group compared with Sham group. Furthermore, nebivolol administration resulted in an increase in p-eNOSSer1177 expression with decreased eNOS phosphorylations at Thr 497 compared with MI. Interestingly, no difference of p-eNOSSer114 expression was observed in all group . As representative results  and semill group .SR59230A and L-NAME are well known as the inhibitors of β3-AR and NOS respectively. To illuminate the mechanism of the protective effect of nebivolol, either SR59230A or L-NAME was administrated with nebivolol. Representative Masson's trichrome staining and quantitative measurement in Representative TUNEL staining in Ser1177 were significantly decreased in MI+Nebivolol+SR group and MI+Nebivolol+L-NAME group. Conversely, SR59230A or L-NAME administrated resulted in a significantly increase of p-eNOSThr495 expression. Moreover, no difference of p-eNOSSer114 expressions was observed in three groups.Representative bloting results and semiquantitative analyses  indicateIn the present study, we found that nebivolol decreased the apoptosis of cardiomyocytes, and enhanced cardiac function after MI injury. Furthermore, the protective effects of nebivolol were abolished by β3-AR inhibition. Overall, this study demonstrates that nebivolol prevents cardiac dysfunction induced by MI via β3-AR-eNOS/nNOS pathway.Nebivolol has recently been shown to cause peripheral vasodilatation, and provide beneficial effects on cardiac dysfunction. Sorrentino et al. The β-adrenoceptors, belonging to the G protein-coupled receptor super family, play an essential role in cardiovascular function. The β1- and β2-ARs, which mediates chronotropic and inotropic effects, are well established. Although the precise cardiac effects remain uncertain, β3-AR is of increasing interest due to its potential as a potential target for cardiovascular disease treatment Previous studies suggested that the protective effects of nebivolol are dependent on β3-AR stimulating Although the mechanism is not clarified, previous studies have demonstrated that the protective effects of β3-AR stimulation were inhibited by NOS inhibitor and could be reversed by excess NO production Ser1177 and decreased p-eNOSThr495 in the infarcted heart. Interestingly, the phosph-eNOS at Ser114 was unaltered with or without nebivolol administration, which was consistent with previous studies. The alterations of eNOS phosphorylation suggested that MI inhibited the eNOS activation which was abolished by nebivolol treatment. Emerging evidence demonstrated that nNOS-derived NO production plays a substantial role in regulating myocardial contraction Three phosphorylation sites, serine residue 1177 (Ser1177),threonine residue 497 (Thr495) and serine residue 114(Ser114), modulate eNOS activity. Phosphorylation at Ser1177 activates eNOS, whereas phosphorylations at Ser114 and Thr 497 inhibit the eNOS activity Our study provides evidence that nebivolol treatment enhanced eNOS activation and increased nNOS protein expression, which was abolished by β3-AR antagonism SR59230A and NOS inhibitors L-NAME respectively. Moreover, nebivolol administration had no effect on the expression of iNOS. These results indicated that nebivolol acts as a dual activator of eNOS and nNOS to generate NO in the myocardium. Furthermore, the cardiacprotective effects of nebivolol were also abolished by L-NAME, indicated that nebivolol prevents cardiac dysfunction induced by MI by eNOS and nNOS which was activated by β3-AR pathway.Although our study bears some clinical relevance, there are many limitations. First, the detailed physiologic and pathologic functions of β3-AR have not been extensively characterized. Moreover, there are still intense controversies about which NOS isoform is the chief player in β3-AR signaling. Further studies defining the exact mechanism are needed. In conclusion, we provided evidence that the administration of nebivolol has a substantial effect on recovery of heart function by eNOS and nNOS activated by β3-AR. These data collectively indicate that nebivolol administration has practical clinical use following myocardial infarction.Methods S1Measurement of blood pressure.(DOC)Click here for additional data file.Table S1Hemodynamic parameters (heart rate and blood pressure) in different experimental groups. Bpm: beats per minute. Data are mean±SD (n = 15). *P<0.05 vs. control group; †P<0.05 vs. baseline group.(DOC)Click here for additional data file.


We show that the history of play in a population game contains exploitable information that can be successfully used by sophisticated strategies to defeat memory-one opponents, including zero determinant strategies. The history allows a player to label opponents by their strategies, enabling a player to determine the population distribution and to act differentially based on the opponent’s strategy in each pairwise interaction. For the Prisoner’s Dilemma, these advantages lead to the natural formation of cooperative coalitions among similarly behaving players and eventually to unilateral defection against opposing player types. We show analytically and empirically that optimal play in population games depends strongly on the population distribution. For example, the optimal strategy for a minority player type against a resident TFT population is ALLC, while for a majority player type the optimal strategy versus TFT players is ALLD. Such behaviors are not accessible to memory-one strategies. Drawing inspiration from Sun Tzu’s the Art of War, we implemented a non-memory-one strategy for population games based on techniques from machine learning and statistical inference that can exploit the history of play in this manner. Via simulation we find that this strategy is essentially uninvadable and can successfully invade  essentially all known memory-one strategies for the Prisoner’s Dilemma, including ALLC , ALLD , tit-for-tat (TFT), win-stay-lose-shift (WSLS), and zero determinant (ZD) strategies, including extortionate and generous strategies. T > R > P > S and often 2R > T + S. A special case known as the donation game is given by T = b, R = b − c, P = 0, S = −c, with 0 < c < b. There are many well-known strategies for the Prisoner’s Dilemma, such as ALLC , ALLD , tit-for-tat (TFT) [The Prisoner’s Dilemma (PD)  is a twoat (TFT)  and win-at (TFT) . The disat (TFT)  has inviat (TFT)  10] [11 [11(RSTPat (TFT)  13] . Res. Resρ/ρt random , instead0 competes against other players. IP0 is nearly as effective against ALLC as ALLD is, and quickly learns to exploit ALLC, but has a slightly smaller fixation probability because of the info gain phase. IP0 also fares well against ALLD, behaving much like TFT in that it cooperates with other (identified) IP0 individuals and defects against ALLD. Outcomes versus ALLD are sensitive to initial population proportion. An invading subpopulation of 10 IP0 has an empirically computed fixation probability of ρ ≈ 0.5 .These values reveal much about how IP0 does not fall prey to the mismatches due to errors that TFT is prone to . (A. (Aσ = 10) as in .Nsim = 10,000 independent simulations were run for each  pair, and p-values for the observed number of successful invasions k were computed under a null hypothesis H0 assuming a neutral rate of fixation θ = 1/N:In most cases, χ  an infogain phase during which an IP0 player chooses its moves to maximize its information yield about a new player, both to assess whether it is another IP0 (self vs. non-self), and in the latter case to estimate its strategy vector; (2) a groupmax phase during which IP0 seeks to maximize its score relative to the opponent group, by either cooperating  or using its current optimal strategy versus the group (if the other player is not an IP0). Note that when multiple IP0 players are present in a population, they operate completely independently; they do not share information or communicate.We implemented a basic information player strategy, called IP0 records the outcomes of its games vs. a given player in terms of  pairs, where A is a possible move (C or D) by itself, B is a possible move by the other player (C or D), nAB is the total number of times game outcome AB has occurred with this player, and mAB is the number of those cases where the other player’s next move was C. Treating each such case as a binomial event with probability pAB = θ (probability of cooperating given game outcome AB), the posterior distribution is p = β (i.e. the Beta distribution assuming a uniform prior p(θ) = 1), the maximum likelihood estimator is θ̂ = mAB/nAB, and the posterior expectation value is IP0 chooses its moves to seek game outcomes AB about which it has the least information . Specifically, if the current game outcome was ab, then it chooses the move A that minimizes the expectation value of nAB:p(B∣ab) = (mab + 1)/(nab + 2) for B = C. In the case of exact ties , the IP0 breaks the tie by computing the MD5 hash value of the game outcomes history string (e.g. CCDC…), and choosing C if its least-significant bit is zero, otherwise D. Note that since this rule depends only on information known to both players (their game outcomes), IP0 can predict what moves the other player would choose if it too were an IP0. .For the first 10 rounds of its play with another player, IP0 seeks to maximize its average relative score vs. the opposing group G  and third (SGI) terms above (see below for details).During this phase, IP group G . Each IP0 computes the likelihood odds ratio for the observed move B of the other player assuming either that it is also an IP0, or that is a member of the opponent group (GP). This is used to update the total log-odds ratio for that player:L is the current log-odds ratio, L′ is the new log-odds ratio, and ε is the error rate (frequency at which a player’s moves are flipped).After each game, IP0 player is predicted by the infogain model. During groupmax phase, it is predicted by a Hidden Markov Model (HMM) [0 recognizes me as an IP0, and hence cooperates with me”); and pgroupmax . The HMM permits a transition between either of these states with 1% probability per round. At the beginning of groupmax phase, the prior probability of the ALLC state is simply set to the current posterior probability that the other player will classify me as an IP, specifically p(ALLC) = 1/(eL−+1), where L is the log-odds ratio the other player would compute from my moves.During infogain phase, the move expected from an IPel (HMM)  consistip(B∣GP) is computed according to 0 has not yet confidently identified any players as GP (see below for details), then this 0 opponents use the same strategy and could be relaxed for games with more than two types.The conditional probability 0 player classifies another player as confidently GP, based on the p-value of its history of moves under the null hypothesis that it is an IP0 playing infogain moves:n is the number of games it has played vs. that player, e is the number of observed mismatches vs. the expected infogain move (during those games), E is the associated random variable, and ε is the error rate. We used α = 0.01, for at most one expected false positive (in a population of at most 100 IP0). During groupmax phase, an IP player classifies each player according to its current log-odds ratio: as an IP0 if L > 0, otherwise as a GP. Finally, it estimates the total number of IPs currently in the population from its posterior expectation value:Li is its log-odds ratio for the hypothesis that player i is IP0 vs. is a GP . When the IP detects birth of a new player, it initializes the new player’s prior log-odds ratio to L < log α), that player’s outcome counts  are saved for inclusion in future computations of the GP strategy vector During infogain phase, an IP0 is in groupmax phase with at least one player, it computes an optimal strategy to use against the opposing group, based on its estimate of the total number of IP0  is the theoretical long-term score for strategy vector p when playing against strategy vector q. We compute S as previously described by [CC, CD, DC, DD}. The chain has a unique stationary distribution s, and the mean of any four-vector f =  with the stationary distribution for two players p and q is given by the Press and Dyson determinant [f gives the scores that player p would receive for outcomes  respectively. Using this expression, IP0 simply searches the 4-dimensional strategy vector space by gradient descent for the p that maximizes the relative score vs. the opponent strategy If an IPribed by . Brieflyerminant D0 and the simulation code used for this manuscript is available at https://github.com/cjlee112/latude.Our implementation of IP


