To examine rehabilitation preferences, participation and determinants for the choice of a certain rehabilitation setting (inpatient vs. outpatient) and setting-specific rehabilitation outcomes.The longitudinal observational study referred to 534 consecutive disc surgery patients (18–55 years). Face-to-face baseline interviews took place about 3.6 days after disc surgery during acute hospital stay. 486 patients also participated in a follow-up interview via telephone three months later (dropout-rate: 9%). The following instruments were used: depression and anxiety , pain intensity , health-related quality of life , subjective prognosis of gainful employment  as well as questions on rehabilitation attendance, return to work, and amount of sick leave days.before as well as after rehabilitation depending on the setting. Inpatient rehabilitees were significantly older, reported more pain, worse physical quality of life, more anxiety and depression and a worse subjective prognosis of gainful employment before rehabilitation. Pre-rehabilitation differences remained significant after rehabilitation. More than half of the outpatient rehabilitees (56%) compared to only one third of the inpatient rehabilitees (33%) returned to work three months after disc surgery (p<.001).The vast majority of patients undergoing surgery for a herniated disc attended a post-hospital rehabilitation treatment program (93%). Thereby two-thirds of these patients took part in an inpatient rehabilitation program (67.9%). Physical, psychological, vocational and health-related quality of life characteristics differed widely The results suggest a “pre-selection” of patients with better health status in outpatient rehabilitation. Gaining better knowledge about setting-specific selection processes may help optimizing rehabilitation allocation procedures and improve rehabilitation effects such as return to work. In Germany, medical rehabilitation predominantly takes place in inpatient rehabilitation (IPR) settings. Outpatient rehabilitation (OPR) is offered comparatively less often In Germany, musculoskeletal disorders such as disc related diseases constitute the largest indication group for medical rehabilitation Yet little is known about the coherence of rehabilitation success and the chosen rehabilitation setting in patients who have undergone herniated disc surgery. Gaining patient-oriented knowledge about setting-specific selection and allocation processes in such an important patient group may have important influence on developing individually tailored setting recommendations and counseling and therefore help improve rehabilitation effects and enable an easier return to working life.The following questions will be addressed:What are the rehabilitation preferences of herniated disc surgery patients regarding generally attending rehabilitation and the choice for a specific rehabilitation setting (IPR vs. OPR)?How do IPR participants differ from OPR participants at the time of the acute hospital stay before rehabilitation and what are determinants for the choice of a certain rehabilitation setting?Are there differences in rehabilitation outcomes regarding inpatient and outpatient rehabilitation settings?This prospective cohort study refers to two measuring points . BaselinBetween April 2007 and October 2008, 620 consecutive nucleotomy patients  of threeThe study has received ethics committee approval of the University of Leipzig (Ethik-Kommission an der Medizinischen Fakultät der Universität Leipzig). At the initial contact, participants were verbally informed on the purpose of the study (including handing out a study information form) and provided written consent to take part in the study.socio-demographic variables such as age, gender, marital status, educational level and whether they had children below the age of 18 years.Patients were asked about Psychological characteristics were assessed with the following assessment instruments: Composite International Diagnostic Interview  illness related variables such as herniated disc location , number of herniated discs and disc surgeries, the existence of other chronic diseases, length of acute hospital stay at the time of disc surgery  and pain intensity : “If 0 is no pain and 100 is the worst, please give me a number that indicates the amount of pain you are having today.”; higher scores indicate higher pain intensity).Patients were asked about health-related quality of lifeThe Short Form 36 Health Survey (SF-36) was used for the assessment of vocational variables: employment status, receiving early retirement pension, amount of sick leave days within the last three months, subjective prognosis of gainful employment  Study participants were also asked for the following Rehabilitation related variables assessed rehabilitation experience in past, whether patients would like to attend rehabilitation after disc surgery and which rehabilitation setting (inpatient vs. outpatient) they would prefer.rehabilitation attendance, and the actual rehabilitation setting (inpatient vs. outpatient).Patients were asked about their actual psychological well-being ), health-related quality of life  were used as measures for effect sizes and applied as appropriate for each statistical test. Effect sizes were interpreted as follows: Cohen's d: small (0.2), medium (0.5), large (0.8); Cramer's V: small (0.10), medium (0.30), large (0.50); Partial Eta-squared (ηp2): small (0.01), medium (0.06), large (0.14) Statistical analysis were performed using the Statistical Package for the Social Sciences (SPSS) Version 20.0 95.1% of the disc surgery patients preferred to take part in a subsequent rehabilitation at the time of their acute hospital stay. 3.8% of the patients declined participation in rehabilitation. 1.1% of the patients were still uncertain about rehabilitation participation at that time.61.4% of the patients intending or still uncertain about rehabilitation participation favoured an IPR setting, 36.6% preferred an OPR setting. 2% of the patients did not report a specific preference for a certain rehabilitation setting.Within a three months period after the baseline interview, 93.0% of the patients eventually took part in rehabilitation, while 7% did not attend a rehabilitation program at that time. 67.9% of the patients who participated in rehabilitation were in an IPR clinic and 32.1% in an OPR centre. 