The theory of games provides a mathematical formalization of strategic choices, which have been studied in both economics and neuroscience, and more recently has become the focus of neuroeconomics experiments with human and non-human actors. This paper reviews the results from a number of game experiments that establish a unitary system for forming subjective expected utility maps in the brain, and acting on these maps to produce choices. Social situations require the brain to build an understanding of the other person using neuronal mechanisms that share affective and intentional mental states. These systems allow subjects to better predict other players' choices, and allow them to modify their subjective utility maps to value pro-social strategies. New results for a trust game are presented, which show that the trust relationship includes systems common to both trusting and trustworthy behaviour, but they also show that the relative temporal positions of first and second players require computations unique to that role. Neuroeconomics brings together research in neuroscience and economics to better understand how actors make decisions by unifying mathematical constructs with behavioural measurements , rock–scissor–paper game (RSPG) and inspection game (ISG), or cooperative games, such as Prisoner's Dilemma game (PDG), equal split game (ESG), ultimatum game (UG), dictator game (DG), investment game (IG) and trust game (TG). In the following, we look at both how these games are formally defined and how game theory analyses these games.simultaneously make a choice from a set of choices. In particular, player 1 must choose a row from the set S1={r1, …, rn}, where n is the number of rows in the matrix. Call this choice player 1's strategy or denote it s1∈S1. At the same time, player 2 must choose a column from the set S2={c1, …, cm}, where m is the number of columns in the matrix. Call this choice player 2's strategy or denote it s2∈S2. The pair of strategies  is called a pure strategy profile. Strategy profiles determine a pay-off or utility for each player, denoted U1 and U2. For example, in the PDG (d), the rows and columns have been labelled cooperate and defect and the pay-offs are displayed in the matrix so that U1 =0 and U2 =3.The MPG, RSPG, ISG and PDG in  the PDG d, the rox, −x). Examples of strictly competitive games include the MPG (a) and RSPG (b). In the MPG, row and column players simultaneously choose heads (H) or tails (T). If they match, the row player wins (+1) and the column player loses (−1). If they do not match, the row player loses and the column player wins. The RSPG is similar except now players simultaneously choose rock (R), scissors (S) or paper (P). If they choose the same, they tie (0), but otherwise rock wins over scissors, scissors wins over paper and paper wins over rock. Optimal strategies involve randomization (or unanticipated play) resulting in uncertainty as to who will actually win. We assume that nature will favour players who can find any inherent advantage in these games, and therefore the brains of these players will be designed to find and exploit these advantages. One well-known system for exploiting advantages is through reinforcement learning  or for one player to win and the other to lose ( the MPG a and RSPand RSPG b. In thelearning .c). If the employee works, he gains 1 no matter what. But work is costly to the employee, who prefers to shirk and gain 2, unless the employee gets caught by the boss and gets 0. The column player, acting as the boss, must decide to inspect (I), the only way to detect shirking, or the boss could choose not to inspect (DI). The cost of inspection is −C. If the boss inspects (I) when the employee works, the boss gains 2−C, but if the boss inspects when the employee shirks the boss loses −C. However, if the boss fails to inspect (DI) when the employee shirks, the boss loses −2, but if the boss chooses not to inspect and the employee works, the boss gains 2. As in the strictly competitive games above, the optimal strategies for the employee and the boss will involve randomization.In the ISG, the row player (acting as an employee) must decide whether to work (W) or shirk (S) c. If thed). If they both cooperate, they do better, with a pay-off  compared with  when they both defect. However, each player's optimal strategy is to always defect and get 3 if the other player cooperates and avoid getting 0 if the other player defects.In the PDG, two players must simultaneously decide to either cooperate or defect d. If thesequentially make a choice at each of the decision nodes, ni, assigned to that player. The strategy sets, S1 and S2, now consist of all the different combinations of choices that a player can make. It is still reasonable to think of the strategy profile  as being chosen simultaneously, but in making these choices players will take into account the sequential order of the moves. In an extensive form game, a strategy profile picks a path from an initial decision node through the game tree to a terminal node {t1, …, tk}, where k is the number of different outcomes in the game. Similar to the strategic form games, players have preferences over the outcomes in the extensive form games, or equivalently over the strategy profiles that produce these outcomes.The ESG, UG, DG, IG and TG in n1 and player 2 to the decision nodes n2 and n3 (a). Player 1 decides whether to offer player 2 an equal split of  or an unequal split of . Player 2 must then decide whether to accept or reject the offer. A play through the game tree is a connected path of branches through the decision nodes that end at one of the terminal (or outcome) nodes labelled as t1 through t4, with a resulting pay-off to player 1 of U1(ti) and to player 2 of U2(ti) for reaching the terminal node ti. For example, in the ESG, the choice of the branch labelled R (reject) by player 1 and the branch labelled A (accept) by player 2 results in the terminal node t4 and the pay-offs U1(t4)=8 and U2(t4)=2.In the ESG, player 1 is assigned to the decision node 2 and n3 a. Playerb). Once player 1 proposes, player 2 can either accept or reject. If player 2 rejects the proposal, both players earn zero; otherwise, the players earn the split proposed by player 1. The same logic applies to the ESG (a), but now the offers are restricted to either  or . Game theory predicts that player 2 should accept any positive offer, and player 1, reasoning this way, should offer player 2 some small amount, such as the proposal of  in the ESG.In the UG, player 1 must propose how to divide a fixed amount of money, say $10 b. Once p the ESG a, but noAlternatively, the evolution of competitive instincts may cause player 2 to reject unequal offers in favour of more equal offers. While a rejection is costly, since both players get nothing, the threat of rejection often will improve a player's terms of trade .c; The DG was introduced as a means to control for the effects of punishment threats in the UG (figure 2d). Whatever amount of money player 1 sends is increased by the experimenter by some amount, say tripled, e.g. if player 1 sends all $10, then player 2 will get $30. Player 2 then decides how much of the tripled money to send back to player 1.In the IG, two players are given some amount of money, say $10. Player 1 is then given the opportunity to send none, some or all of his/her $10 to player 2 d. Whateve). Game theory predicts that player 2 will send nothing back, and that player 1 will realize this and send nothing as well.The TG is a simpler version of the IG where player 1 is restricted to the option of sending nothing or sending all $10, and if the $10 is sent, then player 2 is restricted to either send nothing back or send half of the tripled amount, or $15, back e. Game tAlternatively, the evolution of reciprocity behaviours may cause the second player to feel a social obligation towards player 1's trust, i.e. by sending money. Note that the terms of exchange can be explicit, negotiated in a previous game, or implicit, established by social norms. Consequently, player 2's decision to cheat can be against either an explicit or implicit standard of reciprocity. While both players are clearly better off by extending trust and being willing to reciprocate, their competitive instincts can cause them to get nothing. This again suggests that an ability to deduce the mental state of the other person may help player 1's decisions when to trust and may help player 2's decisions when to reciprocate.d). Furthermore, there are no pure strategy profiles that satisfy (i) and (ii) for the ISG or the MPG. the PDG d. FurtheS1 and S2. In two-person games, with two choices each, we can define a mixed strategy as follows. Let ΔS1={≥0: p1+p2=1}, where we interpret p1 as the probability of playing the pure row strategy r1, and p2 as the probability of playing the pure row strategy r2, then p∈ΔS1 is a mixed strategy for player 1 and, similarly, q∈ΔS2={≥0: q1+q2=1} is a mixed strategy for player 2. Given a mixed strategy profile , we can define the expected utility for player 1 as follows:p*, q*) that satisfies the following conditions:EU1=EU1. Using this result, it is easy to solve for p*= and q*= for the ISG, with 0<C<1, p*= and q*= for the MPG, and p*= and q*= for the RSPG.Note that a pure strategy NE is a mixed strategy NE with all the probability weight on one of the pure strategies. S1={E, ∼E} while player 2 has the strategy set S2={, ,  and }, where the first element of each pair is the choice made at n2 and the second element is the choice made at n3. We can define a mixed strategy profile  and a NE profile  as before. Note that, for the ESG, there are a number of NE profiles including , ) and ). n2 and n3 and ask what player 2’s optimal choice is at each. The answer is for player 2 to always accept or . But given this choice, player 1 should now play E resulting in the SPNE profile ). A similar unique SPNE profile holds for the UG, resulting in player 1 asking and getting 10−e, where e is the smallest divisible monetary unit.We can now write the pure strategy choices for a player in an extensive form game as a choice of branch at each of his/her decision nodes. So, for example, in the ESG, player 1 has the strategy set If a game is repeated a number of times, this can lead to new strategies by players, in particular, where the strategy chosen for the current game is made conditional on previous plays of the game. For example, in the PDG, the tit-for-tat strategy studied extensively by Any game can be turned into an experiment by assigning subjects (either human or non-humans) to the role of one of the players in the game. Subjects make decisions, either by choosing strategies or by choosing an action at each decision node in these roles. Finally, subjects are incentivized by paying them a salient reward for each outcome reached. Treatment conditions can then be varied to observe their effect on strategy choices. There are two important concerns in designing a game theory experiment: the first involves the ability to control or measure individuals' subjective values over outcomes in the game, and the second is the ability to control or account for the expansion of the strategy space due to repeated play of the game.Generally, human subjects are incentivized with a monetary pay-off at each outcome while animals are incentivized with food (or juice) pay-offs. This allows the experimenter to induce a utility function over outcomes that is sufficient for studying pure strategy choices and pure strategy NE. This induction procedure assumes that subjects prefer more of the pay-off to less and that their decision costs of making a choice is low relatively to reward . If suchWhen we consider mixed strategy NE, it is no longer certain that paying money induces the right preferences, since expected utility is a probabilistic weighting of the subject's true utility function. We can solve this problem either by estimating a subject's underlying utility function  or by inWhile many species of animals have been studied in terms of games, the closest evolutionarily to humans are the other primates. First, we consider some of the single-cell firing studies that have been conducted while primates played games against a computer, and then we consider some of the experimental