Rehabilitation following hip arthroscopy is an integral component of the clinical outcome of the procedure. Given the increase in quantity, complexity, and diversity of procedures performed, a need exists to define the role of rehabilitation following hip arthroscopy.(1) To determine the current rehabilitation protocols utilized following hip arthroscopy in the current literature, (2) to determine if clinical outcomes are significantly different based on different post-operative rehabilitation protocols, and (3) to propose the best-available evidence-based rehabilitation program following hip arthroscopy.Per PRISMA guidelines and checklist, Medline, SciVerse Scopus, SportDiscus, and Cochrane Central Register of Controlled Trials were searched.Level I–IV evidence clinical studies with minimum 2-year follow-up reporting outcomes of hip arthroscopy with post-operative rehabilitation protocols described were included.All study, subject, and surgery parameters were collected. All elements of rehabilitation were extracted and analyzed. Descriptive statistics were calculated. Study methodological quality was analyzed using the modified Coleman methodology score.Eighteen studies were included . Labral tear and femoroacetabular impingement were the most common diagnoses treated and labral debridement and femoral/acetabular osteochondroplasty the most common surgical techniques performed. Rehabilitation protocol parameters  were poorly reported. Differences in clinical outcomes were unable to be assessed given heterogeneity in study reporting. Time-, phase-, goal-, and precaution-based guidelines were extracted and reported.The current literature of hip arthroscopy rehabilitation lacks high-quality evidence to support a specific protocol. Heterogeneity in study, subject, and surgical demographics precluded assimilation of protocols and/or outcomes to generate evidence-based guidelines. Strengths and limitations in the literature were identified. Future studies should recognize and report the essentials of rehabilitation following hip arthroscopy. Femoroacetabular impingement (FAI) is a common cause of pain that may lead to osteoarthritis of the hip . Cam andRehabilitation following hip arthroscopy has long been recognized as an integral component of the clinical outcome of the procedure . The widA systematic review was conducted according to preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines using a PRISMA checklist . SystemaSubjects of interest in this systematic review were enrolled in a clinical trial with a minimum of 2 years follow-up following hip arthroscopy (intervention). Specific outcomes of interest regarding post-operative rehabilitation included weight-bearing status, motion, continuous passive motion (CPM), stationary bike, crutches, brace, anti-rotation boots, heterotopic ossification (HO) prophylaxis, and return to sport. Specific surgical outcomes of interest included intra- and extra-articular procedures including arthroscopic femoral osteochondroplasty, pincer acetabuloplasty, labral debridement or repair, loose body removal, articular cartilage surgery, capsular repair/plication or release, iliopsoas release, ligamentum teres debridement, gluteus medius/minimus debridement or repair, iliotibial release or windowing, and greater trochanteric bursectomy. Study and subject demographic parameters analyzed included year of publication, years of subject enrollment, presence of study financial conflict of interest, number of subjects and hips, gender, age, body mass index (BMI), diagnoses treated, and surgical procedures performed. Clinical outcome scores sought were the non-arthritic hip score (NAHS), international Hip Outcome Tool-12 (iHOT-12), hip outcome score (HOS), modified Harris hip score (mHHS), and hip disability and osteoarthritis outcome score (HOOS). Study methodological quality was evaluated using the modified Coleman methodology score (MCMS) . The autp < 0.05 was considered statistically significant.Study descriptive statistics were calculated. Continuous variable data were reported as mean ± SD from the mean. Categorical variable data were reported as frequency with percentages. For all statistical analysis either measured and calculated from study data extraction or directly reported from the individual studies, 2. When present, the mean time from symptom presentation to surgery was 23.1 ± 15 months. Sixty-seven percent of surgeries  were performed in supine position . Mean length of follow-up was 3.2 ± 1.0 years.Eighteen studies were identified for analysis Figure  3, 4, 1–27. Elign = 1,127) and 80%  of hips were diagnosed with FAI and labral tears, respectively. When reported, 67% (n = 634), 5.5% (n = 52), and 28% (n = 28%) were diagnosed with cam, pincer, and mixed FAI, respectively. Other primary diagnoses treated were osteoarthritis , ligamentum teres tear , chondral defects of acetabulum, femur, or both , loose bodies or synovial chondromatosis , and iliopsoas tendon pathology . Labral debridement was the most common surgical technique performed , followed by femoral osteochondroplasty , ligamentum teres debridement , acetabuloplasty rim trimming , labral repair , microfracture of femoral head and/or acetabulum , loose body removal , and iliopsoas release .Fifty-four percent . Study strengths (via MCMS) were length of follow-up, treatment description, and description of rehabilitation protocol. Study limitations were blinding, randomization, number of patients needed to treat analysis, and power analysis and alpha error calculations. MCMS question 13  was adequately described in 4 studies, not adequately described in 14 studies, and not described in 0 studies.Rehabilitation protocols were variably and poorly reported Table . AllowanFour studies Table  recommenClinical outcomes were variably and poorly reported Table . SignifiThe purposes of this systematic review were to determine the current rehabilitation protocols utilized following hip arthroscopy in the current literature, if clinical outcomes are significantly different based on different post-operative rehabilitation protocols, and to propose the best-available evidence-based rehabilitation program following hip arthroscopy. The authors hypothesized that post-operative rehabilitation protocols are infrequently and poorly reported with significant heterogeneity. The authors also hypothesized that there is little to no evidence that supports or refutes specific post-operative rehabilitation protocols and that current protocols are based on theory and biomechanical, rather than clinical, investigations. The study hypotheses were confirmed, thus strengthening the previous assertion by Cheatham et al. that there is a paucity of evidence surrounding post-operative rehabilitation protocols following hip arthroscopy .n = 34) would have also been included , rehabilitation should progress more slowly vs. procedures in which no protection or healing is needed . Avoidance of hip flexor tendonitis is recommended throughout rehabilitation [not only primary hip flexors (iliopsoas) but also secondary flexors ]. Given that the iliopsoas is largely inhibited early after surgery, the activation and over-activation of secondary flexors may occur, thus relegating them to potential inflammatory overuse.Patients undergoing hip arthroscopy are young (mean age 35 years in this review) and active. As such, rehabilitation protocol efficacy should be assessed using patient-reported outcome instruments that are appropriate for use in this patient population. HOS, the International Hip Outcome Tool iHOT-33/iHOT-12), and the Copenhagen hip and groin outcome score (HAGOS) have been recommended to guide therapy progression /iHOT-12,.Return to sport is a very relevant component of the surgical outcome. Too early return may lead to recurrence of pain. Progression through phases of rehabilitation necessitates meeting specific goals and milestones as described above. Passing these thresholds improves the likelihood of safe return to sport. Return-to-sport tests are gaining acceptance in return to play following ACL reconstruction , 36. TheThe limitations of any systematic review are dependent upon the included studies, which it analyzes. Selection bias in this review was minimized by the inclusive nature of study selection. However, bias is also recognized by exclusion of studies with <2 years follow-up. Performance bias was also minimized by the inclusive nature of study selection, allowing all subject diagnoses and surgical treatments available to be included. It is recognized, however, that no study reported subject compliance with rehabilitation, including weight-bearing status, motion restrictions, CPM use, brace or boot use, or return to sports. Heterogeneity in definitions of rehabilitation phases, protocols, goals, precautions, and return to sport variables introduces detection bias. Study design bias is present in the retrospective nature of 17 out of 18 (94%) included studies. Publication bias is present in that the authors excluded medical conference abstracts, non-English language studies, and non-published English language studies.The current literature of hip arthroscopy rehabilitation lacks high-quality evidence to support a specific protocol. Heterogeneity in study, subject, and surgical demographics precluded assimilation of protocols and/or outcomes to generate evidence-based guidelines. Strengths and limitations in the literature were identified. Future studies should recognize and report the essentials of rehabilitation following hip arthroscopy.Shane J. Nho is a paid consultant for Stryker, Pivot Medical, and Ossur; owns stock in Pivot Medical; and receives research support from Arthrex, Linvatec, Smith and Nephew, DJ Orthopaedics, Miomed, Athletico, Stryker, Pivot Medicine, and Allosource. Joshua David Harris is on editorial board for Arthroscopy: The Journal of Arthroscopic and Related Surgery; is a paid consultant for NIA Magellan; and receives royalties from SLACK, Inc. Charles Bush-Joseph is an unpaid consultant for The Foundry and is on the Medical Publications editorial/governing board for the American Journal of Sports Medicine. All other authors have no significant financial conflict of interest. However, the authors confirm that no financial conflict of interest influenced the topic of this manuscript.