3.7% of the patients who favoured an IPR program beforehand actually took part in subsequent OPR 1.7% of these patients did not engage in any rehabilitation. 12.9% of the patients who favoured an OPR program beforehand actually took part in subsequent IPR, and 8% of these patients did not engage in any rehabilitation.OPR patients more often had children below the age of 18 years, were more often single, had a shorter length of acute hospital stay and had participated less often in rehabilitation in their past at Baseline assessment . Howeverp2 = .095) represents a noticeable medium effect, only small effect sizes for anxiety (ηp2 = .023), depression (ηp2 = .026), pain intensity (ηp2 = .031), physical health status (ηp2 = .043) and subjective prognosis of employment (ηp2 = .037) were found. Post-hoc analyses showed that IPR patients had significantly higher depression and anxiety levels, significantly higher pain intensity, a significantly worse physical health status and a significantly worse subjective prognosis of gainful employment at both assessment points (significance level of 0.05). However, post-hoc analysis revealed that IPR patients only showed significantly more days of sick leave at the 3-month follow-up (T1), not at Baseline (T0). In addition IPR patients were significantly less likely to return to work than OPR patients (p2 = .033), strong effects were found regarding time differences for anxiety (ηp2 = .195), depression (ηp2 = .202), physical health status (ηp2 = .492) and days of sick leave within the last three months (ηp2 = .426). In the cases of significant time differences, post-hoc analyses were performed. Post-hoc analyses revealed significant improvements (significance level of 0.05) regarding anxiety and depression levels, physical and mental health within both groups (IPR and OPR). In both groups the amount of days of sick leave significantly increased (significance level of 0.05) within post-hoc analyses when comparing the results from Baseline (T0) and 3-month follow-up (T1).Furthermore the results from repeated measure analyses  of variaThe majority of patients undergoing surgery for a herniated disc attended a post-hospital rehabilitation treatment program. Here two-thirds of these patients took part in an IPR program. Only 7% of the patients did not participate in rehabilitation at all. The presented findings outline that there is almost no difference between the setting-specific rehabilitation preferences during acute hospital stay and the actual rehabilitation participation afterwards. More precisely, most of the patients who wish for IPR at the time of their hospital treatment will in fact attend IPR. The same applies for the preference of an OPR program.Unlike the results of Woischneck et al. While Wolf et al. Regarding illness-related characteristics such as disc location, the number of herniated discs and surgeries in medical history, no setting-specific differences could be found before rehabilitation. The presence of other chronic diseases was also not associated with the choice for a specific rehabilitation setting, which is contradictory to the results of Wolf et al. IPR patients also have a significantly worse prognosis to stay in a job at the time of acute hospital stay than OPR patients. Assuming that IPR patients experience stronger physical and mental impairments before rehabilitation, a worse subjective prognosis of gainful employment might derive from vocational discouragement due to health problems.Regarding rehabilitation outcome three months after disc surgery, it becomes clear that the pre-rehabilitation differences regarding psychological, illness-related and vocational parameters depending on the chosen rehabilitation setting remain stable in the study sample of herniated disc patients.Occupational activity is an essential part of our living environment These findings should not be interpreted as quality differences between IPR and OPR treatments. On the contrary, our findings could show that both groups show significant improvements regarding depression, anxiety, physical and mental health status three months after disc surgery. This improvement is underlined by strong effect sizes. Taking into account that setting-specific group differences were already prevalent before rehabilitation treatment, these findings rather suggest a “pre-selection” of herniated disc patients with better health status in OPR and with poorer health status in IPR settings 2 = 0.125), which implies that there must be other associated factors contributing to the choice for a certain rehabilitation setting. Therefore future studies, that examine the exact allocation procedures for different rehabilitation settings are recommended. Finally, outcome differences might also derive from clinic-specific and not only setting-specific differences, as patients went to different inpatient and outpatient rehabilitation clinics. Event though inpatient and outpatient rehabilitation programs contain the same standard treatments , there might have been clinic-specific differences. In line with this, motivational differences between individuals might have had an impact on how many treatment offers patients took advantage of. Future studies should take these issues into account.A limitation of this study is the relatively short period of time when regarding return to work three months after disc surgery The great majority of herniated disc patients participate in rehabilitation after surgery. Our findings indicate a “pre-selection” of disc surgery patients with better health status in OPR and with poorer health status in IPR settings During the last few years patient orientation and patient participation have gained substantial importance for research and health services Additionally, studies on setting-specific differences regarding the long-term rehabilitation outcome are lacking and therefore strongly recommended. Regarding the long-term perspective of rehabilitation outcome by taking setting specific differences into account may help to improve rehabilitation effects.