studies of primate behaviour in games with other primates.Lee and his colleagues found that neurons in the dorsal lateral prefrontal cortex (DLPFC) and the anterior cingulate cortex (ACC) were involved in encoding strategic choices . The autA number of behavioural experiments involving exchange type games have been run with monkeys. Experiments on human subjects have focused on various two-person forms of classic social dilemma games involving the conflict between self-interest and mutual interest. The most well-known social dilemma is the PDG, but from an economics perspective, more interesting social dilemma games are those that capture human exchange such as the UG, IG and TG. In studying human exchange, it is important to have a control condition, the DG, which can help sort out the role of sympathy as compared with reciprocity.The PDG has been studied extensively with experiments Axelrod , 1997 whNeuroeconomics and social neuroscience experiments have begun to study some of the neural underpinnings of economic game playing. Using fMRI, l-tryptophan, and thus having lower serotonin levels in the brain, were less cooperative in a repeated PDG than subjects who were not. However, lower cooperation was only found on the first day of a two-day study, suggesting that subjects who are l-tryptophan depleted will find ways to adjust back to a more cooperative strategy.In addition, a number of pharmacological interventions have been studied in subjects playing PDG- or PD-like games. For example, In the DG, a person is given $10 by the experimenter and is asked how he/she would like to divide the money between himself/herself and an anonymous person . Using a multiround version of the IG, In another iterated version of the IG, Furthermore, There is recent evidence that greater first player's trust can be induced in strangers by the intranasal administration of oxytocin during interpersonal exchange. a). Subjects were asked to make sequential decisions for monetary pay-offs  presented in a binary game tree (figure 3b). Player 1 can either quit the game by not trusting player 2, resulting in a small equal pay-off for both, or player 1 can continue the game by trusting player 2, hoping to receive a better pay-off. Player 2 can reciprocate the first player's trust, giving them both a higher pay-off, or defect on player 1's trust, resulting in an even higher pay-off for player 2 and a pay-off of zero for player 1. In the control games, partners followed the same timeline as in the TGs, but they did not have to interact with one another and merely had to choose between lower and higher monetary rewards. The design of our experiment allowed us to address the question of which brain regions modulate trust and reciprocity during economic exchange.Previous analyses of the IG and TG games indicate the importance of different systems such as mentalizing, reward systems and social attachment for the neurobiology of trusting and reciprocity. However, little attempt has been made so far to identify the differences of underlying neural architecture for trusting and reciprocating behaviour. We present here additional analyses of the repeated TG experiment studied in figure 3a).Using a general linear model analysis, we first sought brain regions whose BOLD responses were commonly recruited for decisions to trust and reciprocate. We identified two regions, the arMFC and the AI, by performing a conjunction analysis between decisions to trust and reciprocate a.Converging evidence from neuroimaging over the last decade suggests that the arMFC plays a critical role in mentalizing, which is the ability to represent another person's psychological perspective . Accumulating neuroimaging evidence indicates that the right TPJ plays a critical role in social cognition such as perspective taking  and empathy (AI). Both mentalizing (cognitive sharing with another person) and empathy (affective sharing with another person) involve an ability to simultaneously distinguish between different possible perspectives during reciprocal exchange. In addition, trusting behaviour specifically recruited an evaluation system for prospective outcomes (FPC). This more recently evolved system provides a mechanism that enables individuals to weight long-term rewards over immediate short-term gains allowing, therefore, mutual cooperation. The interplay of these neural systems supports reciprocal exchange that operates beyond the immediate spheres of kinship, one of the distinguishing features of the human species.In this paper, we reviewed the results from a number of game experiments that establish a unitary system for forming subjective expected utility maps in the brain, and acting on these maps to produce choices. Game playing in humans involves two major systems: a valuation-choice system for making trade-offs and a shared social system for understanding and sharing mental states. The valuation-choice system has been the target of numerous studies of both human and non-human subjects, resulting in a relatively unified model of decision making involving a reinforcement learning system that calculates the expected utility of different choices, an expected utility map that weights the relative value of different choices and an all or nothing competition to make the final choice. However, more work needs to be done to better understand how neuronal systems learn to construct the underlying decision problems, and how neuronal systems perform backward and forward induction in a multistage decision process such as those made in extensive form games. For example, it is not clear whether strategies themselves are choice variables in the brain or whether strategies are simply stable constructs of the choices made at decision nodes.The shared social system has also been the target of numerous studies, but largely with human subjects. These studies suggest that human subjects use both empathy (shared affect) and mentalizing (shared intentions) to better understand other players in the game and that neural computations that allow shared mental states affect the way games are defined in the brain and, consequently, how experience in games is encoded. While empathy and mentalizing clearly affect the valuation-choice system, there does not exist a biologically plausible computational model of how this occurs.Within the existing paradigms of single-cell recording studies in monkeys and neuroimaging studies in humans, more work needs to be done to develop games that both monkeys and humans can play. So far, little has been done along these lines, and the little that has been done has not controlled for differences in how monkeys or humans are trained or how they make their decisions. Yet, this is important to adequately define, and study, the homologous brain regions that are assumed to exist between monkeys and humans.At the same time, more work needs to be done to develop better game controls for studying the different neuronal computations involved in game play. For example, Furthermore, future theory and research need to gain a better understanding of how the dispositions and behaviours of players affect how they think, feel and behave in economic games. In addition, research should explore how and why certain combinations of partner attributes promote or impede the development of social preferences . NeverthBy working together within the formal construct of game theory to build experiments to study a positive theory of game play, neuroscientists and economists are beginning to develop new insights that benefit both disciplines . One ben


The rats were followed with radiotelemetric blood pressure measurements and echocardiography. At 12 months, the left ventricles of immunized rats had greater wall thickness than control rats. The fractional shortening and dp/dtmax demonstrated preserved systolic function. A decreased E/A ratio in immunized rats indicated a diastolic dysfunction. Invasive hemodynamics revealed increased left ventricular end-diastolic pressures and decreased dp/dtmin. Mean diameter of cardiomyocytes showed hypertrophy in immunized rats. Long-term blood pressure values and heart rates were not different. Genes encoding sarcomeric proteins, collagens, extracellular matrix proteins, calcium regulating proteins, and proteins of energy metabolism in immunized rat hearts were upregulated, compared to controls. Furthermore, fibrosis was present in immunized hearts, but not in control hearts. A subset of immunized and control rats was infused with angiotensin (Ang) II. The stressor raised blood pressure to a greater degree and led to more cardiac fibrosis in immunized, than in control rats.We immunized Lewis rats with the second extracellular-loop peptides of the human α1A-AR-AB cause diastolic dysfunction independent of hypertension, and can increase the sensitivity to Ang II. We suggest that α1A-AR-AB could contribute to cardiovascular endorgan damage.We show that α Phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies from patients and from rabbit 1A-AR-antibodies result in protein kinase C alpha activation and transient extracellular-related kinase (ERK1/2) phosphorylation. The antibodies also exerted acute effects on intracellular Ca2+ in cardiomyocytes and contracted mesentery artery segments 1-AR, Jahns et al immunized rats and showed that agonistic autoantibodies caused idiopathic dilated cardiomyopathy in the same rats. Furthermore, passive transfer also caused disease α1-adrenergic signaling, namely α1A-, α1B-, and α1D-AR. All subtypes were expressed in cardiac tissue but differ in the amino acid sequence of the second extracellular loop. Zhou et al immunized rats with a second extracellular loop peptide from the α1D-AR subtype epitope jun, and matrix metalloproteinase 2 (MMP2) expressions in the heart. We immunized our animals against the second extracellular loop of α1A-AR. This report is the first showing the in vivo relevance of α1A-AR-AB (as opposed to α1D-AR-AB) to our knowledge. We investigated the effects on blood pressure by radiotelemetry and on cardiac function by invasive hemodynamic measurements with a conductance catheter and echocardiography. Cardiac molecular pathways influenced by α1A-AR-AB signaling were investigated by gene expression array analyses. Furthermore, we tested the hypothesis whether immunized rats react more sensitive to angiotensin (Ang) II.Three different receptor subtypes mediate α1A-AR. Eighteen rats were immunized by subcutaneous injection  dissolved in 1 mL saline at 0, 2, and 4 weeks. The animals were boosted monthly over 12 months. Eighteen control rats received saline. For Ang II infusion, osmotic pumps  were implanted under isoflurane anesthesia in the animals (n = 6 per group) 12 months after first immunization. The animals received 200 ng Ang II/kg/min for 14 days . Local authorities  approved the animal protocol that complied with criteria outlined by the American Physiological Society.Experiments were performed in 36 male Lewis rats aged 8 weeks. We prepared a synthetic GWRQPAPEDETICQINEEPGYVLFSAL-AmidxTFA/salt  peptide corresponding to the second extracellular loop of human α1A-AR-AB were detected by peptide ELISA . Rat sera (100 µL), 3 or 12 months after first immunization, were added (dilution 1∶1000). As second antibody, we used rabbit anti rat IgG fc horseradish peroxidase (HRP) conjugated . The reaction was detected by tetramethylbenzidine (TMB) as substrate for the enzyme HRP.Rat α1A-AR (CHO/α1A-AR) were carried out as earlier described 1A-AR cells for 10 min. We checked specificity by inhibiting with 1 µM of α1-AR antagonists prazosin or urapidil. The development of AT1-AR-AB, β1-AR-AB, or β2 AR-AB during immunization or Ang II treatment was excluded by cardiomyocyte contraction assay in presence of the antagonists.Neonatal rat cardiomyocyte contraction assay and the detection of extracellular regulated kinase 1/2 (ERK1/2) phosphorylation in CHO cells stably transfected with human αRats were anesthetized with 2% isoflurane and kept warm on a heated platform. Temperature and ECG were continuously monitored. Cardiac function and morphology were assessed by echocardiography with a VisualSonics Vevo 770 High-Resolution Imaging System with the use of a high-resolution (37.5 MHz) transducer. The telemetry system  and the implantation