These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants ( Each dre.g., nilutamide, OHF) and translocation, especially in prostate cancer cells (LNCaP and C4-2) as compared to breast cancer lines. This was also true when the antagonists were combined with DHT , together with a set of BPA analogs using the platform we described above. BPA and some of its derivatives have been previously shown to affect AR action [i.e., MDV3100) used in combination with DHT.As part of a larger series of studies examining the activity of EDCs on NR activities , we testR action –22, mostR action , 22. TheThe DHT treatment-containing group was primarily comprised of compounds that do not have much impact on antagonizing DHT across cell lines; however, there are few interesting observations. First, when used individually, nilutamide, OHF, and RU486 were present in this cluster indicating they cause similar increases in AR nuclear levels as DHT. RU486 was particularly interesting because its effect upon nuclear AR levels was cell type specific, with no effect in T47D and maximal effect in LNCaP. Two other cell type-specific compounds were identified:1) bisphenol Z (BPZ), a BPX that was antagonistic only in LNCaP and C4-2 prostate cell lines; and, 2) 4-octylphenol (4-OP), which had antagonistic activity only in MCF-7 and LLC2 breast cancer cell lines.Collectively, our experimental interrogation of compound effects upon AR nuclear levels is thus capable of classifying ligands, and is helpful in identifying cell type-specific responses.From the cluster analysis in Figure Because many EDCs are capable of affecting other nuclear receptors, we tested BPAP effects upon modulating the levels of several endogenous NRs  in breast cancer cells Figure . As expeBecause the total AR protein in 22Rv1 is comprised of both a mutated full-length AR and constitutively active AR splice variants, we sought to determine if BPAP was capable of reducing all AR species across AR variant expressing cell lines . For thivs. LNCaP cells by immunofluorescence. The results indicate that whereas BPAP had an IC50 of ~15 μM in 22Rv1, no activity was apparent in LNCaP cells. Time-course analysis showed that, by 8 hrs, AR protein levels are already significantly reduced in 22Rv1 cells, while the 24 hrs time point showed the largest response.We then performed a six-point dose-response Figure  and timeNext, to further validate the effect of BPAP on AR and AR-V7 in CRPC models, we performed immunoblot analysis of whole cell extracts of LNCaP  . As shown in Figure g., EDN2 ) and oth) Figure . As expei.e., up to 72 h, Figure 50 , and that cell cycle arrest may be the main initial mechanism of action. Signs of cell death became more evident after 48 hrs or by increasing BPAP concentration above 20μM.We next determined the effect of BPAP upon the cell cycle and growth of 22Rv1 cells. We treated cells with different concentrations of BPAP for 24 hrs, pulsed them briefly with EdU to identify which cells are actively engaged in DNA synthesis, and then performed dual labeling with Click-iT EdU kit and anti-AR antibody . Figure We then performed a 21-day soft-agar anchorage-independent growth assay and found that treatment with BPAP greatly reduced the number of colonies as determined by crystal violet staining Figure ; of noteIn conclusion, we have identified BPAP as a novel AR down-regulator in CRPC cells that causes cell cycle arrest, and time- and dose-dependent reduction in cell number and soft-agar colony formation.Because of the cell cycle arrest and cell number reduction caused by BPAP, and because it is affecting AR levels, a major player in prostate cancer cell metabolism –27, we qin vitro. BPAP causes cell cycle arrest and metabolic changes in keeping with its action as a down-regulator of AR. This endocrine disruptor thus represents an interesting new compound with potential for structure/activity studies to develop new lead candidate moieties for CRPC.In conclusion, utilizing HTM/HCA, we have identified the Bisphenol A analog, BPAP, as a novel molecule capable of reducing the levels of AR (both full-length and variants) in CRPC tumor cell models The Androgen Receptor (AR) has been the top drug target in prostate cancer for decades, as it plays central roles in the development and progression of such tumors , 5, 28. endogenous AR, without the need for engineered cell systems. After demonstrating the capability of this multi-cell line platform for classifying known AR antagonists and determining their cell specific activities, we tested a small number of endocrine disrupting compounds, thus beginning to exploit the highly varied chemical space derived from the chemical industry, a largely untapped resource for drug discovery. AR is a well known endocrine disruptor target ; furthermore, a BADGE (Bisphenol A Diglycidic Ether) marine sponge metabolite (EPI-001) has been shown to be effective in reducing CRPC growth via non-canonical mechanisms [In this study, we were interested in utilizing a single cell–based high throughput microscopy platform to characterize modulators of AR levels and localization by comparing the effects of small molecules on endogenous AR across six prostate and breast cancer cell lines. Importantly, this effort was designed to study the effects of compounds on chanisms .In our primary screen, we identified several EDCs with androgenic or anti-androgenic activity (at least in terms of AR nuclear levels and stabilization), however, one BPA analog, Bisphenol AP (BPAP), stood out in our analysis as it caused marked reduction of AR levels specifically in 22Rv1 cells. BPAP is commonly used as a plasticizer and a component of flame retardants in many industrial products, and is a bona fide environmental contaminant, especially in wastewater .50~20μM) and would require SAR experiments to optimize its potency and possibly selectivity  or a protein degradation mechanism (and highlighting that BPAP blocks AR gene transcription). While BPAP is not a very potent compound , it wasIn conclusion, we developed a high throughput, microscopy based platform to classify the effect of compounds on endogenous AR and we identified BPAP, a bisphenol A analog, as a molecule that is capable of causing AR (both full-length and variants) down-regulation in CRPCs resulting in cell cycle arrest and metabolic defects. Further studies will be needed to fully unravel BPAP mechanism(s) of action and therapeutic potential via SAR studies.Cell lines  were cultured as described by ATCC. Cells were plated either on optical quality 384-well glass bottom plates  or plastic bottom plates  using a TitreTek robot . LNCaP95 were a kind gift from Dr. Stephen R. Plymate (University of Washington) and GFP-AR-V7:PC-3 were obtained from Dr. Marco Marcelli (Baylor College of Medicine), single cell cloned and used as in . After 2Cells were fixed in 4% paraformaldehyde for 30 minutes on ice and quenched with 0.1 M ammonium chloride. Permeabilization was performed using 0.5% Triton-X100 for 30 minutes. Cells were blocked with 5% milk in TBS-T buffer and then incubated at 4°C overnight with the following antibodies: AR  and AR-V7, . Following 3x washes, cells were incubated for 30 minutes with Alexa Fluor dye-tagged secondary antibodies  at room temperature. Cells were washed and a post-fixation step was performed. Finally, cells were incubated for 5 minutes with 1 μg/ml DAPI. All immunolabeling in multi-well plates was carried out robotically using a BioMek NX instrument .EdU staining, after a 5 minute pulse, was performed following manufacturer's instructions . For mitochondria staining we used Mitotracker Red CMX , cells were exposed to the dye for 30 minutes. JC-1  treatment was performed in live cells following manufacturer's instructions.384 well plates were imaged on an IC200  or InCell 6000  high throughput microscope, using 40X/0.95 NA lenses. Images were background subtracted using a rolling-ball method and segmented using the DAPI channel applying a locally adaptive mean threshold. Objects that had an area of <20 pixels were considered noise and discarded. A distance transform was applied to the nuclear objects and used in a seeded watershed routine to split touching nuclei and produce the final nuclear masks. Approximate cell masks were obtained by dilating a circular region around the nuclei. Regions with cell masks touching the edge of the image were discarded. Finally, a set of morphological and intensity based measurements , 18 wereThe metabolic phenotyping was performed as previously reported . Briefly