The pathophysiology of migraine is incompletely understood, but evidence points to hyper-responsivity of cortical neurons being a key feature. The basis of hyper-responsiveness is not clear, with an excitability imbalance potentially arising from either reduced inhibition or increased excitation. In this study, we measure centre-surround contrast suppression in people with migraine as a perceptual analogue of the interplay between inhibition and excitation in cortical areas responsible for vision. We predicted that reduced inhibitory function in migraine would reduce perceptual surround suppression. Recent models of neuronal surround suppression incorporate excitatory feedback that drives surround inhibition. Consequently, an increase in excitation predicts an increase in perceptual surround suppression.Twenty-six people with migraine and twenty approximately age- and gender-matched non-headache controls participated. The perceived contrast of a central sinusoidal grating patch  was measured in the presence and absence of a 95% contrast annular grating . For the static grating, similar surround suppression strength was present in control and migraine groups with the presence of the surround resulting in the central patch appearing to be 72% and 65% of its true contrast for control and migraine groups respectively (t(44) = 0.81, p = 0.42). For the drifting stimulus, the migraine group showed significantly increased surround suppression (t(44) = 2.86, p<0.01), with perceived contrast being on average 53% of actual contrast for the migraine group and 68% for non-headache controls.In between migraines, when asymptomatic, visual surround suppression for drifting stimuli is greater in individuals with migraine than in controls. The data provides evidence for a behaviourally measurable imbalance in inhibitory and excitatory visual processes in migraine and is incompatible with a simple model of reduced cortical inhibitory function within the visual system. Migraine is an episodic neurovascular brain disorder that causes significant burden to both individuals and society Visual symptoms are common in migraine either in the form of aura, or photophobia, or less specific symptoms such as blur and accommodative dysfunction Migraine pathogenesis is incompletely understood, however, most current models of the disease process invoke a mechanism of brain hyperresponsivity  A key aspect of visual processing that requires a balance of inhibitory and excitatory neural networks is the modulation of neuronal responses depending on the surrounding context. Visual neurons, at various stages of the visual system, respond to stimuli presented within their classical receptive field, however, the magnitude of the response is regulated depending on the surrounding image features In this study, we explore the balance between inhibition and excitation in migraine by measuring visual performance for a well-studied perceptual analogue of centre-surround neuronal responses: the Chubb illusion  We hypothesized that reduced cortical inhibition in migraine would result in decreased perceptual surround suppression on the contrast-contrast task, whereas, a general increased level of neural excitation would increase the effect of the surround on central responses. This seemingly counterintuitive prediction of an increase in suppression from elevated neural excitability results from models of surround suppression that incorporate contrast dependent feedback excitation to drive lateral inhibition Ethics approval for the project was obtained from the Human Research Ethics Committee of the University of Melbourne. Prior to participation, written informed consent was provided in accordance with a protocol approved by our institutional human research ethics committee and in accordance with the tenets of the Declaration of Helsinki.t(44) = 1.70, p = 0.10) or gender  = 1.43, p = 0.23) between these groups.The migraine group consisted of 26 participants: 12 fulfilling the International Headache Society's All participants were required to have best corrected visual acuity of 6/7.5 or better and to have refractive errors less than ±5.00 D sphere and ±2.00 D astigmatism. Participants were required to be free from systemic disease known to affect visual or cortical function and were also not permitted to be taking medications known to affect visual or cortical function. Normal findings in a comprehensive eye examination conducted as part of the study were also required. This examination included slit lamp biomicroscopy, ophthalmoscopy of the macula and optic nerve and an intraocular pressure measurement with applanation tonometry (less than 21 mm Hg was required for inclusion).Prophylactic medications for migraine were not permitted and participants were tested at least 4 days since the end of their last migraine in order to allow recovery from the episode and washout of any medications taken to relieve migraine symptoms. Participants in the migraine group completed the MIDAS (migraine disability assessment) questionnaire to enable a basic measure on the current impact of migraine on their lives by scoring the impact of headaches over the past 3-months on tasks of daily living Stimuli were presented on a gamma-corrected, 21-inch monitor  and generated with a ViSaGe system  using custom software written in Matlab 7 . Participants indicated responses using a button box  and viewed the monitor binocularly from a distance of 100 cm using a chin and forehead rest, with their required refractive correction for the viewing distance.The static and motion tasks were investigated in separate runs, the order of which was randomized between participants. Participants completed trials within a session of approximately 2 hours in duration, with practice trials to familiarize themselves with the task and rest breaks permitted as required. A schematic representation of the stimuli used for the experiments is shown in ‘no-surround’ condition, participants discriminated a difference in contrast between two central target gratings. For the ‘surround’ condition, it was determined how performance changed when the target was presented within a surrounding annulus ; and b) the spread of the Gaussian σ which provides an estimate of contrast discrimination precision for each observer. An estimate of bias was calculated for each observer, which was determined as the [PSE for the annular condition – PSE for the target patch alone]  the mean of the Gaussian p>0.05) therefore the results for the MA and MO groups were pooled for comparison against controls.Statistical analyses were performed using SPSS 16.0  with t-tests or repeated measures, mixed design ANOVA analyses as appropriate. Huynh-Feldt adjustments were used for non-spherical data. No significant difference between migraine groups was demonstrated on any measure  and 95% confidence intervals of the mean. The presence of the surround resulted in the target patch appearing to be 72% and 65% of its true contrast for the control and migraine groups respectively and these values were not significantly different (t(44) = 0.81, p = 0.42).p>0.05).For the migraine participants, we examined whether the shift in PSE (PSE surround – PSE no surround) correlated with any of the following migraine characteristics: MIDAS score, frequency of migraines, time since last migraine, and age at first migraine. None of these correlations were significant . Transcranial magnetic stimulation responses in V5 are different compared to non-headache controls An alternate consideration is whether the pattern of results is explicable by a non-visual mechanism such as differences in attention, aversion or some other non-visual difficulty in performing the drifting relative to the static task. Previous research shows that migraine groups find high contrast patterns of between 2–4 c/deg more aversive than non-headache controls Perceptual performance measures in people with migraine have the potential to inform about differences in neural activity in the migrainous brain, the neural subsystems involved, and the ability to monitor the progression of neural changes in the build up and resolution phases of migraine events depending on the specific type of change. Our study reveals that migraine increases the strength of perceptual centre-surround suppression for drifting stimuli at times between migraine events, when participants were asymptomatic. Further research is required to ascertain whether the strength of surround suppression varies in a systematic fashion relative to migraine event timing and to understand the underlying mechanisms for the drifting stimulus specific nature of our findings.