procedure is described in detail by Brockway et al. http://www.ebi.ac.uk/arrayexpress/experiments/E-TABM-725.Total RNA was extracted from the cardiac apex  of three immunized and three control rats using the RNeasy Purification Kit . RNA was treated by deoxyribonuclease I (Qiagen). Two µg RNA of cells were transcribed in cRNA with One-Cycle Target labeling and Control Reagents . Non-pooled microarray experiments were performed using Rat Genome 230 2.0 Arrays . Gene expression and RT-PCR experiments were carried as previously described.Cardiac tissues were fixed in paraformaldehyde and embedded in paraffin. The 5 µm thick sections were deparaffinized, rehydrated, and stained by the Trichrome-Masson-Goldner or 0.1% Sirius red saturated in picric acid. Histomorphological analysis and cardiomyocyte diameter was determined on elastica van Gieson and hematoxylin-and-eosin–stained, 4 µm-thick sections of tissue placed in 5% formalin. Cardiomyocyte diameter was determined perpendicular to the outer contour of the cell membrane at the nucleus level in 15 representative myocytes of the section as desribed by van Heerebeek et al For analysis of echocardiography, telemetric, and hemodynamic measurements Mann-Whitney-test was used at P<0.05. Data are expressed as mean ± SD. Continuous variables (blood pressure and heart rate) were analyzed by repeated measures analysis of variance with appropriate corrections.1A-AR-AB activity one month after first peptide injection persisting over the immunization process of 12 months. Urapidil inhibited the activity  and the myosin heavy chain beta (β-MHC) were upregulated. Further, the collagen type I maintaining tissue structure and collagen type IV, a component of basal lamina that allows interaction with integrins and favors cell adhesion, were increased expressed. The collagen IV binding glycoprotein laminin was also upregulated. Other genes coding for proteins involved in the Ca2+ signaling were represented. Among them were the cardiac ryanodine receptor 2 (Ryr2), the cardiac ATPase 2 Ca2+-transporting, slow-twitch (Atp2a2), and the L-type calcium channel (Cacna1c). Important components of the energy metabolism like muscle glycogen phosphorylase and the peroxisome proliferators-activated receptor-gamma, co-activator 1, alpha (Ppargc1a) a master regulator of metabolic function responsible for fatty acid uptake and oxidation and oxidative phosphorylationGenes coding for proteins of the sarcomere and extracellular matrix were differentially expressed in the heart . Thus, tα-MHC (Fold change (FC) = 3.5), β-MHC (FC = 2.8), collagen, type I, alpha 1 (FC = 1.7), and Cacna1c (FC = 2.0). The cardiac expression of α1A-, α1B- or α1D-AR subtype was not differential in immunized and control rats one year after immunization. Sirius red and Trichrome-Masson-Goldner staining were used for fibrosis detection. More fibrosis occurred in immunized rats compared to the controls .We tested whether or not immunized rats react more strongly to Ang II. Ang II further increased the chronotropic response of αcontrols . We usedcontrols . The ext1A-AR, namely α1A-AR-AB. The α1A-AR-AB in our model did not increase telemetrically measured blood pressure. However, they clearly caused cardiac target organ damage and resulted in a model of diastolic dysfunction. Finally, stimulation of the system with an additional stressor (Ang II) resulted in worsened target-organ damage in the α1A-AR-AB-producing animals compared to controls. The immediate question is, “how do the results reported here differ from those of Zhou et al 1A-AR, while Zhou et al used a sequence corresponding to α1D-AR. These receptors are different. We investigated Ang II provocations, while they did not. We focused on target-organ damage. Finally, we monitored endorgan damage with echocardiography, invasive hemodynamics, histological analysis, radiotelemetrically determined blood pressure measurements, and by gene expression array.The important findings in our study were that active immunization results in production of activating antibodies to the α1A-AR-AB that we detected by both cardiomyocyte contraction assay and ELISA. The blocking experiments underscored the specificity of these antibodies. The antibodies were capable of initiating α1A-AR signaling as documented by our phospho-ERK1/2 experiments. We documented clear-cut differences in cardiac function by two independent techniques, one relying on direct cardiac catheterization that revealed a diastolic pathology. Cardiac structure was substantially different between the groups, as shown by echocardiography and by light microscopy. Our notion that a much more sensitive blood pressure measurement than the tail-cuff technique would show a difference in blood pressure between groups, proved not to be the case. Nonetheless, the Ang II experiments showed clearly that when a driving force for hypertension is applied, the presence of α1A-AR-AB clearly aggravates target organ damage and blood pressure. Finally, we explored new mechanistic pathways.The immunized rats developed specific αOur gene expression analyses showed different compensatory mechanisms in structure and metabolism to maintain the cardiac function. In contrast to the failing rat heart with a shift in myosin isoform from α to β-MHC 1D-AR-AB immunization study Ppargc1a) activates multiple genes that are responsible for fatty acid uptake and oxidation and for oxidative phosphorylation Ppargc1a deficiency Myocardial remodeling implies an alteration in the extracellular matrix composition and distribution. Accordingly, we found in our array analysis the upregulation of collagen type I and IV in immunized rat hearts. Collagen I and III maintain the tissue structure, transmit forces throughout the myocardium and contribute to the elastic properties of the myocardium 2+ regulating proteins, such as ATPase, Ca2+ transporting, slow-twitch (Atp2a2) coding for the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), the cardiac ryanodine receptor 2, and the L-type calcium channel, alpha 1 C subunit (Cacnac1c) were all up-regulated. The overexpression of SERCA2a in diseased hearts has been shown to result in the recovery of contractility 1-AR-autoantibodies from patients or rabbit α1A-AR-AB to neonatal cardiomyocytes affected intracellular Ca2+ at two different levels, namely the acute, short-term elevation of intracellular Ca2+, and the increased transcript expression of Cacna1cCacna1c. A link between increased L-type calcium channel, alpha 1 C subunit levels and hypertrophy has also been demonstrated for the human heart Another important observation is the fact that genes coding for important Ca1A-AR-immunized rats were only 12 months old, had no hypertension, and no signs of heart failure, whereas the SHR rats were older and had signs of pressure-induced diastolic heart failure. Interestingly, Wallukat et al have shown that SHR develop autoantibodies against the β1 adrenergic receptor that permanently stimulate the receptor, while α1-AR-AB have not been found Rysa et al. performed DNA microarray analysis in 12 month-old SHR with manifest hypertrophy, compared to 16–20 month-old SHR with diastolic dysfunction and transition to heart failure 1-adrenergic receptors are important to both developmental cardiomyocyte growth and pathological hypertrophy. The α1A-AR-AB production induced hypertrophy by causing fibrosis and cardiomyocyte hypertrophy. Patel et al 1A-AR showed a greater increase in myocardial fibrosis post-myocardial infarction, compared to the non-transgenic control animals. Transgenic lines with an even higher expression level developed progressive cardiac fibrosis.The α1-AR subtype is involved predominantly in vasoconstrictive responses to sympathomimetic agonists has not been answered. The studies with knockout mice indicate that all subtypes play a role in the blood pressure response to α1-agonists and that the dominant contractile α1-AR is different in different vascular beds. Although we did not observe an increase in blood pressure by α1A-AR-AB, our results do not justify the conclusion that the α1A-AR is not involved in the blood pressure control. Our results are in line with results reported by Tanoue et al 1A-AR in maintaining basal blood pressure, whereas other subtypes such as α1B-AR and α1D-AR were more important in the pressor response to catecholamines.In spite of subtype-selective agonists and antagonists and gene knockout and transgenic overexpression approaches, the question of which α1-AR-AB-producing rats developed an increased LVEDP and diminished dp/dtmin in the face of preserved ejection fraction and fractional shortening. This state-of-affairs is termed “diastolic dysfunction” and is a precursor for diastolic heart failure. Half the patients with heart failure fall into this category, notably older women. The prognosis of the condition is no better, if not worse, than systolic heart failure. All medication trials to date have been disappointing. The α1-AR blockers have not been studied in detail in the context of diastolic dysfunction. Two smaller studies reported positive effects of α1-AR blockers on echocardiographic parameters of diastolic dysfunction. To our knowledge, this experimental model is the first to show that α1A-AR receptor stimulation can cause diastolic dysfunction independent of any change in blood pressure. De Blois et al showed that chronic α1-AR stimulation increases smooth muscle cell DNA replication is in arterial wall, leading to remodeling after vascular injury 1A-AR-AB model. We believe that these changes, independent of any blood pressure changes we were able to detect, resulted in the diastolic dysfunction that we observed.Our immunized α1A-AR-AB, whereas phenylephrine infusion, as reported by Patel et al, did not Our Ang II experiments showed that Ang II markedly aggravated the already-present cardiac fibrosis. We used Ang II as a stimulus to further induce vascular dysfunction. The sympathetic nervous system and renin-angiotensin-aldosterone system act synergistically to elevate or maintain blood pressure. Ang II signaling plays a critical role in modulating many of the stimuli and signals that govern arterial aging, arterial structural, and vascular functional and adaptational responses. Ang II also potentiated the chronotropic response to α1A-AR-AB treated with chronic α1-AR blocker therapy. An additional desirable control group could consist of chronic phenylephrine infusion (for 1 year). Acute infusion experiments could elucidate the issue of baroreceptor reflex blood pressure buffering capacity or resetting that remains unanswered from our study. However, our acute experiments showed that the effects of α1A-AR-AB could be blocked pharmacologically. Chronic experiments could have allowed us to speculate with greater confidence on a possible role of α1-AR blockade to alleviate diastolic heart dysfunction and remodeling.Limitations in our study are the fact that we did not include a long-term treated control group, namely immunized rats producing α1A-AR-AB after immunization caused diastolic dysfunction. Our animal model suggests that α1A-AR-AB could play a role in target-organ damage; however α1A-AR-AB are probably not initiators of hypertension. These findings have implications for the notion of viewing agonistic autoantibodies as primary treatment targets in human diseases 1-AR blocker therapy. These agents may warrant a closer look, particularly in terms of diastolic dysfunction.We elucidated agonistic autoimmunity-induced target-organ damage and showed that αFigure S11A-AR-antibodies (AB) or phenylephrine (PE) caused an increase in cardiomyocyte contraction. This effect was not further potentiated by the combination of AB and PE. The combination of AB and Ang II resulted in a further raise of contraction.Cardiomyocyte contraction assay. The incubation of cardiomyocytes with Î±(0.13 MB DOC)Click here for additional data file.Table S1Primer and probe sequences used for TaqMan RT-PCR.(0.05 MB DOC)Click here for additional data file.Table S2Physiological parameters of immunized and control rats.(0.03 MB DOC)Click here for additional data file.Table S3Echocardiography of immunized and control rats 12 months after first immunization and after Ang II treatment.(0.04 MB DOC)Click here for additional data file.