NCI-H292 epithelial cell line was used to determine the contribution of β2-AR signaling to CSE-induced MUC5AC production by treatment with β2-AR antagonists propranolol and ICI118551 and β2-AR-targeted small interfering RNA. The effect of propranolol on airway mucus hypersecretion was examined in a rat model exposed to CS. MUC5AC expression was assayed by real-time PCR, immunohistochemistry and ELISA. β2-AR and its downstream signaling were detected by western blot analysis. We found that pretreating NCI-H292 cells with propranolol, ICI118551 for 30 min or β2AR–targeted siRNA for 48 h reduced MUC5AC mRNA and protein levels stimulated by CSE. However,inhibiting the classical β2AR–cAMP-PKA pathway didn’t attenuate CSE-induced MUC5AC production, while silencing β-arretin2 expression significantly decreased ERK and p38MAPK phosphorylation, thus reduced the CSE-stimulated MUC5AC production. In vivo, we found that administration of propranolol (25 mg kg−1d−1) for 28 days significantly attenuated the airway goblet cell metaplasia, mucus hypersecretion and MUC5AC expression of rats exposed to CS. From the study, β2-AR–β-arrestin2–ERK1/2 signaling was required for CS-induced airway MUC5AC expression. Chronic propranolol administration ameliorated airway mucus hypersecretion and MUC5AC expression in smoking rats. The exploration of these mechanisms may contribute to the optimization of β2-AR target therapy in chronic obstructive pulmonary disease.Progression of chronic obstructive pulmonary disease is associated with small airway obstruction by accumulation of inflammatory mucous exudates. However, the mechanism of mucin hypersecretion after exposure to cigarette smoke (CS) is still not clear. In this study, we explored the contribution of β The normal mammalian airway epithelium produces and is coated by mucins such as MUC5B and MUC5AC, which protect the airway epithelia against exogenous insult. However, excessive epithelial goblet cell metaplasia and mucus hypersecretion contribute to the pathology of many respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis. In COPD, mucin hypersecretion contributes to airway obstruction, accelerated decline of lung function and increased hospitalization and mortality in vitro and in vivoCigarette smoking is the primary cause of COPD and induces the expression and production of MUC5AC 2-adrenoceptor (β2-AR) is the most common adrenergic receptor subtype expressed in various types of lung cells, including airway epithelial cells. However, the role of β2-AR in airway mucus hypersecretion is still unknown. In an asthma murine model, the β-agonist formoterol inhibits airway goblet cell hyperplasia and MUC5AC protein expression, whereas administration of other β-agonists fails to improve mucus metaplasia 2-AR signaling has been proven to be required for the full development of 3 cardinal features of asthma: mucous hypersecretion, airway hyperresponsiveness and the presence of inflammatory cells in the lungs and chronic administration of ICI118551 or nadolol reduced inflammation and mucous metaplasia, that may contribute to airflow obstruction and airway hyperresponsiveness of asthma2-AR is involved in regulating lung inflammation and mucus secretion.β2-AR may be involved in the development of pathological changes associated with smoking. In the present study, we hypothesized that β2-AR signaling is required for airway MUC5AC expression and β-blocker treatment could improve CS-induced mucus hypersecretion by attenuating MUC5AC production. We explored the contribution of β2-AR and downstream signaling to MUC5AC expression in NCI-H292 cells, a mucin-expressing cell line from human airway epithelia, stimulated by CS extract (CSE) which is commonly used to mimic the effects of CS. We further examined the effect of chronic propranolol administration on MUC5AC production in rats exposed to CS.Cardiovascular disease is the most common co-morbidity of COPD because of the risk of smoking-induced atherosclerosis 2-AR antagonist), H89 (PKA inhibitor) PD98059 (ERK inhibitor) and SB203580 (p38MAPKK inhibitor) were from Sigma-Aldrich . Rp-8-Br-cAMPs and primary antibodies (Abs) for PKA, β-arrestin1, β-arrestin2, GAPDH and mouse or rabbit horseradish peroxidase-conjugated secondary Abs were from Santa Cruz Biotechnology . Primary Abs for phospho-PKA, extracellular signal-regulated kinase 1/2 (ERK1/2) and phospho-ERK1/2 were from Cell Signaling Technology . Primary Abs for MUC5AC and β2-AR were from Abcam .CSE, used to mimic the effects of CS, was purchased from Murty Pharmaceuticals  and was prepared using a Phipps-Bird 20-channel smoking machine designed for FTC testing −1 each), at 37°C in a humidified 5% CO2 atmosphere and subcultured twice weekly. Confluent cells were incubated in serum-free DMEM for 12 h, then washed 3 times with 10% phosphate buffered saline (PBS), placed under serum-free conditions and exposed to the indicated concentration of CSE and inhibitors. Control cells were incubated with medium alone for the same period. After 24-h stimulation, samples of a cell-conditioned medium and/or cell lysates were collected by brief centrifugation and stored at −80°C. Most experiments, except for cell viability assays (96-well microplate), were involved 6-well plates or 35-mm dishes  with 1×106 cells/well.NCI-H292 cells, a mucin-expressing cell line from human airway epithelia, were obtained from the Cell Resource Center . Cells were maintained in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin  as in This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Peking University (permit No.: LA2010-004). We housed 32 male Sprague-Dawley rats (7 weeks old) in divided cages (4 each) under specific pathogen–free conditions of the Animal Center of Peking University Health Science Center, Beijing. Animals were raised at room temperature with a natural light-dark cycle and had free access to food and water. All rats were anaesthetized by 20% uratan  for surgery, overdose of uratan for euthanasia, and all efforts including comfortable bedding, improved surgical intubation and standard surgical procedure, were made to minimize suffering. From weeks 1–16, rats in Group S and Group S/P were exposed to whole-body cigarette smoke in inhalation chambers  generated from commercial cigarettes  at 20 cigarettes’ inhalation for 2 h followed by a 4-h recovery, repeated twice a day. Rats were exposed to CS 6 days per week. The study protocol was showed in details as −1), then respiration was maintained by a computer-controlled small-animal ventilator connected to the rat through a tracheal cannula. Respiratory function was measured as described Rats were anesthetized by intraperitoneal injection of 20% urethane  were counted by use of a Coulter counter. The remaining BALF was centrifuged  and supernatants were stored at −80°C. The left lobe of the lung was preserved in 4% paraformaldehyde for histology. Tracheal samples 2–4 mm in diameter excised from the trachea were preserved in 3% glutaric dialdehyde for scanning electron microscopy and transmission electron microscopy. The right posterior lobe of the lung was preserved in liquid nitrogen for mRNA and protein analysis.Tissues were embedded in paraffin and stained with hematoxylin and eosin or periodic acid-Schiff (PAS). Images were acquired before any measurements and analyzed by a pathologist blinded to treatment. Pathological changes in small airways (≤2 mm in diameter) were assessed by inflammation scores as described Tracheal samples 2–4 mm in diameter were excised from the right principal bronchus of 3 rats under urethane anesthesia, taking care to avoid trachea cannula damage. Samples of untreated control mucosa were immediately immersed in the appropriate fixative as described and sent to the cytology and pathology labs of Peking University Health Science Center for conventional electron microscopy examination. Specimens were coated with gold-palladium in a Hummer II sputter-coating apparatus and examined under a JEOL JSM-5600LV scanning electron microscope. For transmission electron microscopy, mucosal slices were examined under JEOL JEM-1400 electron microscopes.After paraformaldehyde fixation, paraffinized 5-µm sections of lung tissue underwent immunohistochemical staining. For MUC5AC, the primary antibody was mouse polyclonal anti-MUC5AC . After incubation at 4°C overnight, the biotinylated secondary goat anti-mouse antibody was applied (1∶500) for 30 min at room temperature and DAB chromogen was applied for 1 min.−ΔΔCTTotal RNA was extracted from frozen lung tissues or cultured NCI-H292 cells by the Trizol method (Takara Bio Inc. Japan). Real-time PCR involved use of Lightcycler480  with SYBR Green assay after reverse-transcribing 1 µg RNA by use of the Reverse Transcription System . All data were normalized to mRNA levels of GAPDH. The primer pairs and expected lengths are in At 24 h after NCI-H292 cells were stimulated with CSE, cell culture media was collected for determining MUC5AC concentrations by use of an ELISA kit . Total mucin and MUC5AC level in BALF was assayed by use of rat mucin and MUC5AC ELISA kits .4P2O7, 10% glycerol, 0.1 M NaCl, 5 mM EDTA, pH 8.0, 0.1% SDS, 0.02 M Tris-HCl, pH 7.4, 0.05 NaF, 1 mM Na3VO4, 100 µg ml−1 phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin). Cell lysates underwent protein determination by use of a Pierce BSA protein assay kit . Proteins (50 µg) were loaded onto 10% SDS polyacrylamide gel and electrophoretically transferred to nitrocellulose membrane  that was incubated with antibodies according to the manufacturer’s protocol; immunolabelled bands were visualized by use of the SuperSignal West Pico chemiluminescence kit . Autoradiographs were quantified by densitometry by use of Image J software. Bands were normalized to GAPDH expression.Cells were harvested and lysed with lysis buffer .Data are presented as mean ± SEM. One-way ANOVA and Newman-Keuls multiple comparison test were used for normally distributed data as appropriate. A t-test was used to compare two different groups of treatment to each other. For multiple comparisons, two-way ANOVA was used. 2-AR antagonists propranolol or ICI118551 with or without CSE stimulation to explore the contribution of β2-AR to the CS-induced airway MUC5AC production. CSE at 0.1 mg ml−1 increased MUC5AC mRNA level and protein secretion in cells, and preincubation with antagonists reversed the increase , allowing it to bind to CRE sites2AR–cAMP–PKA pathway, we pretreated cells with the cAMP analogue Rp-8-Br-cAMPs and PKA inhibitor H89. Inhibiting the cAMP–PKA pathway did not decrease the CSE-induced MUC5AC production (2AR–cAMP–PKA independent.