Sexual differentiation of the nervous system occurs via the interplay of genetics, endocrinology and social experience through development. Much of the research into mechanisms of sexual differentiation has been driven by an implicit theoretical framework in which these causal factors act primarily and directly on sexually dimorphic neural populations within the central nervous system. This review will examine an alternative explanation by describing what is known about the role of peripheral structures and mechanisms  in producing sex differences in the central nervous system. The focus of the review will be on experimental evidence obtained from studies of androgenic masculinization of the spinal nucleus of the bulbocavernosus, but other systems will also be considered. We now have overwhelming evidence that early gonadal hormones do indeed organize sex differences in the central nervous system (CNS), and we believe that these sexual dimorphisms contribute to sex differences in behavior [because there is good evidence for the periphery, including the peripheral nervous system as well as non-neural tissues (e.g. muscle), contributing to sex differentiation of the CNS [any of the known sex differences in the nervous system has remained elusive despite intensive investigation. Nonetheless, to the extent to which we have answers to the site of action question, there appear to be as many or more cases of indirect actions of steroid hormones on the CNS via peripheral structures.One of the most vexing problems facing those who study sexual differentiation of the nervous system was apparent right at the beginning. Phoenix and colleagues  are widebehavior -6. Nonet the CNS . The goaThe SNB is a neuromuscular system that mediates copulatory functions associated with the phallus. When the the SNB system is disabled via target muscle ablation, intense reflexive erection of the glans penis known as “cups”, efficient ejaculation, and the efficient deposition and removal of seminal plugs are impaired -11. ThesIn rats, both SNB motoneurons and their target muscles are larger in males than females due to testosterone action both during a perinatal sensitive period -14 as weSex differences in the SNB result from androgenic action on many morphological aspects of motoneuron and muscle (reviewed thoroughly in ). Site oTfm see [Ar mutation [Ar gene is located on the X chromosome, allowing for some interesting experimental preparations. Genetic Tfm male rats (XTfmY) have a complete loss of Ar function, but produce high levels of testosterone. These Tfm males have a feminine SNB system on all measures examined, including motoneuron number [Tfm allele (XTfmXwt) have also been used to address site of action in the androgenic masculinization of the SNB system. Due to X chromosome inactivation, and indirect rescue of SNB motoneurons, androgenized females possess a mosaic of SNB motoneurons, approximately half of which express the X with the Tfm mutation while the remainder express the X chromosome with a wt Ar allele. Androgenic effects on SNB motoneurons that are direct should only occur in those cells expressing wt Ar, whereas indirect effects should occur in both cells types. This type of mosaic analysis has been used to provide evidence of direct activation of SNB soma size and gene expression via actions on motoneuronal Ar, and also that masculinization of SNB motoneurons occurs independently of motoneuronal Ar  transgene, which comprises a promoter from a skeletal myocyte-specific actin isoform (ACTA1) driving expression of AR, in rats [Tfm males. This experiment provided a strong test of the predictions made by the prevailing model of androgenic rescue of the SNB at that time, which held that testosterone acts on ARs within myocytes to masculinize the target muscles as well as rescuing SNB motoneurons via a retrograde neurotrophic mechanism. Much to our surprise, we were not able to verify either of these predictions. Instead, expressing AR in myocytes had no measurable masculinizing effect on either target muscles or motoneurons in rats [Ar has been genetically deleted selectively within myocytes [Ar, these mice nonetheless possess a masculine  target musculature. Similarly, these mice have masculine, but reduced SNB motoneuron number [Ar knockout in this model is uncertain, both in terms of how completely Ar was deleted when measured in adulthood [Ar deletion occurred prior to the sensitive period for androgenic masculinization of the SNB. These results may therefore either be interpreted as being consistent with a necessary role of myocyte Ar in the androgenic masculinization of the SNB, or they may be interpreted as a modulatory role for myocyte Ar in this process.We therefore attempted to rescue the  in rats . The ini in rats . This pumyocytes . Despiten number . One impdulthood , and alsin situ in nerve [On balance, these studies indicate that myocytes indeed play a role in the androgenic masculinization of the SNB, but they do not act alone. This conclusion raises the question of which cells are involved. Skeletal muscle is composed of a variety of cell types other than myocytes, including: adipocytes, endothelial cells, fibroblasts, satellite cells and Schwann cells. Perhaps because myocytes have been such an exclusive focus of research, we have limited knowledge of these other cell types as candidates. Among these cells, both Schwann cells ,55 and sin nerve . UnfortuHSA-AR rats. In this case, a rescue experiment concerning the SNB was not feasible, as HSA-AR Tfm males do not develop a masculine SNB [HSA-AR males showed an enhanced response to androgen manipulations: sham-operated or castrated and androgen-maintained