Background and Purpose. Inpatient rehabilitation in countries other than the United States (US) has been described as a time where patients are often not engaged in intensive physical activity. The purpose of this case report is to explore the amount and intensity of physical activity provided in inpatient rehabilitation after stroke in the US. Methods. This study presents a case report of a person admitted to an inpatient rehabilitation unit after sustaining a first stroke. A customized data collection tracked type of activity, activity intensity and social interaction every 5 minutes during the rehabilitation day. Results. 74 percent of the day was spent in low intensity, often seated, physical activity; 14% of the day was spent resting or sleeping. Only 2.91% the day was spent in moderate or high intensity activity with a mobility focus. Conclusions. Consistent with other studies, this case report suggests a relatively low physical demand to rehabilitation delivered in inpatient stroke rehabilitation. This case begins to raise questions about optimized rehabilitation parameters for acute stroke rehabilitation. Amount and duration of rehabilitation therapy delivered after stroke is largely influenced by regulations guiding reimbursement from federal funding programs. Inpatient rehabilitation is viewed as being beneficial to people in the early stages of stroke recovery . The comOn average, rehabilitation units in the United States provide 1.5–1.6 thirty minute sessions of physical, occupational and speech therapy daily , 4. PhysAn emerging body of evidence from stroke centers in other countries characterizes the acute rehabilitation window as lacking in activity intensity. A small study comparing the time use of people recovering from stroke in Belgium and Switzerland found discrepancies between hours spent in therapeutic activities (2.5 versus 4 hours), with PT the predominant therapy provided by both countries. In both countries, the bulk of the care was provided in the patient's room. The intensity of the PT services was not documented . The finThe prospective payment system (PPS) used by the Centers for Medicare and Medicaid to reimburse care in inpatient rehabilitation facilities (IRFs) was passed as part of the Balance Budget Act in 1997 and implemented in 2002 . While tUndertreatment may have significant long-lasting implications for the stroke survivor. Recovery of independent ambulation is a critical component of stroke rehabilitation, and those who recover ability to ambulate live longer . RecoverIf inpatient physical activity dosage with an emphasis on walking is critical to recovery of ambulation then an observation of the stroke rehabilitation environment may assist the rehabilitation team in optimizing an environment that will maximize outcomes. The purpose of this case report is to describe the physical demands of a person recovering from stroke in the early days of stroke recovery, admitted to an inpatient rehabilitation unit in the United States within 2 weeks of experiencing onset of first stroke. As a second objective the social interaction of the stroke rehabilitation is also reported. This case report was developed to test methods for a larger study designed to explore the relationship between physical activity intensity in inpatient stroke rehabilitation and mobility outcomes. To improve sensitivity of the testing materials, the person selected for the case had to be completing the first week of inpatient rehabilitation and meet the criteria of: (1) medical stability meaning not confined to bed rest or restricted mobility for medical reasons, (2) able to participate in some mobility training both seated  and upright (gait or pregait activities), (3) require speech therapy services, and (4) have family involved in discharge planning. A physical therapist collaborator identified the first individual meeting this criteria after methods had been developed to participate in this case report. Data collection occurred in an inpatient stroke rehabilitation facility in a local teaching hospital representing the only stroke rehabilitation center accredited by the American Stroke Association in the geographic area. At the time of the data collection, EA was a 67-year-old married man admitted to a local inpatient rehabilitation center/Stroke Unit four days prior to participating in the data collection. After the study was described, he gave his assent to participate. EA was diagnosed with a first ischemic right middle cerebral artery infarct 12 days prior to admission and 16 days prior to data collection. At the time of data collection, according to the initial examinations of the occupational therapist and physical therapist assigned to EA, he required minimal assistance and set up with self-cares if completed in a seated position. He required moderate assistance with all upright mobility including pregait activities and supervision with seated wheelchair mobility over short distances. He had active use of the affected lower extremity (LE) and some voluntary activity of the affected upper extremity (UE), but did not spontaneously use the affected extremity for self-care including brushing teeth and shaving. He was right hand dominant. He was described as having normal receptive language skills, but slurred speech and mild short-term memory limitations by the speech pathologist. Discharge goals for EA were to return home with support, able to complete seated self-care skills with set up and requiring minimal assistance for upright tasks including transfers. He was expected to require minimal assistance for household level ambulation using an assistive device and to be independent with wheelchair mobility over even and uneven surfaces for short distances. Therapists anticipated his discharge to be 2 weeks from the date of data collection. A modification of methods used by Bernhardt et al.  was usedFour evaluators recorded EA's activity intensity and social contacts from 7:30 AM until 5:00 PM on a weekday in the first week of inpatient rehabilitation. Observations were collected every ten minutes between 7:30–9:00 and every 5 minutes from 9:00 AM until 5:00 PM to increase sensitivity of the observations. Observations between 9:00 and 10:00 were completed by all four raters to assess interrater reliability (percent agreement) of the tool; otherwise all observations were by a single rater. At each observation the activity category, intensity of the activity, and presence of others was noted on the scoring form according to the template presented in the appendix. The raters did not interact directly with EA. For observations of EA in the room, the observer witnessed the activity from outside the room. For observations in the unit, observations were made from a distance that did not allow for physical or verbal interaction with EA. The observation was recorded as unavailable if EA was off the unit, or if any person providing care or socializing with EA requested privacy. The highest level of activity witnessed at the observation moment (as opposed to the highest level of activity in the 5 minute observation window) was recorded. In total 103 observations were recorded; EA was off the unit for 3 observations resulting in 100 observations with data.Interrater reliability was evaluated using percent agreement; raters agreed 89% of the time on behavioral ratings indicating good reliability of the recording tool. Activity data are reported in Most (73.79%) of EA's day is spent in low-intensity activity typically involving sitting and engaging in self-care activities, cognitive rehabilitation, or upper extremity activities. Only 2.91% of EA's day is spent in activity that may present a significant aerobic challenge with a focus on mobility training. EA spent relatively little time resting in bed but not sleeping, but spent a large portion of the day sitting and resting. This may suggest that there is down time during the day where a transfer back to bed to rest may not be productive due to the rehabilitation schedule . This time window may present opportunity for increasing physical activity participation independent of traditional therapy times. While relatively inactive, the participant was rarely alone. EA was interacting with at least one other individual for 67% of the observations. Of this time, 55.5% of the observations recorded interactions with the therapy staff, 34.4% with family members and 10.1% with nontherapist/nonprofessional hospital staff. Two or more persons were present with the subject during 25% of the person-present observations. There are two issues facing optimizing of dosage of rehabilitation therapies and nontherapeutic activity: the dosage necessary to improve the cardiovascular conditioning of the patient to increase independent engagement in daily living skills and the dosage necessary to improve the functional mobility of the patient. The activity pattern for EA suggests that there is considerable “down time” during the rehabilitation day. Some of that down time provides needed rest between therapy sessions. However some time, especially that associated with the “seated but resting” category, may reflect a window that could be capitalized on to increase activity, especially that activity which could increase cardiovascular endurance. Independent seated physical activity  may be able to be increased in a way that does not require additional staff supervision or increase risk of injury due to falls. Although EA receives rehabilitation services consistent with reported norms, it appears that the bulk of the rehabilitation delivered is provided at a relatively low intensity with an emphasis on seated activities. This is a bit surprising given EA's discharge goal of “some household ambulation.” Other studies have reported more inactivity in the stroke recovery process, however, the intensity of services provided seem to be consistent with other reports , 7. The examination of EA's activity intensity profile provides a window into the types of interventions delivered, and secondarily into the dosage of interventions targeted specifically to practice of ambulation. The relatively low-activity intensity suggests few upright-, gait-, or pregait-related activities were delivered. Although one explanation for the low physical activity intensity in this case may be attributed to EA's risk of injury with unsupervised upright activity, the activity profile suggests that therapeutic activities may not be challenging the ambulation goal. It is possible that the activity pattern reflects the prioritization of safety over upright mobility. As the therapists did not identify independent upright ambulation as the primary mobility goal after discharge from the stroke unit, a focus on seated mobility may have dominated the rehabilitation sessions. While independent seated mobility may be a very meaningful outcome for assuring safety at discharge, if this goal limits practice of upright ambulation (and thus discharge outcome) perhaps these strategies should be reconsidered. Identifying ideal intensity for rehabilitation is a difficult challenge for therapists designing a comprehensive rehabilitation program for a person in the early stages of stroke recovery. Dosage and intensity are informed by many factors; some specific to the individual, some based on the experience of the therapist and team, and some based on resources and externally imposed regulations. In the absence of clear guidelines informing ideal intensity, however, the rehabilitation team should explore whether activity intensity in and outside of formalized rehabilitation sessions is adequate to advance behavioral change desired. This case study begins to ask the question as to whether intensity of physical activity is adequate to sustain the long-term behavioral change and cardiovascular conditioning needed in stroke recovery. This particular case begs some interesting questions. EA spent approximately 13 percent of the rehabilitation day either sleeping or resting. While this number is not as high as reported in other studies, perhaps this down time needs further description to optimize outcomes. Additionally, absent more time to utilize, perhaps an increase physical activity of individual therapy sessions could be examined. A comprehensive assessment of rehabilitation from admission to the rehabilitation unit through discharge from rehabilitation should be considered to adequately assess the intensity of dosage of rehabilitation throughout the entire course of formalized stroke recovery. This case also demonstrates some limitations to the understanding of stroke recovery. Although the impact of low intensity functional training in the post-acute window of stroke recovery appears to support improvement in functional performance without clear change in underlying cardiorespiratory fitness , 15, wheStroke rehabilitation is complex. Interventions promoting functional recovery need to be balanced with interventions designed to assure safety of the stroke survivor and primary caregiver, and interventions that optimize reduction of stroke. What may be best in the acute phases of stroke recovery may not be best in subacute phases of stroke recovery. These changing and sometimes competing needs are delivered in an environment defined by shorter lengths of stay and increased responsibility on the family to provide care. The acute recovery window needs further study to support the rehabilitation team in providing optimized care in a changing resource environment.In the United States, inpatient rehabilitation after stroke is characterized by a short, intense bout of therapy provided with the goal of optimizing probability of discharge to home. In the case observed, the physical intensity of the rehabilitation seems relatively low compared to the mobility and self-care expectations of the patient upon return home. The relative inactivity raises questions about adequacy of activity dosage necessary to meet rehabilitation goals.