βoduction . Therefo2-AR is a well-known G protein–coupled receptor (GPCR) and has many potential sites of phosphorylation. β-arrestin can uncouple the receptor from G protein, thus terminating desensitization and internalization of the receptor 2-AR activation induced airway epithelial MUC5AC production by CSE via a β-arrestin–MAPK pathway. We pretreated cells with 2 sets of β-arrestin-targeted siRNA, β-arrestin1 siRNA and β-arrestin2 siRNA. CSE-stimulated MUC5AC expression was significantly decreased in cells pretreated with β-arrestin2 siRNA but not β-arrestin1 siRNA . Among the 4 lung function indexes, propranolol decreased the peak expiratory flow, intra-airway pressure and intra-airway pressure slope . As compared with air-exposed rats, CS-exposed rats showed increased airway mucus, and propranolol treatment significantly decreased airway luminal mucus accumulation and total mucins in BALF . 3) Propranolol inhibits β2-AR downstream signaling molecules. β2-AR signaling was found required for the full development of mucous metaplasia, and inhibition of all β2-AR signaling but not biased agonism was responsible for the beneficial effects of chronic inverse-agonist treatment on mucous metaplasia 2-AR is the most common β-AR subtype expressed in various cell types in the lung including airway epithelial cells. β2-AR level has been found increasing along the human airway, and the density in the subsegmental bronchus and lung parenchyma was approximately two-fold higher than that of muscarinic acetylcholine receptors in the same region. β1-AR was also detected in lung parenchyma but not in the bronchus, which suggests the inhibition was mainly mediated through β2-AR We further established a rat model with a prominent feature of airway inflammation and mucus secretion by CS exposure and evaluated after chronic low-dose propranolol intervention. Propranolol, one of non-selective β-blockers, is the most commonly prescribed classes of cardiovascular medication. Despite increasing evidence that BBs are safe and can actually be beneficial in patients with COPD, their use in this population is limited 2-AR 1-AR to MUC5AC production is not involved here. However, our previous study found that treatment with metoprolol, a β1-AR selective antagonist, did not improve airway mucus hypersecretion in a rat model of CS exposure (data not shown). The protective effect of propranolol in the rat model exposed to CS may be mainly achieved by inhibiting β2-AR signals. 3) In vitro systems usually lack complex interactions with other cell types and the disease process can be affected by the complex cytokine milieu established by inflammatory cells in addition to CS exposure. Thus, the findings in vitro may not exactly reflect responses in vivo.Our study contains some limitations. 1) Mainstream CS is a complex mixture inhaled into the respiratory system. Approximately 4,700 substances have been identified in fresh tobacco smoke. Many factors influence CS-stimulated phosphorylation of ERK1/2, such as epidermal growth factor receptor, toll-like receptor or NADPH oxidase, and interactions between these factors and β2-AR activation on airway mucus hypersecretion. Some pathological signals of β2-AR are initiated under CS exposure and proper application of β-blockers may, to a point, benefit airway disease. The exploration of these mechanisms may contribute to the optimization of β2-AR target therapy in smoking associated airway disease, like COPD.In conclusion, our findings indicate a detrimental effect of βFile S1Dose- and time-dependent effect of CS extract (CSE) on cell viability and MUC5AC production from NCI-H292 cells.(DOCX)Click here for additional data file.File S22-siRNAs.Transfection and knockdown efficiency of β(DOCX)Click here for additional data file.File S3The body weight of rats in all 3 groups at different time.(DOCX)Click here for additional data file.File S4Effect of propranolol on pulmonary pathological parameters in rats exposed to cigarette smoke.(DOCX)Click here for additional data file.File S5Effect of propranolol on lung function.(DOCX)Click here for additional data file.


The androgen receptor (AR) pathway plays a central role in prostate cancer (PCa) growth and progression and is a validated therapeutic target. In response to ligand binding AR translocates to the nucleus, though the molecular mechanism is not well understood. We therefore developed multimodal Image Correlation Spectroscopy (mICS) to measure anisotropic molecular motion across a live cell. We applied mICS to AR translocation dynamics to reveal its multimodal motion. By integrating fluorescence imaging methods we observed evidence for diffusion, confined movement, and binding of AR within both the cytoplasm and nucleus of PCa cells. Our findings suggest that in presence of cytoplasmic diffusion, the probability of AR crossing the nuclear membrane is an important factor in determining the AR distribution between cytoplasm and the nucleus, independent of functional microtubule transport. These findings may have implications for the future design of novel therapeutics targeting the AR pathway in PCa. The androgen receptor (AR) regulates genes involved in the development and maintenance of the male phenotype, and also plays a role in the growth and survival of prostate cancer (PCa). AR is a ligand-activated transcription factor belonging to the class I subgroup of the nuclear receptor superfamily3The interactions of AR with DNA and protein, as well as molecular crowding and intracellular structural barriers, can give rise to complex kinetic behavior of AR in a ligand-dependent manner. Alteration of AR nuclear/cytoplasmic localization and of its interactions with other molecules are tightly associated with PCa growth, progression, and resistance to therapy811121314Recently developed microscopy techniques have greatly improved our understanding of nuclear receptor kinetics, including AR451617181918194Imaging correlation spectroscopy (ICS) is an attractive approach to probe intracellular dynamics at wide spatiotemporal scales. It works well with genetically encoded fluorescent proteins, and requires minimum perturbation of the biological system. We specifically applied raster image correlation spectroscopy (RICS)22While RICS provides isotropic diffusion information, to further investigate the anisotropic as well as slower component of AR dynamics, we needed an extra dimension. For this purpose, we developed a novel technique that we named multimodal Image Correlation Spectroscopy (mICS) to extract the isotropic as well as anisotropic AR motion from image stacks by fitting correlations to a two-component function. mICS analysis is based on spatiotemporal image correlation spectroscopy (STICS)252628293031In the present study, we quantitatively measured AR kinetics across the cell and the changes due to agonist ligand binding by combining RICS and mICS analysis. We observed diffusion, confined movement, and binding of AR within both cytoplasm and nucleus. Combining our experimental data with simulations, our findings suggest that with the presence of diffusion in the cytoplasm, the probability of AR crossing the nuclear envelope is a key factor in determining AR translocation, independent of the presence of functional direct transport in the cytoplasm. This suggests that taxane-induced AR translocation blockade may involve mechanisms other than arresting dynein-mediated AR transport.We used a clonal PC3 cell line stably expressing GFP-AR as our model system to study AR kinetic changes due to the presence of AR agonists. PC3 is a metastatic human prostate cancer cell line with low AR expression, and was favored in this study to limit interference from non-fluorescent endogenous AR. To verify the functionality of our model system, we analyzed AR expression levels and AR-dependent gene transcription in our cell line. Western blot quantification revealed a 2-fold overexpression of AR in our PC3 GFP-AR cell line compared to endogenous AR within the LNCaP cell line see , suggest3p < 0.02 for nucleus and rp < 0.14 for cytoplasm, with no identifiable correlation peak). We conclude that the majority of GFP-AR was not bound to microtubules and was likely free to diffuse throughout the cytoplasm. The relatively uniform distribution of AR persisted over the course of agonist-stimulated AR translocation, as shown in AR and microtubules were previously shown through fixed cell immune-staining to have partial co-localization2/s. There was also no significant difference in AR diffusion rates in the cytoplasm or nucleus To measure the diffusion coefficient of GFP-AR, we applied RICS within the cytoplasm and nucleus at different stages during AR translocation. AR diffusion coefficients showed no significant changes during the course of Cl-4AS-1 treatment  resulted in weak correlation, whereas slower diffusion gave rise to the isotropic component in the correlation image, and the amplitude of this isotropic component decreased with longer delay time. We further demonstrated the correlation behavior with free GFP diffusion in the cell, fluorescent beads diffusing in glycerol solution, and Hoechst dye that strongly bound to DNA in the nucleus To test whether the GFP-AR anisotropic motion in the nucleus after agonist treatment could be explained by its binding to a structure (DNA) that moves at a time scale detectable by mICS, we simultaneously acquired signals from GFP-AR, Hoechst (to indicate DNA location), and NLS-RFP . Chromat40Based on our observation of diffusive AR, AR binding, as well as previously reported AR microtubule transportTo compare with simulation results, we generated a kymograph from the nuclear envelope to the membrane using the same dataset shown in 33in vitro experiments resulted in a relatively delayed AR concentration change. We then simulated a varying nuclear permeability that increased linearly with time  and the distinct response of agonist ligand binding. In the present study, we implemented different imaging techniques at several spatiotemporal scales to assess AR intracellular motion, and integrated them with simulations to provide a dynamical picture of AR translocation at the molecular scale. We detected the existence of both diffusing and binding components of AR, as