transgenic males had greater dendritic extent than sham-operated or androgen-maintained wt males. This effect was eliminated by castration without androgen maintenance, which equalized dendritic extent between wt and transgenic males [We and our collaborators also studied site of action in androgenic activation of SNB dendrites using line SNB . Insteadline SNB . In contic males . Soma siic males . These rHSA-AR rats. We investigated the possibility that androgens can influence body composition via myocyte AR[AR within myocytes does indeed increase myocyte size, but curiously, it is also sufficient to decrease adipose tissue [AR on spinal motoneurons.These results most obviously support a neurotrophic mechanism whereby the peripheral cells (myocytes) increase dendritic extent of innervating motoneurons. Target musculature is suspected to exert retrograde neurotrophic influence via brain derived neurotrophic factor (BDNF) on androgenic activation of dendritic extent . Howeveryocyte AR. Androgee tissue . Becausee tissue ,65. It tAlthough the main focus of study in the sexual differentiation of the SNB has been on an intrinsic factor (i.e. androgens), there is reason to believe that extrinsic factors may also contribute to this process. Interactions with conspecifics, and especially with the dam, have been investigated as potentially mediating some aspects of organization of the system. Maternal behavior directed towards pups is unequal between male and female pups . The salex copula penile reflexes in adulthood, and higher levels of stimulation reversed these deficits. Secondly, perineal stimulation of neonatal males results in neural activity and oxytocin release in the vicinity of the SNB [This idea of a sensorineural mechanism mediating maternal effects on organization of the SNB is supported by several lines of evidence. Firstly, some of these effects of maternal anosmia can be reproduced by artificially rearing pups, and reversed by providing licking-like tactile stimulation . Low lev the SNB . Nonethe the SNB , central the SNB , and alt the SNB . It woulExtrinsic influences have also been implicated in activation of the SNB. Several studies have been conducted to evaluate the role of sexual stimulation on the SNB. In the first study, gonadally intact adult males were either given access to sexual partners, or only to sexual cues without coital contact, or given no social partners for 4 weeks . In thisOne sometimes comes across a spoken or unspoken objection that the SNB is simply different from other sexual dimorphisms, and represents an exception to, rather than an example of, a CNS dimorphism. Certainly there are salient differences between the SNB and the few cases of brain dimorphisms about which we have some mechanistic understanding: the SNB is AR regulated rather than estrogen receptor regulated , the SNIn addition to its targeted effects on SNB morphology and sexual behaviour, maternal licking in general contributes to many other aspects of development, including reproductive behavior and stress response ,73. Thistabula rasa upon which sexual differentiation of behavior is a consequence of unequal sensation and experience produced by sex differences in peripheral structures. For example, according to this argument, males are better able to engage in male-typical insertive sexual behaviors because they possess intromissive genitals.Historically, the argument has been made (e.g. ) that seTrpc2, an ion channel mediating VNO chemosensation, showed increased male typical copulatory behavior (mounting). The authors concluded that females therefore possess the neural circuits necessary for male typical copulation and that the VNO  serve to suppress these behavioral responses. The authors go on to speculate that sex differences in behavior may generally arise from sexually differentiated peripheral structures, with both sexes retaining a sexually bipotential brain throughout life [More recently, a similar argument was made on empirical grounds ,83. In tout life ), and alout life ,85). Furout life  may haveout life , and obsout life .Sex differences in behavior and/or neural structure have been seen as relative, rather than absolute, from the earliest study . From thAr mutant mice (described below) that are puzzling according to a traditional view of exclusively local differentiation of the brain.Nonetheless, there is good reason to think that the VNO is important for sexual differention of brain and behavior. There is evidence that the for sexually dimorphic VNO  and mainGonadotropin releasing hormone neurons are another set of peripheral cells that interact with, and in fact contribute to, sexually dimorphic CNS structures. These neurons migrate into the CNS from the primordia of the VNO  beginninTfm mice [Tfm mutants, males with global genetic deletion of Ar exhibit profound deficits in masculine sexual behavior, even following treatment with estrogens or androgens [Tfm or equivalent mutants, precisely because they lack the peripheral structures  that are essential for some classic measures of behavioral masculinization , and alAr in neurons seems to reproduce only a relatively mild version of the copulatory deficits observed in loss of function Ar mutations such as Tfm or global Ar knockout [Ar was inactivated selectively in neurons using a Nes promoter. Both studies report a reduction in the proportion of mutant males mating with stimulus females and attacking intruder males relative to wt male controls. In one study an increase in latency to initiate intromissions, thrusts and ejaculations was observed even in males that did copulate [Ar for sexual differentiation, because of potential