The chemopreventive effects of resveratrol (RSV) on prostate cancer have been well established; the androgen receptor (AR) plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity.The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+) cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(-) cells serving as controls. AR(+) cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP) assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE).AR in the AR (+) stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment.We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding. Prostate cancer is one of the biggest threats to men's health in the western world and it accounts for the second largest number of male cancer deaths in the United States Androgen is an important regulator of prostate gland development and function, including proliferation, differentiation, maintenance et al found that RSV effects on AR activity are concentration dependent; AR activity is enhanced at low concentration of RSV and is repressed at high concentrations et al reported recently that RSV represses AR target gene expression, at least partially, by enhancing AR degradation in a time- and dose-dependent manner Environmental factors, including nutritional and dietary factors, play fundamental roles in the development of prostatic cancer as well as other cancers As a first step elucidating the molecular mechanisms of the chemopreventive effects of RSV on prostate cancer development, experiments were designed to clarify whether RSV regulates AR target gene expression by repressing AR transcriptional activity. For this purpose, AR cDNA was integrated into the genome of the AR-negative HeLa cell line to make an AR-positive cell line, AR(+), in which the expression of AR is not affected by RSV. Since AR is constitutively expressed in AR(+) cells, this enables us to specifically analyze the repressive effects of RSV on AR transcriptional activity. The AR-negative cell line, AR(-), was established by infecting the same parental HeLa cells with empty vector DNA and serves as a control. With this system, we demonstrated that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Further, we show that the repressive effects of RSV on AR transcriptional activity are not due to changes in nuclear translocation or DNA binding.Resveratrol was purchased from Sigma  and a stock solution (1 mM) was made by dissolving RSV in DMSO . The solution was stored at −20°C in the dark. The synthetic androgen R1881 was purchased from Sigma Inc. and dissolved in ethanol to make a stock solution (10 mM). AR antibodies N-20 and N-19 were purchased from Santa Cruz Biotechnology . Anti-Flag antibody was purchased from Sigma . The PSA-enhancer-Luc reporter, containing a 6.1 kb DNA fragment corresponding to the human PSA enhance plus promoter 2. For treatment with either agonists or antagonists, LNCaP cells were culture in phenol red-free RPMI 1640 medium with 10% charcoal-stripped FBS and 1% antibiotics at 37°C under 5% CO2 for at least 3 days. After this, cells were incubated in fresh medium containing charcoal-stripped FBS supplemented with either 10 nM of the synthetic androgen R1881 or different concentrations of RSV for the specific time periods. AR(+) and AR(-) cells were maintained in DMEM with 10% FBS 1% antibiotics at 37°C under 5% CO2. For treatments, the cells were also transferred and kept in phenol red-free medium with charcoal-stripped FBS for 3 days before addition of either androgen agonist or RSV. Since the androgen agonists and antagonists were dissolved in ethanol and RSV was dissolved in DMSO, corresponding amounts of ethanol or DMSO were added to cells in separate dishes to serve as negative controls of treatments.The LNCaP cells were maintained in RPMI 1640 medium supplemented with 10% (wt/vol) fetal bovine serum (FBS) and 1% antibiotics at 37°C under 5% COHuman AR was amplified by PCR with AR-specific primers flanking the open reading frame (ORF). For cloning purposes, the restriction sites XhoI and BamHI were added to the 5′- and 3′- primer, respectively. Both the amplified AR and the retroviral pOZ-N vector were digested with XhoI and BamHI and the AR was cloned downstream of the Flag-epitope. DNA sequencing was conducted to assure that the AR was correctly inserted in the vector. The pOZ-N retroviral vector expresses a bicistronic mRNA encoding the Flag and therefore the Flag-tagged AR is expressed. The virus DNA was transfected into 293T cells using FuGene 6 Transfection Reagent . Packaged viruses were collected from the transfected 293T cells, filtered through a 0.45 um filter, and used to infect AR-negative HeLa cells. The transduced cells also express interleukin-2 receptor subunit IL-2R,  which seImmunostatining was performed as previously reported A luciferase reporter construct containing the PSA-enhancer region was transiently transfected into cells using Lipofectamine 2000 reagent according to the manufacturer's instructions (Invitrogen). Transfected cells were allowed to recover for 6 hours before the R1881 and/or RSV were added, and were then grown overnight. Cells were harvested and lysed in Luciferase Lysate Buffer (Promega) and luciferase activities were measured by Luminometer. All experiments were conducted in triplicate.Total RNA was isolated using the RNeasy mini kit (Qiagen), according to the manufacturer's specifications. Total RNA from each sample was reverse-transcribed with random primers using a StrataScript reverse transcriptase kit (Stratagene) followed by either semi-quantitative or real-time PCR. Our standard PCR procedures are as follows: In a 25 ul of reaction, DNA was denatured at 94°C for 2 min and followed by 30 cycles of 94°C for 45 sec, 62°C for 45 sec and 72°C for 45 sec. After the last cycle, reactions were incubated for an additional 5 min at 72°C to ensure that all DNA strands were extended to the ends. PCR products were separated by electrophoresis on 1% agarose gel and visualized under UV light. The intensities of DNA bands were estimated by the Image-J program.Whole cell lysates, cytoplasmic and nuclear extracts were obtained using the nuclear extract kit  according the manufacturer's instructions. Protein concentrations were estimated by Bradford reagents and equal amounts of total proteins were separated on a 7.5% SDS-PAGE gel. The proteins were transferred to nitrocellulose membrane  using the BioRad Blotting System according to the manufacturer's instructions. Staining with Ponceau Red was done to confirm equal transfer of protein in all lanes. Blots were blocked for 2 hours in 5% non-fat milk and incubated with antiserum overnight at 4°C. After washing three times in TBST, the blot was incubated with the second conjugated antibody. The blot was detected by Supersignal West Pico Chemiluminescent Kit (Pierce). The same membrane was stripped and re-probed for either β-actin or GAPDH as internal controls.9 cells in 150 mm dishes were first treated with PBS containing 1% formaldehyde for 10 min, washed twice with cold PBS, and incubated with 100 mM Tris-HCl (pH 9.4)/10 mM DTT at 30° for 20 min. The cells are then rinsed twice with cold PBS and re-suspended in 600 µl of Buffer A  by pipetting. After a brief spinning, the pellets are re-suspended in Buffer B  containing protease inhibitors by vigorous pipetting. After centrifugation at 4000 rpm for 5 min, the nuclear pellets were resuspended in Buffer C  with freshly added protease inhibitors. The nuclear lysates were then sonicated to break the chromatin into fragments with average lengths of 0.5–1.5 kb. Immunoprecipitation was then conducted by adding specific antibodies. Equal amounts of rabbit or mouse normal IgG were used as negative controls for polyclonal and monoclonal antibodies, respectively. Precipitated DNA was used as a template for PCR amplification with primers specific to the promoter region of PSA gene. Forward primer, 5′-TGCCAGGGCCTATTTTGAATC-3′. Reverse primer is 5′-AGAGCCTGAGTGAAGACCCATAAG-3′. The PCR conditions were as described above.ChIP assays were conducted as described previously The general theme of RSV's effects is that this phytochemical, similar to other dietary components with chemopreventive effects, inhibits cancer cell growth and enhances apoptosis LNCaP cells are AR-positive, androgen-responsive cells and RSV has been shown to affect both AR and AR target gene expression in these cells In order to differentiate between the effects of RSV on AR transcriptional activity from its effects on AR expression, we wanted to establish a cell line in which AR expression is unaffected by RSV. For this purpose, the AR open reading frame (ORF) was first amplified by RT-PCR using mRNA purified from LNCaP cells as template. Sequence analyses showed that neither XhoI nor BamHI restriction site were in the ORF of AR. For cloning purposes, we added the XhoI and BamHI sequences to the upper and lower PCR primers, respectively. As shown in To verify that AR is specifically expressed in the AR(+) cell line, western blots were conducted. Whole cell lysates were prepared from both AR(+) and AR(−) cells, and equal amounts of total protein were separated by electrophoresis on a 7.5% SDS-PAGE gel. Antibodies against both AR and the FLAG-tag were used for western blotting . A singlThe first response of AR to androgen stimulation is dissociation from heat shock protein complex and translocation to the nucleus Next, we want to demonstrate that the artificially expressed AR in the AR(+) cells possesses transcriptional activity. First, we conducted a simple luciferase reporter assay. The plasmids containing the PSA-enhancer cloned up-steam of the Luciferase gene were transiently transfected to both AR(+) and AR(−) cells. Transfected cells were allowed to recover for 6 hours before the synthetic androgen R1881 was added, and were then grown overnight. Luciferase activities in the lysates from cells with different treatments were measured. As shown in In order to demonstrate that the above observed effects on AR target gene expression were resulted from the expressed AR, we compared the PSA mRNA levels in AR(+) and AR(-) cells with and without hormone treatment. As shown in AR expression in the AR(+) cells is controlled by the CMV promoter and its expression is constitutive. This enables us to analyze the effects of RSV on AR transcriptional activity without interference from the changes in AR mRNA and protein levels. We first conducted western blot to check the AR protein levels in AR(+) cells after treatment with RSV. As shown in Furthermore, we wanted to demonstrate that the effect of RSV on AR transcriptional activity occurs by affecting AR-target gene (PSA) expression. The AR(+) cells were cultured in medium with 10 nM of R1881 and 50 µM of RSV, and fractions of cells were collected at different time points as indicated in In order to understand the mechanisms of the RSV effects on AR transcriptional activity, we examined the hormone-stimulated nuclear translocation of AR with and without RSV treatment for two hours. As shown in Although it has been well established that RSV serves as a potent chemopreventive reagent in several cancers including prostate cancer, the underlying molecular mechanisms are largely unknown et al found that the effects of RSV on AR activity are also concentration dependent; RSV enhances AR activity at low concentrations and represses AR activity at high concentrations et al reported recently that RSV represses AR target gene expression, at least partially, by enhancing AR degradation in a time- and dose-dependent manner Similar to other cancers, prostate tumorigenesis develops with complex etiologies. The chemopreventive effects of RSV on prostate cancer are multi-faceted as well In order to distinguish the effects of RSV on AR regulated gene expression, we established an AR positive cell line, AR(+), from the AR-negative HeLa cell line. The FLAG-tagged AR is recognized by both AR and FLAG antibodies, and more importantly, the overexpressed AR behaves in a manner similar to the AR expressed endogenously (see below). However, since it is driven by the strong cytomegalovirus (CMV) promoter Since the parental cells used for the establishment of the AR(+) cell line are not of prostate origin The AR(+) cells were made by transfecting the retroviral pOZ vector containing the human AR open reading frame which is integrated into the genome randomly. Insertion-induced interruptions of certain endogenous genes are therefore unavoidable. However, when a population of cells is used, the effects from the insertion-induced interruption are minimal. Thus, results from such experiments, especially when the AR(−) cells are used as a negative control, should represent AR-mediated effects specifically. For the same reason, individual AR(+) cells or colonies derived from individual AR(+) cells would not be recommended in studying AR functions even if AR(−) cells are used as controls.It is important to note that because the parental AR-negative HeLa cells are not of prostate origin We want to understand the molecular mechanisms underlying the chemopreventive effects of RSV on prostate cancers, and to apply this knowledge to further development of more potent chemopreventive reagents. By using the unique AR(+) cell line, we demonstrated that RSV amended the expression of AR target genes by affecting AR transcriptional activity. This is consistent with experiments using LNCaP cells