well as the increased binding and confined motion in the presence of agonist ligand. We estimated that RICS and mICS capture roughly 65–75% of AR population, whereas the rest of the population is relatively immobile within the measurement time frame (~1 minute) see .In this article we highlighted the existence of multimodal AR kinetics in prostate cancer using a model system. The PC3 cells we used lack endogenous AR and originate from metastatic neuroendocrine cancer. Thus there are caveats in generalizing these results to clinical disease. Importantly, using additional cell lines that correspond to different stages of prostate cancer  is warranted. Adding to the complexity, even the widely used AR + prostate cancer cell line LNCaP may have multiple AR genetic variants, and its androgen responsiveness is sensitive to passage number and culture conditionsIn addition to the conventional confocal imaging, the ICS methods demonstrated in this work can be applied to study a broad array of biological systems with minimum perturbation and routine fluorescent sample preparations. The technique is particularly useful when the labeled particles under study are too dim or too dense to be individually tracked, and can capture motions of different natures. The mICS analysis we developed not only allows us to retrieve the isotropic component more accurately, but we can also decipher the biophysical origins of the anisotropic component. Using a confocal microscopy system, the spatiotemporal resolution that can be achieved with mICS is determined by two major factors: (1) the fluorophore brightness and photon collection efficiency that dictate the pixel dwell time, which limits the speed of acquisition, and (2) spatial coverage and desired pixel resolution, which dictate the number of pixels that need to be acquired. Going forward, implementing camera-based fast acquisition and multi-color acquisition can potentially offer a more detailed view of the interaction of AR with different cellular components, with higher spatiotemporal resolution.et al.et al. reported both fast and slow components  of the retinoic acid receptor (RAR), a structurally similar nuclear receptor, in the nucleusUsing multiple fluorescence microscopy assays, Van Royen However, we observed neither evidence of dominant AR-microtubule binding nor significant direct transport in the cytoplasm, despite previous reports of AR association with dynein and increased AR-dynein interaction upon ligand induced AR nuclear translocationet al.4445Although both treating with dynein inhibitor and taxanes showed some degree of AR translocation blockade  and Penicillin- Streptomycin . Cells were maintained at 37 °C in a humidified incubator with 5% carbon dioxide. Prior to imaging, cells were seeded on 35 mm glass bottom dish coated with poly-D-lysine (MatTek). Cells were switched to Phenol red free RPMI  media supplemented with charcoal:dextran stripped FBS and L-Glutamine the night before imaging.PC3, HeLa, and LNCaP cell lines were obtained from ATCC. All were maintained in RPMI1640  supplemented with 10% heat-inactivated GemCell® pLKO.1-puro-CMV-TurboGFP™ Positive Control Transduction Particles  to transduce cells, followed by FACS. GFP-AR transient transfections of HeLa cells were performed using pEGFP-C1-AR plasmid and FuGENE one day before experiments.To produce the PC3 GFP-AR stable cell line, PC3 cells were transfected with the pEGFP-C1-AR plasmid )Live cell microtubule labeling was done by adding 100 nM SiR-tubulin (Spirochrome) to the dish and incubating overnight. NLS-RFP transfection was done by adding 15 μl CellLight Nucleus-tagRFP BacMam 2.0 (Life Technologies) to the dish and incubating overnight. Hoechst 33342 (Thermo Fisher) was added 30 minutes prior to imaging at final concentration of 1 μg/ml. The synthetic AR agonist Cl-4AS-1 (Tocris) was applied to cells at final concentration of 20 nM; Ciliobrevin A (Tocris) was applied at final concentration of 10 nM; Paclitaxel  was applied at final concentration of 1 μM.2 level, and humidity throughout the experiment. Images were obtained using 63 × 1.15 NA water immersion objective mounted on Zeiss LSM-780 inverted confocal microscope (Carl Zeiss). Hoechst signal was excited at 405 nm and collected at 407–485 nm; GFP signal was excited at 488 nm and collected at 495–557 nm; NLS-tagRFP signal was excited at561 nm and collected at 567–735 nm; SiR-tubulin signal was excited at 633 nm and collected at 637–753 nm.Cells were maintained on the microscope stage in an environmental chamber with controlled temperature, CODepending on the spatial and temporal resolution required, acquisition parameters were adjusted accordingly: For cellular level translocation, 512 × 512 pixels were acquired for over 100 frames at the speed of 15 to 20 s/frame. For RICS data acquisition, 128 × 128 pixels were acquired for 100 to 200 frames with pixel size of 80 nm and pixel dwell time of 6.3 μs; images were acquired continuously. For mICS data acquisition, 256 × 32 pixels were acquired for 1000 frames with pixel size of 100 nm and pixel dwell time of 1.27 μs; images were acquired continuously with frame rate of 49.6 ms. For comparing the effect of Ciliobrevin A and paclitaxel, 3 × 3 tile scans of 512 × 512 pixels (415 nm/pixel) were acquired.For FRAP experiment, a total of 30 pre-bleach images were acquired , followed by a single square photobleach (100% ATOF). 80 frames were acquired immediately after photobleaching. The immobile fraction was determined as described in Day and SchaufeleCell lysates containing 20 μg of protein were separated by 10% SDS–PAGE  and transferred to PVDF membranes . The blots were probed with polyclonal antibodies against AR (H-280) . Horseradish peroxidase-linked sheep antibody against rabbit IgG  and mouse IgG  were used as secondary antibodies. The signal was detected using enhanced chemiluminescence . Quantifications were performed using ImageJ 1.46r.® 96 Microplate Luminometer (Promega).Reporter gene assays were accomplished with the constructs pGL4.72 ARE2 TATA, kindly provided by Andrea Koehler and Helmut Klockerp) was calculated according to Steensel et al.51For each value of pixel shift (x), Pearson’s correlation coefficient . In brief, we first temporally high-pass filtered the images by subtracting a moving average of 10 frames from the time lapse images to remove slow motion that may be due to cell movement. Then we computed the intensity-normalized spatial correlation within each frame using:The detailed steps for performing RICS data acquisition and analysis was performed as previously describedI is the intensity, ξ and ψ are the spatial increments in the x and y directions respectively, and the angle bracket denotes an average over all pixels. We averaged these image correlations across all frames and then fit this average to a 2D diffusion model to retrieve the diffusion coefficient. The RICS model is formulated as:Here RICS is broken up into two parts: G, which is related to molecular dynamics, and S, which is related to scanning optics, plus the background term b. N is the number of particles in the focal volume; γ depends on the shape of the illumination volume; D the diffusion coefficient; τp the pixel dwell time; τl the time between lines; δr the pixel size; and ω0 the beam waist.Here Get al.An overview of mICS analysis is shown in i) is the spatial average intensity at t = ti.where f. We fitted the resulting correlation image for each lag time with a two-component (isotropic + anisotropic) Gaussian mixture model including a spatially uniform noise term ε:where ξ and ψ are the x and y spatial increments respectively, and the angle bracket indicates the average over all available spatial locations in both x and y directions. wherewithThis model includes an anisotropic term that is not needed if the motion is actually isotropic. Thus we first determined whether the data are well fit by the purely isotropic model. We performed a one-sided likelihood ratio test between model fits with and without the anisotropic term included. If the likelihood ratio test p-value is less than 0.05 we included the anisotropic component in the model. Otherwise we defined the motion as isotropic.2) as the goodness of fit. Data for model fits with R2 ≤ 0.1 were typically without spatial structure and we considered them to be noise 2 > 0.1 we proceeded to evaluate the amplitudes of the Gaussian components (a1 and a2). We displayed a model component (isotropic or anisotropic) only if its corresponding amplitude is greater than 10−4.We then assessed the maximum likelihood model’s fit and the correlation magnitude to decide whether the correlation function had a significant Gaussian component. We applied two criteria. First, we used the coefficient of determination . The software is available upon request.improfile to extract the intensity profile from edge of nucleus to edge cell. Repeating the operation on different time points produces the intensity profile as a function of time.Intensity profiles are extracted from a time-lapse image of 32.5 minutes . A Matlab script uses the function We devised a 1-dimensional Monte-Carlo simulation for AR cytoplasm to nucleus translocation to better understand the role of various molecular kinetics and nuclear permeability.At time 0 (in the environment of no ligand presence), we assume all AR is uniformly distributed within cytoplasm. The distance from cell membrane to nucleus was set to 50 μm. A total of 500 AR were simulated for 1800 seconds at time step equals to 1 second. We assume in the cytoplasm, at each time point AR can either choose diffusion, stand still How to cite this article: Chiu, C.-L. et al. Intracellular kinetics of the androgen receptor shown by multimodal Image Correlation Spectroscopy (mICS). Sci. Rep.6, 22435; doi: 10.1038/srep22435 (2016).