problems with the efficiency of Ar deletion discussed previously.More striking is that selective genetic deletion of knockout ,97. In tcopulate . In the Ar. This suggests that ARs other than those in neurons (and presumably those outside the CNS) mediate the essential features of sexual differentiation of mouse copulatory behavior. If we extend this thinking, we might also suspect that other sociosexual deficits exhibited by Tfm or Ar knockout animals might result from other peripheral deficits in masculinization of, for example, genitals, chemosensation, or pheromone production.On balance, these studies suggest that neural deletion of AR interferes with masculinization of copulatory behavior, although it does not prevent it entirely in the manner of global loss of function of The principle of accepting the simplest explanation for phenomena is undeniably useful in science. In the case of neural dimorphisms that are caused by androgens, the simplest explanation is of course that androgens act directly on the cell populations in question. Perhaps unfortunately for those who study this problem, when this assumption was put to the test for the first time, using the SNB, it proved inconsistent with the data. The results of studies in the SNB have identified at least three potential mechanisms whereby the periphery might influence the CNS: endocrine, neurotrophic and sensorineural. The less intuitive explanation, that differentiation occurs indirectly, and in some cases via peripheral structures, therefore becomes a necessary complexity in at least some cases.Ar within neurons [However, it is not worth abandoning the hypothesis that androgens contribute to CNS sex differences directly via actions on neurons. Other studies of the SNB, including those dealing with site of androgen action in activating soma size and gene expression, have yielded results that are consistent with this hypothesis. Furthermore, the site of action in androgenic differentiation of any brain region is simply unknown and so the question remains open. Finally, there are findings that suggest local, neural effects, such as the lack of masculinization of aggressive behavior in males with selective genetic deletion of  neurons .either the periphery or central mechanisms explain sexual differentiation of the CNS. Rather, as has been the case for the SNB, both peripheral and central mechanisms contribute to any given sex difference. Multiple mechanisms can come about because there are multiple sexually differentiated features, each of which may have a different mechanism of action. For example, SNB motoneuron number is determined by androgen action on muscle whereas some gene expression appears to be determined by androgen action directly on SNB motoneurons , and weAR, androgen receptor; BDNF, brain derived neurotrophic factor; CNS, central nervous system; SNB, spinal nucleus of the bulbocavernosus; Tfm, testicular feminization mutation; VNO, vomeronasal organ; wt, wild type.The authors have no competing interests to declare.All authors were involved in the conception of the review, as well as drafting and revising it. All authors read and approved the manuscript.


We examined the contribution of altered β-AR functioning in AA to understand these disparate findings. Twenty-one or 28 days after disease induction, we examined β2-AR expression in spleen and draining lymph nodes (DLNs) for the arthritic limbs using radioligand binding and western blots and splenocyte β-AR-stimulated cAMP production using enzyme-linked immunoassay (EIA). During severe disease, β-AR agonists failed to induce splenocyte cAMP production, and β-AR affinity and density declined, indicating receptor desensitization and downregulation. Splenocyte β2-AR phosphorylation (pβ2-AR) by protein kinase A (pβ2-ARPKA) decreased in severe disease, and pβ2-AR by G protein-coupled receptor kinases (pβ2-ARGRK) increased in chronic disease. Conversely, in DLN cells, pβ2-ARPKA rose during severe disease, but fell during chronic disease, and pβ2-ARGRK increased during both disease stages. A similar pβ2-AR pattern in DLN cells with the mycobacterial cell wall component of complete Freund's adjuvant suggests that pattern recognition receptors  are important for DLN pβ2-AR patterns. Collectively, our findings indicate lymphoid organ- and disease stage-specific sympathetic dysregulation, possibly explaining immune compartment-specific differences in β2-AR-mediated regulation of cytokine production in AA and rheumatoid arthritis.Adjuvant-induced arthritic (AA) differentially affects norepinephrine concentrations in immune organs, and  The sympathetic nervous system (SNS) via its modulation of the immune system is an important player in determining the disease onset, progression, and severity in rheumatoid arthritis (RA) and animal models of RA , 2. Autoβ2-AR receptors and functional responses of peripheral blood mononuclear cells (PBMCs) to sympathomimetic agents compared with nonarthritic, age-matched controls [β-AR functions, these findings indicate that sympathetic signaling in targeted immune cells is dysregulated in RA and animal models of RA. SNS dysregulation in RA patients is supported by reports of reduced numbers of controls –12. The controls . Diminiscontrols  and in acontrols . In RA pβ2-ARs. β-ARs are G coupled-protein receptors that classically use the cAMP/protein kinase A (PKA) signaling pathway. The expression of β2-AR on target cells is dynamically regulated, in part by ligand availability, but also intracellularly by PKA, β-arrestins, and different GRKs [Sympathetic nerves signal immune cells in lymphoid organs primarily through the release of norepinephrine (NE). The primary