In conventional multi-trauma care service (CTCS), patients are admitted to hospital via the accident & emergency room. After surgery they are transferred to the IC-unit followed by the general surgery ward. Ensuing treatment takes place in a hospital's outpatient clinic, a rehabilitation centre, a nursing home or the community. Typically, each of the CTCS partners may have its own more or less autonomous treatment perspective. Clinical evidence, however, suggests that an integrated multi-trauma rehabilitation approach ('Supported Fast-track multi-Trauma Rehabilitation Service': SFTRS), featuring: 1) earlier transfer to a specialised trauma rehabilitation unit; 2) earlier start of 'non-weight-bearing' training and multidisciplinary treatment; 3) well-documented treatment protocols; 4) early individual goal-setting; 5) co-ordination of treatment between trauma surgeon and physiatrist, and 6) shorter lengths-of-stay, may be more (cost-)effective.This paper describes the design of a prospective cohort study evaluating the (cost-) effectiveness of SFTRS relative to CTCS.The study population includes multi-trauma patients, admitted to one of the participating hospitals, with an Injury Severity Scale score > = 16, complex multiple injuries in several extremities or complex pelvic and/or acetabulum fractures. In a prospective cohort study CTCS and SFTRS will be contrasted. The inclusion period is 19 months. The duration of follow-up is 12 months, with measurements taken at baseline, and at 3,6,9 and 12 months post-injury.Primary outcome measures are 'quality of life' (SF-36) and 'functional health status' . Secondary outcome measures are the Hospital Anxiety & Depression Scale, the Mini-Mental State Examination as an indicator of cognitive functioning, and the Canadian Occupational Performance Measure measuring the extent to which individual ADL treatment goals are met. Costs will be assessed using the PROductivity and DISease Questionnaire and a cost questionnaire.The study will yield results on the efficiency of an adapted care service for multi-trauma patients (SFTRS) featuring earlier (and condensed) involvement of specialised rehabilitation treatment. Results will show whether improved SFTRS logistics, combined with shorter stays in hospital and rehabilitation clinic and specialised early rehabilitation training modules are more (cost-) effective, relative to CTCS.Current Controlled Trials register (ISRCTN68246661) and Netherlands Trial Register (NTR139). In the Netherlands, with a population of approximately 16 million people, every year about 99.000 persons are admitted to hospital after an accident, whereas 880.000 people visit the Accident & Emergency department (A&E) after an accident -4. Many Medical care for trauma patients is a combined responsibility of hospitals, ambulance services, trauma centres, rehabilitation clinics and GHOR . This co-operation between teams of specialists is called 'trauma care chain' (TCC). In the present study multi-trauma is defined as having 2 or more injuries of which at least 1 is life threatening, including trauma with an Injury Severity Scale (ISS) score > = 16; complex multiple injuries on both lower extremities; a combination of 1 upper and 1 lower extremity injury, the latter of which can not be used in load-bearing; or complex pelvis/acetabulum fractures. Several tools for rating trauma severity have been designed . The ISSIn conventional multi-trauma care service (CTCS) each of the partners has its own more or less autonomous treatment perspective, depending on the professional's individual treatment views and experience. Clinical evidence, however, suggests that an integrated multi-trauma rehabilitation service approach or 'Supported Fast track multi-Trauma Rehabilitation Service' (SFTRS), featuring:1) Earlier involvement of the rehabilitation physician in the hospital;2) shorter stay in hospital and earlier transfer of multi-trauma patients to a specialised trauma rehabilitation unit;3) an earlier start of both specific physical and psychosocial treatment provided by a multidisciplinary team;4) early individual goal setting;5) early start of psychological and social counselling;6) an integrated co-ordination of treatment between the trauma surgeon and the rehabilitation physician, and7) a shorter stay in a trauma rehabilitation unitmay be more (cost-) effective. Such SFTRS approach may lead to less secondary complications associated with immobilisation, which would negatively influence recovery and quality of life. Early personalised goal setting and early treatment of depression are known to positively affect outcome. SFTRS may lead to faster reintegration into society. Early return to work and active support from the multidisciplinary rehabilitation team will lead to a more stable social network, and the patient becoming less reliant on professional care in the long term. Also, the SFTRS may reduce the length of stay of multi-trauma patients in a hospital. Furthermore, earlier rehabilitation treatment in a specialised rehabilitation unit may also reduce the length of stay in the rehabilitation clinic, thus reducing costs of hospital/clinic consumption. At this moment it is not possible to make a precise calculation of these savings. Since earlier discharge also means that patients take part in society and work earlier, costs related to production losses and patient & family costs are expected to be lower.The main objective of this study is to examine the effectiveness, costs and cost-effectiveness of an integrated care service for multi-trauma patients, called 'Supported Fast track multi-Trauma Rehabilitation Service' or SFTRS.The general research question is:Which of 2 rehabilitation services, i.e. 'Conventional multi-Trauma Care Service' (CTCS) or 'Supported Fast track multi-Trauma Rehabilitation Service' (SFTRS), is most (cost-) effective from a societal point of view?Sub-questions are:- What are the effects of the SFTRS on generic quality of life in multi-trauma patients as compared to the CTCS?- What are the effects of the SFTRS on functional health status as compared to the CTCS?- What are the costs to health care and to society of the SFTRS as compared to the CTCS?- What is the cost-effectiveness of the SFTRS as compared to the CTCS?The general hypothesis of this study is that SFTRS is more (cost-) effective than CTCS.This study is a prospective, multi-centre, non-randomised clinical trial in which two multi-trauma rehabilitation services will be contrasted, i.e. 'Conventional Trauma Care Service' (CTCS) and 'Supported Fast track multi-Trauma Rehabilitation Service' (SFTRS). The patients will be followed for 12 months  as well as at the Netherlands Trial Register (NTR1391).Multi-trauma patients admitted to one of the accident & emergency departments (A&E) of the participating hospitals are included. Multi-trauma is defined as having at least 2 or more injuries of which at least 1 is life threatening, including a) trauma with an Injury Severity Scale score ISS > = 16, b) complex multiple injuries on both lower extremities, c) a combination of one upper and one lower extremity injury, the latter of which can not be used in load-bearing, or d) complex pelvis/acetabulum fractures  The rehabilitation physician from the rehabilitation centre is routinely involved at a very early stage post-trauma. This allows an early start for multidisciplinary rehabilitation treatment;b) Early transfer (within five days after having been added to the waiting list from the rehabilitation centre) to a centralised, specialised trauma rehabilitation unit equipped with facilities for early training programs;c) Early individual rehabilitation goal setting;d) Close co-operation and exchange of views and experiences between the trauma surgeon and the rehabilitation team by, for example, monthly clinical sessions and individual patient visits by the trauma surgeon in the first weeks after discharge;e) Well-documented treatment protocols for multi-trauma patients for both the hospital and rehabilitation centre phases.Three phases can be identified in the treatment of multi-trauma patients:1 Early rehabilitation phase2 Stage II rehabilitation phase3 Discharge or post-discharge phaseIn the early rehabilitation phase, the patient is not allowed to mobilise weight bearing. Consequently, the physiotherapist is concerned with maintaining joint mobility, muscle strength, sitting balance, condition and training transfers as well as treatments with non-weight-bearing conditions such as hydrotherapy and non-weight-bearing gait training. There are 10 sessions per week of 30 minutes each. In addition, fitness, gymnastics, table tennis, swimming, bowling, hand bike, wheelchair training, and archery are given. There are 2–3 sessions per week for each treatment modality of 60 minutes each. The occupational therapist advises on bed posture, mattress types, aids for independent daily self-care, wheelchair-dependency training and meaningful activities that can be performed while wheelchair-bound. In addition, the wheelchair accessibility and wheelchair friendliness of the patient's home are studied. If necessary, written and oral advice on temporary and long-term adaptations to the home is given and support is given and the patient is helped to apply for financial support so that the patient can return home as soon as possible. At first, this would be for a day or two at the weekend, supervised by an occupational therapist, but would later become permanent. With regard to work, the patient's job is analysed and the patient's workplace is visited. There are 4 sessions per week of 30 minutes each. The social worker and the psychologist will see every multi-trauma patient within the first week after admission. The social worker helps the patient to return home by dealing with the family and offering advice and support to the patient on financial matters, transport facilities. The social worker also contacts the employer and company doctor to look into the possibility of reintegrating the patient into their present job. The psychologist will examine the patient with regard to such conditions as mood disorders, posttraumatic stress syndrome (PTSS), acceptance