Allergic rhinitis (AR) is the most common allergic disease but little is known about the difference of local immune responses in children and adults with AR.To compare local immune responses between children and adults with AR and nonallergic rhinitis (NAR), and to investigate whether the association of local and systemic immune responses is different between the two age groups.Fifty-one patients with chronic rhinitis were enrolled and grouped into children  and adults . Diagnosis of AR was based on symptoms, skin prick tests and serum specific IgEs. Nasal lavage (NAL) fluids were collected from all subjects and used to measure the levels of total IgE, specific IgEs to house dust mites (Dp and Df), and cytokines . Flow cytometry was used to measure inflammatory cell types in NAL fluids.P < 0.05). Nasal eosinophils % (P = 0.01) was significantly increased only in children with AR. Local-systemic correlations of total IgE  and eosinophil %  between the peripheral blood and NAL fluids were found only in children. Moreover, children had correlations between total IgE and eosinophil % in the peripheral blood  and in NAL fluids .AR had significantly increased local levels of total IgE and specific IgEs to Dp and Df compared with NAR in both age groups (Elevated local IgE is a common feature of AR in children and adults. Local measures in NAR showed naïve state of immune response which disagree with the hypothesis of local allergic rhinitis. Children showed intense local inflammation and close local-systemic interactions compared to adults supporting pediatric AR as a distinct feature. Allergic rhinitis (AR) is the most common allergic disease with a high economic burden . AR has AR can be diagnosed by history and clinical symptoms, and through the demonstration of specific IgE to common inhalant allergens by skin prick tests (SPTs) and in vitro assays . When thNasal lavage (NAL) is a non-invasive procedure to collect nasal secretions, which provides information on local nasal immunity in patients with rhinitis. House dust mite-specific IgE was observed in the nasal secretions of adults with NAR as well as those with AR . The finAR can develop during early childhood and the prevalence decreases with age , 14. AllThe aims of this study were to compare local immune responses between children and adults with AR and NAR, and to investigate whether the association of local immune response with systemic parameters is different with aging process.We enrolled 51 consecutive patients (27 children and 24 adults) with AR or NAR between October 2013 and February 2014. Exclusion criteria included chronic rhinosinusitis , fungal sinusitis, asthma, aspirin intolerance, history of nasal surgery, and paranasal sinus tumors. Adults  were comorbid with septal deviation and children  were comorbid with adenotonsillar hypertrophy. None of the subjects used antibiotics or nasal corticosteroids within 4 weeks.All patients had rhinitis symptoms, including nasal obstruction, rhinorrhea and sneezing, and underwent assays for serum total IgE and complete blood count (CBC). SPTs were performed to determine their sensitivity to commonly inhaled allergens and the reaction was regarded as positive if the mean wheal diameter was 3 mm or greater . The dia≥ 13 years and children aged 7–12 years to understand objects, benefits, and harms of this study. And we obtained informed consents from both children and their parents. We used questionnaire reporting their nasal symptoms  with four-point scale  [Two Consent forms were prepared for children aged  severe) . The totNasal lavage was performed as described previously . BrieflyNon-specific antibody binding was blocked by incubating cells with TruStain FcX  on ice for 15 minutes. The cells were subsequently incubated with monoclonal antibodies  on ice fDermatophagoides pteronyssinus; Dp and Dermatophagoides farinae; Df) and superantigens  in NAL fluids. To monitor the allergic response, we also measured eosinophil cationic peptide (ECP) in NAL fluids. All analyses were performed using the ImmunoCAP system . IgE concentrations were expressed as kU/L and ECP as μg/L [To evaluate local antibody production, we measured total IgE and specific IgEs for house dust mites , IL-10, IL-17A, interferon-γ (IFN-γ) , and tumor necrosis factor-α  were measured in NAL fluids using commercially available ELISA kits, according to the manufacturers' protocols.P < .05) was analyzed using the Mann-Whitney U test. The relationships between systemic and local allergic parameters were examined using Spearman correlation analysis. Receiver-operating characteristic (ROC) curve analysis was used to determine the AR cutoff in adults and children with optimal sensitivity and specificity. All statistical analyses were performed using Graph-Pad Prism ver. 5.0  and IBM SPSS ver. 21.0 .Because of the relatively small sample size and the non-normal distribution, continuous variables are presented as medians with/without an interquartile range instead of as means with/without standard deviations. Statistical significance (P = 0.011) but older in children (P = 0.043) than their respective NAR group. Sex (%), TNSS, and QOL were not significantly different between AR and NAR in either age group. House dust mites (Dp and Df) were the most common allergen in children (100%) and adults (73.3%) with AR. There was no correlation between TNSS and nasal IgE levels regardless of rhinitis types and age groups (not shown). The volumes of NAL fluids recovered after washing the nasal cavity with 10 mL of saline were not significantly different among the groups.The clinical characteristics of the study subjects are summarized in P = 0.016) and blood eosinophil % (P = 0.001) were significantly increased in AR compared to NAR in children  and a decrease in macrophages (P = 0.005) in AR compared with NAR (P = 0.01), Dp-specific IgE (P = 0.001) and Df-specific IgE (P = 0.001) in AR compared with NAR. Macrophages (%) had negative correlations with total IgE , Dp-IgE , and Df-IgE  in NAL fluids. However, there was no difference in the levels of specific IgE to superantigens (SEA and SEB), ECP, and other T cell cytokines (including IL-10) in NAL fluids between the subgroups in children.In children, flow cytometry of the NAL fluids showed a significant increase in eosinophils (with NAR . CAP anaP = 0.019), Dp-specific IgE (P = 0.006) and Df-specific IgE (P = 0.003) in AR compared with NAR  were significantly higher in children than in adults, but the levels of serum total IgE were not different between the two groups (P = 0.827). In NAL fluids, eosinophil % (P = 0.036) were significantly increased in children compared to adults (P = 0.021) and IFN-γ (P = 0.038) were significantly increased in children compared to adults , and specific IgEs to Dp  and Df  measured in NAL fluids from children and adults. However, nasal eosinophils %  was significant for the diagnosis of AR only in children.ROC curves of the local immune parameters were constructed to evaluate their ability to diagnose AR in children and adults based on their cut-off values, sensitivity, and specificity . The areThis study analyzed cellular and humoral immune responses in NAL fluids from children and adults with AR and NAR, and their local-systemic associations. Additionally, we investigated whether children with AR have a different immunologic phenotype from adults with AR. Our findings can be summarized as follows: (1) Increased production of total and allergen-specific IgEs was found in the nasal airways of AR compared with NAR in both age groups. (2) In AR, children had higher levels of eosinophilic inflammation in both peripheral blood and the nasal airway than adults. (3) Local-systemic correlations of total IgE and eosinophil % were significant only in children.AR is a hypersensitivity disease mediated by specific IgE when exposed to inhalant allergens . IgE is Increased levels of total IgE and specific IgEs to house dust mites were found in the NAL fluids from children and adults with AR compared to levels measured in their respective NAR groups. Our findings support the fact that local IgE is a key molecule that drives nasal eosinophilic inflammation in patients with AR , 26. HowLocal and systemic levels of total IgE and eosinophils showed significant associations in children but not in adults. Local eosinophilic inflammation may precede systemic eosinophilic inflammation, as local IgE is the source of serum IgE. Consistent with previous findings , 28, theSpecific IgEs for inhalant allergens (e.g. house dust mite) can be locally produced and spill over into systemic circulation . Flow cyLAR is characterized by the presence of nasal Th2 inflammation with local production of specific IgEs and/or a positive response to a nasal allergen provocation test without evidence of systemic atopy . PreviouRecent studies have shown that innate immune cells, like macrophages and dendritic cells, can induce Th2 responses. Macrophages can affect the production of IL-4 or immunoglobulin and the class switching of lymphocytes in the initiation of mouse AR . In our Enhanced Th2 cytokine responses and lower levels of IFN-γ from Th1 cells have been reported in NAL fluid from patients with AR –41. HoweIn conclusion, elevated local IgE is a common feature of AR in children and adults. Local measures in NAR showed naïve state in immune perspectives having very low levels of all cytokines and immunoglobulins which disagree with the hypothesis of “local allergic rhinitis”. In addition, children showed intense local inflammation and close local-systemic interaction compared to adults which support the phenomenon of “immunosenescence” and also suggest pediatric AR as a distinct feature.