adrenergic receptors (ARs) expressed on lymphocytes are ent GRKs . β2-AR function is regulated by PKA and GRK through β2-AR phosphorylation (pβ2-AR) at different serines [β2-AR (pβ2-ARPKA) alters receptor conformation, impairing Gs and enhancing Gi protein coupling to induce transient activation of extracellular signal-regulated kinases (ERK 1/2) [β2-AR by GRK (pβ2-ARGRK), specifically GRK2, leads to β-arrestin binding to the β2-AR [β2-AR, further impeding Gs protein coupling [β2-AR are dephosphorylated and recycled to the plasma membrane or degraded [ serines –18. Dese serines –21. PKA-e β2-AR , alterindegraded , 25. β2-AR signaling can induce sustained ERK 1/2 signaling in a β-arrestin- [β2-ARGRK occurs in an agonist concentration-dependent manner by GRK 2, 5, and 6 to induce different receptor functions [β2-AR and subsequent binding to β-arrestin [β2-AR that result in β2-AR-induced ERK 1/2 signaling. Additionally, rrestin- , 20, 26 rrestin- , 28. pβunctions , 28, 29.arrestin , a scaffarrestin –33. In tarrestin , 34, 35.arrestin –39 may pβ2-AR signaling in immune cells from these two lymphoid organs. Consistent with this hypothesis, in rats challenged to induce AA, a model of RA, we find that TH1 versus TH2 cytokine production differs depending on the source of immune cells cultured ex vivo [β2-ARs in peripheral blood leukocytes from RA patients, no studies have examined the extent to which leukocyte β2-ARs in animal models of RA mimic the receptor changes observed in RA. It is also not clear if leukocytes from relevant lymphoid organs, where autoreactive T cells differentiate, also demonstrate changes in β2-AR indicative of altered signaling and how such changes impact immune functions in different lymphoid organs in RA or animal models of this disease. In the present study, we tested the hypothesis that disparate TH cell cytokine profiles in different lymphoid organs reflect disease-induced and organ-specific changes of β2-AR functions. We examined β2-AR numbers, affinity, signaling, and phosphorylation patterns in immune cells from spleens and DLN of AA rats challenged with complete Freund's adjuvant (CFA) and non-AA rats challenged with components of the CFA  or saline vehicle. We report here immune organ-dependent changes in β2-AR expression, intracellular signaling, and receptor phosphorylation in AA rats and nonarthritic rats treated with one of the components of CFA, mineral or the heat-killed bacterial cell wall in saline. Previously, our laboratory has reported reduced and elevated NE concentration in the spleen and draining lymph nodes (DLN) of adjuvant-induced arthritis (AA) rats, respectively, compared with non-AA controls . These f ex vivo . DespiteMycobacterium butyricum (M. butyricum) and mineral oil were obtained from Difco  and Sigma-Aldrich Company , respectively. CFA and M. butyricum in saline were prepared as previously described [125I)CYP (2200 Ci/mMole) were purchased from R&D Systems and Amersham International , respectively. OptEIA kits for the detection of interferon-γ (IFN-γ) were obtained from BD Pharmingen . Antibodies recognizing the β2-AR (no. sc-569) and pβ2-AR at serines 345 or 346  or 355 or 356  were purchased from Santa Cruz Biotechnology . CGP-12177, propranolol, and all chemicals, reagents, and buffers used for the western blotting were purchased from Sigma-Aldrich  unless stated otherwise. Dried and heat-killed escribed . All tisad libitum, and placed on a 12 h-on 12 h-off light schedule. Rats were allowed to adjust to conditions in the Banner Sun Health Research Institute's AAALAC accredited vivarium for one week prior to starting the experiments. For AA rats, food (Purina Lab Diet 5001) was placed in the bottom of the cage, and water was supplied using long-stemmed sipper tubes to ensure easy access to food and water. All rats were observed to eat and drink throughout the study. Animals were weighed weekly and observed daily to verify adequate weight gain and overall good health. Other than the development of arthritis, the general health of the animals was maintained during the course of the experiments. Protocols for the use and care of animals in this study were approved prior to beginning the experiments by the Banner Health Research Institute's Institutional Animal Care and Use Committee and complied with NIH guidelines for the humane use and care of research animals.Male Lewis rats (200–250 g) were obtained from Charles River Laboratory . Rats were housed two per cage in plastic bottom cages with soft CareFRESH bedding , allowed access to water and food pellets n = 4 per group per sacrifice day) based on the treatment they received. The experiment was repeated twice per time point. No differences were seen between the experiments so the data for the two experiments was collapsed giving an n = 8 per group, per time point. Rats were given 0.1 mL (1) complete Freund's adjuvant (CFA); (2) mineral oil (MO); (3) M. butyricum ; or (4) sterile endotoxin-free saline by intradermal injection into the base of the tail. Only rats that received CFA developed AA. Control rats treated with MO or SMB served to control for the CFA vehicle or the effects of mycobacterium challenge in the absence of inflammatory arthritis, respectively. Saline-treated rats controlled for the stress of the injection and handling. A single preparation of CFA and SMB was used for this study. All CFA-immunized animals developed arthritis with similar timing of disease onset and severity. After overdose-induced anesthesia with 8% chloral hydrate, rats were sacrificed on day (D) 21 or 28 after CFA challenge or control treatments. The DLN  and spleens were harvested, and single cell suspensions