problems and cognitive problems. The latter requires extensive neuropsychological testing. In addition, individual and group psychological counselling and specialised treatment for PTSS are given. If necessary, the rehabilitation specialist can refer the patient to a consultant in psychiatry, a consultant in neurology, a consultant in internal medicine, a consultant in rheumatology and/or a consultant in urology, who is affiliated to the rehabilitation centre.In the Stage II rehabilitation phase, new treatment aims are added by the physiotherapist. These might include a gradual individual weight bearing scheme, co-ordination training and functional training. There are 7 therapy sessions per week of 30 minutes. In addition, fitness, gymnastics, table tennis, swimming, rowing, cycling and archery are given. This is offered in 2–4 sessions per week for each treatment modality of 60 minutes each.The occupational therapist continues with the treatment goals as mentioned for phase I and trains the patient to perform household tasks, hobbies, etc in a home-like environment. There are three sessions per week of 30 minutes each. In addition, group therapies such as occupational therapy and recreational therapy are given 2–4 times per week each.The social worker and the psychologist continue the work mentioned in phase I, depending on the individual needs of each patient.In the discharge phase, the patient is prepared for living at home and is referred to local physiotherapists, specialised sport clubs and mental health care professionals.CTCS is provided in several centres. Multi-trauma patients are admitted to hospital via the A&E department. After possible surgery, they are transferred to the IC-unit, followed by the hospital's nursing ward, where the patient may stay for several days or weeks. The trauma surgeon, as chief consultant, decides whether or not a rehabilitation physician will be consulted during hospitalisation. Next, ensuing treatment may take place in the hospital's outpatient clinic, in a rehabilitation centre, in a nursing home or with a local GP or physiotherapist. Van Vree and co-workers  reporteThe effectiveness of multi-trauma rehabilitation interventions and its constituting elements has been established in numerous studies. Recently, Holtslag , in his Baseline measurements will be administered as soon as possible post-injury (= T0). Further measurements will be taken at 3 months = T1), 6 months (= T2), 9 months (= T3) and 12 months (= T4) post-trauma  the following variables will be recorded: age, gender, medical history, mediation usage, diagnosis, ISS score, date and time of trauma and Glasgow Coma Scale (GCS).As soon as possible post-injury the following data are collected: trauma treatment, possible complications  and time in hospital. The following additional data are recorded upon admission in the rehabilitation centre: individual rehabilitation treatment aims, pre-trauma psychosocial status and pre-trauma employment status.Outcome measures are presented in table In the current study the primary outcome measures are FIM, measuring quality of life and SF-36, measuring functional health status. In several studies it was found that in multi-trauma patients quality of life and functional recovery do not solely depend on injury severity and complications , but alsAt an international and interdisciplinary consensus conference in 1999 about the assessment and application of quality of life (QoL) measures after multiple trauma, experts clinicians and methodologists agreed on the SF-36 as generic tools for QoL assessment across all trauma patients . In the The Canadian Occupational Performance Measure (COPM) is an individualised client-oriented measure to assess the evolution of self-perception of skills in patients across time ,35. The The Hospital Anxiety and Depression Scale (HADS) gives clinically meaningful results as a psychological screening tool, in clinical group comparisons and in correlational studies with several aspects of disease and quality of life. It is sensitive to changes both during the course of diseases and in response to psychotherapeutic and psychopharmacological intervention. Finally, HADS scores predict psychosocial and possibly also physical outcome . The HADIn studies comparing the effectiveness of different treatment regimes, differences in treatment credibility and expectancy may influence the outcome. In the proposed study the credibility/expectancy questionnaire (CEQ)  will be General considerations:For the economic evaluation the main research question is:From the viewpoint of the society is another organisation of professional care service for trauma patients (i.e. SFTRS) compared to CTCS preferable in terms of costs, effects and utilities?Based on this main research question several sub-questions are relevant:1) What are the costs of SFTRS compared to CTCS preferable in terms of costs, effects and utilities?2) What are the extra effects  of SFTRS compared to CTCS preferable in terms of costs, effects and utilities?We hypothesise that SFTRS is associated with a reduction in health care and patient costs, and an improvement in quality of life, compared to CTCS. We expect SFTRS to be cost-effective from a societal perspective. Assessments of the quality of life and costs will take place at T1 through T4.In the cost identification, the following costs are considered:Health care costs: cost of the intervention program and other health care resources both by the patient and the caregiver.• Patient and family costs: informal care, paid domestic help, transportation, over the counter medication, and other out-of-pocket expenses.• Production losses: absenteeism, presenteeism (loss of productivity while at work), and compensation mechanisms for both the patient and the caregiver, if relevant.Measurement of volumes:• Hours spent on the intervention program will be recorded on a pre-structured form by the acting healthcare professionals.• All other health care costs and patient & family costs will be recorded in a cost questionnaire.• Production losses will be measured using the patient modules of the PRODISQ ,44.The PRODISQ will be used together with the costs questionnaire, every 3 months at baseline and T1 through T4.For the valuation of health care costs and patient & family costs, an update of the Dutch manual for costing in economic evaluations  will be Both generic Quality of life (QoL) and utilities are derived from the SF-36. An overall utility score for population based QoL can be obtained, which facilitates comparisons with other interventions, i.e. the social tariff of the SF-36 -33.The primary outcome measure for the cost-effectiveness analysis will be FIM. The primary outcomes measure for the cost-utility measure will be utilities based on the SF-36 social tariff.The time horizon is 12 months. Ratios will be determined, based on incremental costs and effects of SFTRS compared to CTCS. The cost-effectiveness ratio will be stated in terms of costs per improvement on the FIM. The cost-utility ratio will focus on the net cost per QALY gained. Bootstrap re-sampling techniques ,47 are uSensitivity analyses will be performed for the costs that turn out to have the largest impact on the differences in total costs between SFTRS and CTCS. In these analyses both the variance in volumes and prices will be considered. The range over which uncertain factors are thought to vary will be assessed by calculating a minimum and maximum .In non-randomised comparative studies, variations in case mix between centres can influence the interpretation of outcome data . TherefoWhen patients drop out of the study, the reason for their withdrawal will be recorded. Drop-outs may bias the treatment effect evaluations. Therefore, the following regime will be applied:- Missing T4 measurement: 'last-observation-carried-forward' principle will be applied.- Missing T3 or T2 measurement: linear interpolation of data using data from adjacent time points (e.g. T1 and T4) for imputation.- Missing T0 or T1 measurement or more than 2 missing measurements: discarding of patient data and influx of additional patient in order to meet n = 82 per group.The main objective of this study is to examine the effectiveness, the costs and the cost-effectiveness of an integrated Supported 'Fast Track' Rehabilitation Service for multi-trauma patients (SFTRS) involving dedicated early rehabilitation intervention programs.As there are no publications found about contrasts in (cost-) effectiveness between different multi-trauma rehabilitation services it is important to investigate whether a new rehabilitation service is more effective than the conventional service.During the conceptualisation of the study design several choices had to be made. The major ones will be discussed.First, randomisation in this study is not possible because of practical and ethical reasons. The distance between the two centres is approximately 150 km. Since this study includes acute (complex) multi-trauma patients who need to be take care of as soon as possible it is not justified to transport patients for hours to accommodate to any randomisation procedure.Second, in literature the 'severe multi-trauma' patients are frequently reported as having at least 2 or more injuries of which at least 1 is life threatening (ISS > = 16). However, during a pilot study it became apparent that part of the multi-trauma patients suffering from complex multiple injuries and who were admitted to the rehabilitation centre had an ISS < 16. Clinically speaking, it concerned patients suffering from one of the following injuries: a) complex multiple injuries on both lower extremities; b) a combination of one upper and one lower extremity injury, the latter of which could not be used in load-bearing; or c) complex pelvis/acetabulum fractures. Multi-trauma patients, who often suffer from skull and brain injuries, generally have an ISS > = 16. However, especially in complex pathology of the locomotor system, necessitating intensive rehabilitation training, the AIS system often scores less. Given the fact that these patients do participate in CTCS and SFTRS, indicating that they do belong to the target population in accordance with the other inclusion criteria, these patients have been added to the project population. This classification of multi-trauma patients is in accordance with the revised AIS coding ("AIS upgrading") which is currently being elaborated internationally.In the present study the definition of multi-trauma reads: "Multi-trauma is defined as having at least 2 or more injuries of which at least 1 is life threatening, including a) trauma with ISS > = 16, b) complex multiple injuries on both lower extremities, c) a combination of one upper and one lower extremity injury, the latter of which can not be used in load-bearing, or d) complex pelvis/acetabulum fractures".In conclusion, this paper describes the design of a prospective, multi-centre, non-randomised clinical trial that will investigate the (cost-) effectiveness of a new Supported Fast Track multi-Trauma Rehabilitation Service (SFTRS). The inclusion of the patients will start in October 2008 and will continue until May 2010. The results of this study will give evidence whether the new 'fast track' rehabilitation service for multi-trauma patients is more effective than the conventional care service, and thus should be introduced nation-wide.The authors declare that they have no competing interests.SK is the main researcher and has conceptualised this paper together with HS. HS has written the protocol for this study and is also the project leader of this study. BH, HS and PB originated the idea for the study. SE has co-written the study protocol regarding the cost aspects. HS and BH are the supervisors of SK. The promotor of SK is PB.