Androgen deprivation therapy (ADT) has been the standard care for patients with advanced prostate cancer (PC) since the 1940s. Although ADT shows clear benefits for many patients, castration-resistant prostate cancer (CRPC) inevitably occurs. In fact, with the two recent FDA-approved second-generation anti-androgens abiraterone and enzalutamide, resistance develops rapidly in patients with CRPC, despite their initial effectiveness. The lack of effective therapeutic solutions towards CRPC largely reflects our limited understanding of the underlying mechanisms responsible for CRPC development. While persistent androgen receptor (AR) signaling under castration levels of serum testosterone (<50 ng/mL) contributes to resistance to ADT, it is also clear that CRPC evolves via complex mechanisms. Nevertheless, the physiological impact of individual mechanisms and whether these mechanisms function in a cohesive manner in promoting CRPC are elusive. In spite of these uncertainties, emerging evidence supports a critical role of prostate cancer stem-like cells (PCSLCs) in stimulating CRPC evolution and resistance to abiraterone and enzalutamide. In this review, we will discuss the recent evidence supporting the involvement of PCSLC in CRPC acquisition as well as the pathways and factors contributing to PCSLC expansion in response to ADT. Prostate cancer (PC) is a major male health risk in the developed world . The disi.e., castration and estrogen administration caused regression of metastatic PC [Although our knowledge of the androgen-dependent nature of prostate epithelium was established long before 1941 [tatic PC . Since ttatic PC . Althougtatic PC ,9,17. WeDespite castration-resulted reduction of circulating testosterone, the intratumoral levels of androgens in CRPCs are not lower than those in hormone naive tumors ,19. In lNCOA2 was reported in 24.3% metastatic PCs and is associated with AR transcription activity [Persistent AR signaling in CRPC is also facilitated through AR coactivators. FOXA1 is a pioneer factor that plays a role in AR recruitment to chromatin, a process that is required for AR to transactivate target genes ,28. Elevactivity ,30.in vitro and in vivo (xenograft tumors) [A major mechanism leading to persistent AR signaling is AR amplification and mutations. Amplification of the AR gene at Xq11-12 was first reported in 30% (7/23) of CRPCs . A signi tumors) ,36. Knoc tumors) . AR-V7 w tumors) . Moreove tumors) . CollectAR gene. More importantly, the functional status of persistent AR signaling in CRPC is demonstrated by the effectiveness of abiraterone and enzalutamide in treating patients with CRPC [Several lines of evidence, as discussed above, support a critical role of persistent AR signaling in CRPC development, including the transition from the paracrine to autocrine dependency on androgens, upregulation of AR coactivators, and the recurrent amplification and mutations in the ith CRPC ,12.However, it is also clear that complex mechanisms regulate the resistance to ADT and to the second-generation anti-androgens. Genetically, copy number alterations occur in numerous genomic loci besides AR amplification in metastatic PC . PCs are+/α2β1hi/CD133+) were isolated from primary PC tissues; these cells showed high levels of clonogenic ability [+α2β1hiCD133+ surface profile and these cells were able to regenerated heterogeneous xenograft tumors representing the primary prostate tumors [+ subpopulation isolated from several cultured PC cell lines was more tumorigenic than its isogenic CD44− populations [Definitive demonstration of the prostate cancer stem cell (PCSC) properties  remains a challenge for the PC research community. However, accumulative evidence supports the existence of PCSCs or prostate cancer stem-like cells. PCSLCs bearing a specific surface antigen profile  705 (pSTAT3) and consequently pSTAT3-mediated transactivation of target genes ,95. In aJAK2 and STAT3 genes are among the SNPs in the top 9 genes linked to increased risk of PC and PC progression [+/α1β2hi/CD133+) from high-grade primary PC, as well as the prevention of human CRPC tumor growth in mice via inhibition of either IL-6 or STAT3 [+, α1β2hi, SOX2+, and NANOG+ with elevated pSTAT3 in xenograft tumors. The expression of pSTAT3 also associates with these stem factors in primary PC. Blocking IL-6 via soluble IL-6R abolished this PCSLC expansion [in vivo, and in vitro evidence all support a critical role of the IL-6/JAK/STAT3 pathway in promoting PCSLC growth under androgen signaling inhibition. PCSLCs then drive PC evolution towards CRPC , JAK1, and pSTAT3 in CRPCs were found to associate with poor prognosis . Single gression . Elevatigression ,109,110,gression . An upregression . Elevategression . These ror STAT3 . Furtherxpansion . These rxpansion ,116. As rds CRPC . This coThe Wnt/β-catenin pathway plays a critical role in tissue homeostasis by sustaining the tissue stem/progenitor cells . For exaConsistent with the contributions of PCSLCs to CRPC development, evidence supports a role of the Wnt/β-catenin pathway in stimulating resistance to ADT. In seven pairs of patient-derived advanced PC with and without ADT treatment for 22 weeks, RNA sequencing identified the Wnt signaling as a top pathway activated in ADT treated tumors . Exome sin vivo tumorigenic events were accompanied with an increase in PC cells positive for the well-established PCSLC markers ALDH+ and CD133+ [in vitro assay for self-renewal), and the expression of CD133, CD44, and ABCG2 in LNCaP and LNCaP C4-2 cells [+ cell subpopulation, Oct4 and ABCG2 expression [+ PCSLC subpopulation, miR-320 expression was substantially reduced along with increases in β-catenin expression. In aggregate, the functional connections of Wnt activation with increases in tumor generation, metastasis, self-renewal, standard PCSC markers , and pluripotency genes (OCT4 and NANOG) strongly support an important role of the Wnt/β-catenin signaling in promoting PCSLCs.The Wnt/β-catenin signaling contributes to PCSLCs. Activation of the Wnt pathway via inhibition of GSK3β promoted LNCaP C4-2B and DU145 cell-derived xenograft tumor growth and C4-2B cell-produced bone metastasis. These d CD133+ . In supp-2 cells . On the pression . ConversSOX2 plays an essential role in maintaining adult stem cells in epithelial tissues , and is in vitro and in vivo (xenograft tumors) expressed elevated SOX2 [SOX2 is also critical to sustain the self-renewal status of PCSLCs. Epidermal growth factor receptor (EGFR) maintains the self-renewal of PCSLCs largely through the stimulation of SOX2 expression; knockdown of SOX2 abolished EGFR signaling-initiated PCSLCs . LNCaP cted SOX2 . As wellted SOX2 . AR signted SOX2 . Furtherted SOX2 ,141. Theted SOX2 . In esseNANOG and one of its pseudogenes NANOGP8 decreased DU145 cells’ sphere forming ability in vitro and tumorigenesis capacity in vivo [In addition to SOX2, NANOG is also an important pluripotency gene . A clear in vivo . DU145 s in vivo .TMPRSS2-ERG and TMPRSS2-ETV1) in PC [TMPRSS2-ERG genomic breakpoints, which led to the generation of TOP2β-mediated DNA double stranded breaks and the subsequent genomic fusion [While AR maintains the normal functions of prostate and promotes prostatic epithelial cell differentiation , its rol1) in PC . It has c fusion . Additioc fusion  and ERG c fusion ,150. Colc fusion . This moAR gene. It has been reported that 20% of regions in each of 10 primary PC samples and 28% of areas in individuals of 14 CRPC tissues displayed hypermethylation in the 5′ regulatory region of the AR gene [+ cells from 0.2% to 87% among individual mCRPC tissues with 31% of tumors containing >50% of AR+ cells and 41% of mCRPCs displaying <10% of AR+ cells [As it has recently been elegantly reviewed, an important contribution of CSCs to tumor evolution is their derived heterogeneity . Therefo AR gene . Intratu AR gene . Intratu AR gene . ImmunohR+ cells .+ cells might not always translate into PSA+ tumor cells. For instance, among the 30 rapid autopsy mCRPC tissues, low percentages of AR+ tumors could express high percentage of PSA+ cells  and vice versa  [+;PSA− and AR−;PSA+ tumor cells in both primary and CRPC samples has been recently confirmed [AR gene did not contain mutations in other components of the AR pathway, like FOX1A and NCOR1/2, there were mCRPC tissues with co-alterations in AR and FOXA1, NCOR1, or NCOR2 [The observed intratumoral AR heterogeneity suggests an interesting possibility that AR may function differently under different cellular context. Despite PSA being a well-demonstrated AR target, ARof PSA+) . The exionfirmed . While tor NCOR2 . Furtheror NCOR2 . Howeveror NCOR2 .n = 8, time to progression <20 months) than those with progression time >20 months (n = 9) [In support of the above possibility, limited evidence suggests intratumoral AR heterogeneity being associated with the kinetic of CRPC development. For example, in a quantification of AR staining intensity in 200 nuclei for each of the 17 CRPC samples, variations of AR staining were significantly higher in individuals with a rapid kinetics of cancer progression under ADT ( (n = 9) . CollectNANOG gene via association with the gene’s promoter [Indeed, evidence suggests a direct role of AR in facilitating ADT-induced PCSLC expansion. Crosstalk between the Wnt/β-catenin pathway and AR has been demonstrated to enhance CRPC ,161. AR promoter . Ectopicpromoter . Howeverpromoter . NonetheDespite intensive research for mechanisms leading to CRPC in the past few decades, our understanding remains limited. Nonetheless, it has been noted that persistent AR signaling under ADT levels of serum testosterone <50 ng/mL) clearly contributes to CRPC, as evident by the short-term survival advantage offered by abiraterone and enzalutamide in CRPC patients ,12. Howe0 ng/mL ci.e., how ADT, abiraterone, and enzalutamide stimulate PCSLC expansion and by what mechanisms PCSLCs drive resistance to the cytotoxic effects imposed by these approaches. Numerous factors and pathways are able to facilitate CRPC acquisition, including AR, PI3K/AKT/PTEN, HIF, IGF-1, Wnt, IL-6, SOX2, NANOG, and many others [It might be crosstalk between both processes  that expy others . It is p