Androgen ablation is one of the viable therapeutic options for patients with primary hormone (androgen)-dependent prostate cancer. However, an antibiotic brefeldin A (BFA) has been shown to exhibit the growth inhibitory effect on human cancer cells. We thus investigated if BFA might inhibit proliferation of androgen-responsive prostate cancer LNCaP cells and also explored how it would be carried out, focusing on cell cycle and androgen receptor (AR).Androgen-mediated cellular events in LNCaP cells were induced using 5α-dihydrotestosterone (DHT) as an androgenic mediator. Effects of BFA on non-DHT-stimulated or DHT-stimulated cell growth were assessed. Its growth inhibitory mechanism(s) was further explored; performing cell cycle analysis on a flow cytometer, assessing AR activity by AR binding assay, and analyzing AR protein expression using Western blot analysis.1 cell number, indicating a G1 cell cycle arrest. This was further confirmed by the modulations of specific cell cycle regulators , revealed by Western blots. In addition, the growth inhibition induced by BFA was accompanied by a profound (~90%) loss in AR activity, which would be presumably attributed to the significantly reduced cellular AR protein level.DHT (1 nM) was capable of stimulating LNCaP cell growth by ~40% greater than non-stimulated controls, whereas BFA (30 ng/ml) completely inhibited such DHT-stimulated proliferation. Cell cycle analysis showed that this BFA-induced growth inhibition was associated with a ~75% reduction in the cell number in the S phase and a concomitant increase in the G1 cell cycle arrest and the down-regulation of AR activity/expression, possibly accounting for its primary growth inhibitory mechanism. Thus, it is conceivable that BFA may provide a more effective therapeutic modality for patients with hormone-dependent prostate cancer.This study demonstrates that BFA has a potent growth inhibitory activity, capable of completely inhibiting DHT (androgen)-stimulated LNCaP proliferation. Such inhibitory action of BFA appears to target cell cycle and AR: BFA led to a G Although androgens are essential for the development and growth of normal prostate, they are also responsible for the development of benign prostatic hyperplasia and prostate cancer . AndrogeAntiandrogens ,6 such ain vitro screen of human tumor cells conducted by the National Cancer Institute (USA) also confirmed that BFA had a potent antiproliferative activity on several strains of prostate cancer cells -R1881  for 2 h at 37°C in the presence or absence of 100-fold excess of unlabeled DHT. Following incubation, cells were washed extensively with PBS to remove unbound radioactive ligand and then solubilized in 0.3N NaOH-ethanol  for 5 min. Those solubilized cells were transferred to the scintillation vials for measuring the radioactivity incorporated into AR (i.e. a ligand-AR binding) using a scintillation counter. Specific AR-binding was then normalized by subtracting non-specific binding (background) and expressed by cpm/106 cells.The protocol was adopted from the method of Turcotte et al.  with minCell lysates were prepared from control and BFA-treated cells by freezing-thawing in liquid nitrogen as described earlier. Either 100 μl aliquots of spent media or 10 μg of cell lysates were used for Tandem-E PSA assay . The quantitative measurement of PSA was performed by following the manufacturer's assay procedures.t test. A value of p < 0.05 was considered significant.All data were presented as mean ± SD (standard deviation) and statistical differences between the groups were assessed with the unpaired Student's LNCaP cells were first cultured with the varying concentrations (0-50 ng/ml) of BFA for 72 h, and their effects on cell growth were assessed by cell count. Cell growth was significantly inhibited by BFA in a dose-dependent manner, resulting in a 45, 78, and 95% growth reduction with 20, 30, and 50 ng/ml of BFA, respectively Fig. . The modp < 0.05) in the S phase cell number (while a ~13% decrease in the G1 cell number) was observed in DHT-treated cells  in the S phase cell number, concomitant with a ~20% increase (p < 0.05) in the G1 cell number, was seen with sole BFA as well as BFA/DHT treatments , BFA (30 ng/ml), or BFA/DHT combination (in CS-FBS medium) for 72 h. Compared with control cells, a >35% increase  for 72 h were subjected to Western blot analysis, followed by densitometric quantification. The expressions of CDK2, CDK4, and cyclin D1 were significantly (>60%) reduced or down-regulated (compared with controls), while p21WAF1 expression was up-regulated in BFA-treated cells  of AR protein following DHT, BFA, or BFA/DHT treatments (in CS-FBS medium) for 72 h was analyzed using Western blots. Although AR expressions in both control and DHT-treated cells were similarly high and apparent, BFA remarkably down-regulated its expression by > 90% Fig. . This stMoreover, a nearly complete disappearance or degradation of AR with BFA led us to assume that BFA might have primarily targeted AR at the early time point. Such a possibility was then tested by treating the cells with BFA 30 ng/ml) for 6, 12, or 24 h, and AR expressions were analyzed on Western blots and quantified with a scan densitometer. The results showed that the AR level decreased to ~60% (p < 0.05) of the initial level after only 6-h BFA treatment and steadily declined to 47% and 35% (p < 0.05) at 12 h and 24 h, respectively Fig. . Taken t0 ng/ml fp < 0.05 compared with controls) inhibited PSA secretion throughout the experiments. In contrast, DHT dramatically accelerated PSA secretion up to a maximal 4.6-fold (p < 0.001 compared with controls) at 48 h and then its level considerably plunged by 72 h . However, this stimulatory effect of DHT (on PSA secretion) was almost completely abolished by BFA, bringing its PSA levels down to nearly the same as those in BFA alone. Thus, these results illustrate that BFA is capable of inhibiting PSA secretion, one of androgen-regulated cellular events, presumably through abolishing DHT stimulatory action due to AR degradation (down-regulation of AR expression) induced by BFA.As BFA appears to profoundly down-regulate AR expression, it was of interest to examine whether BFA might also affect other androgen-mediated cellular events (via AR) such as secretion of PSA, which is a useful biomarker for prostate cancer and under the androgenic control . LNCaP cin vitro. BFA demonstrated a potent growth inhibitory activity on these cancer cells and completely suppressed DHT-stimulated cell growth as well . In other words, if transcriptional activation of AR gene was somehow turned off by BFA, no AR mRNA would be transcribed, and no AR protein would be subsequently translated. This is another possible notion that the inhibition of AR transcription by BFA could lead to the inhibition of AR preceding inhibition of AR transcription. As PSA secretion follows its synthesis that is directly under the androgenic (DHT) regulation via "AR", the inhibition of PSA secretion indicates that no PSA gene activation (required for PSA synthesis) would have been followed because no AR transcription (required for AR synthesis) had been carried out due to the inhibition of its transcriptional activation by BFA. In other words, no AR protein would be synthesized and available for activation of PSA gene, resulting in no PSA synthesis and secretion. This may then support the notion that the inhibition of DHT-stimulated PSA secretion by BFA could mainly result from the inhibition of AR transcription.In the meantime, our separate study of BFA effect on PSA secretion Fig.  may alsoAR degradation (through proteasome) or its transcriptional inhibition remains uncertain at present. Further studies are thus warranted and required.Taken all together, BFA-induced growth inhibition appears to be primarily attributed to the greatly reduced AR protein level, although whether "it" would result from independent prostate cancer cells [Nevertheless, the fact remains the same that BFA has a direct impact on AR, down-regulating its protein expression as well as its biological activity. This is indeed the primary mechanism, along with a cell cycle arrest, to induce the growth inhibition by BFA in androgen-responsive prostate cancer cells. In addition, previous studies also described the BFA-induced growth inhibition in androgen-er cells ,16 and per cells . Therefo1 arrest and down-regulating AR activity/expression, respectively. In particular, inactivation of AR due to its degradation through BFA may primarily account for the inhibition of androgen-stimulated LNCaP cell growth. Thus, BFA could be considered a useful, effective adjuvant in ongoing androgen ablation therapy for hormone-dependent prostate cancer. Further investigations are warranted.In summary, BFA has a potent growth inhibitory activity, interfering with the androgen-mediated growth pathway in LNCaP cells. Specifically, BFA appears to target cell cycle and AR, inducing a GThe authors declare that they have no competing interests.SR is a primary investigator in charge of performing all experiments and drafting the manuscript; CA and SA serve as assistants for SR to help set up and run experiments ; MC is the department chairman, providing us with all his support for this project; and SK is responsible for designing experiments, analyzing the data , and editing/finalizing the manuscript. All authors read and approved the final manuscript.