were prepared. D21 and D28 represent acute and severe disease, respectively.Rats were randomly assigned to 1 of 4 groups  was administered by intraperitoneal (i.p.) injections twice a day in a total volume of 250 μL per injection (n = 8). Control arthritic rats were given i.p injections of the same volume of vehicle . Terbutaline or vehicle treatments were started on day 12 after immunization, the time of disease onset, and continued until sacrifice. The animals were sacrificed using an overdose of 8% chloral hydrate 10.0 mL/kg body weight. On day 28, DLN and spleen were dissected and collected in preparation for culturing immune cells. A separate experiment was performed to evaluate whether altered The onset and progression of arthritis was apparent based on the development of inflammation and swelling of the hind and fore limbs. The inflammatory response in arthritic rats was assessed by routine methods, as previously described . DorsoplX-rays were evaluated using a grading scale, as previously reported . In brieμm nylon mesh  to remove the extraneous connective tissue. The collected cells were centrifuged at 3,000 g for 7 min and then resuspended in 5 mL of a NH4Cl hypotonic buffer for 3 min to lyse the red blood cells. The immune cells were washed 2X with 10 mL HBSS, centrifuged, and resuspended into complete media . Splenocytes were counted using a hemocytometer and then brought to a final concentration of 2 × 106 cells/mL in complete media. Single cell lymph node suspensions were prepared by teasing apart the harvested lymph nodes in 5 mL HBSS using fine forceps. These cells were then triturated with a 5 mL pipette to disperse immune cells and washed with an additional 5 mL HBSS. Cells and tissue debris were separated by filtering the cell suspension through a 70 μm nylon mesh. The cells were pelleted and resuspended in 5 mL HBSS. The cells were pelleted and resuspended into complete media, counted using a hemocytometer, and brought to a final concentration of 2 × 106 cells/mL.After anesthetic overdose, the spleens and DLN were aseptically removed. Spleens were placed into stomacher bags containing 10 mL Hank's balanced salt solution (HBSS) and homogenized for 2 × 30 sec. Spleen cells were triturated with a 10 mL pipette, then washed with an additional 10 mL of HBSS, and passed through a 70 −5 M isoproterenol (ISO) or forskolin, a direct activator of adenylyl cyclase that bypasses the G-coupled protein receptors, in 1.0 mL of HBSS containing 1.0 mM IBMX (a cAMP phosphodiesterase inhibitor) at 37°C for 10 min. The reaction was then stopped by addition of 3 mL ice cold RPMI 1640 to the tubes. Samples then were centrifuged at 4°C for 8 min at 1,000 rpm, and the supernatants decanted. The cell pellets were resuspended in 0.5 mL 50 mM sodium acetate buffer. Splenocytes were lysed by two cycles of freezing (dry ice) and boiling (100°C water bath) for 5 min each. The samples were centrifuged for 10 min at 3,000 rpm to remove cellular debris, and the supernatants were collected and stored at −70°C until cAMP analysis by EIA according to the manufacturer's instructions.To measure cAMP production, spleen cells were incubated in the presence or absence of 10n of 8 per group) on D21 after challenge. Binding studies were completed using the ligand (125I)cyanopindolol ((125I)CYP), a β-AR antagonist with equal affinity for the β1- and β2-AR subclasses. Spleen cells used for β-AR binding assays were resuspended to 5 × 106 cells/mL RPMI 1640. (125I)CYP (2200 Ci/mMole) was diluted in 1% ethanol, 5 mM HCl, and 0.2% bovine serum albumin (BSA). Assays were performed in duplicate in 13 × 100 mm polypropylene tubes containing 1 × 106 spleen cells with 8 concentrations of (125I)CYP ranging from 15.8 to 333 pM. Nonspecific binding was determined using parallel assays incubated in the presence of 10−6 M CGP-12177, a hydrophilic β-AR antagonist. Tubes were incubated at 37°C for 60 min in a shaking water bath (100 oscillation/min) to ensure that equilibrium was reached. The reaction was terminated with the addition of 3 mL of ice cold hypotonic buffer  for 20 min to lyse the cells. The reaction mixture was filtered using a cell harvester  and bound radioactivity collected on Whatman fiberglass filters (GF/B) . Filters were washed with 16 mL (4 × 4 mL) of ice-cold Tris-EGTA buffer to remove the unbound radioligand. Filters were removed, placed in 12 × 75 mm tubes, and counted in a gamma counter (Whatman) at 82% efficiency.Radioligand binding studies were performed on whole splenocytes from CFA- and saline-treated rats  and affinity (KD) were determined using an iterative nonlinear regression curve fitting program, Prism, version 4, to a model of a single class of homogenous binding sites. Data were transformed into linear form by Scatchard analysis. Lines of best fit were generated using the Bmax and KD to determine the X and Y axis intercepts. The maximal number of binding sites per cell was calculated based on simple stoichiometric assumptions (1 molecule of ligand binding to 1 receptor site) and expressed as sites/cell. Specific binding was defined as the difference between binding of the radioligand at each concentration in the absence or presence of 1 6 cells were lysed by addition of 1 mL of ice cold RIPA buffer  containing a cocktail of protease inhibitors  and phosphatase inhibitor . Cell samples were placed on ice, vortexed every 10 min for 30 min, and then centrifuged at 14,000 rpm for 5 min. Supernatants were collected for western blot analysis and Bradford protein determination. Samples (20 μg protein) were treated with reducing agent, denatured at 95°C for 5 min, and then cooled on ice. The samples were loaded onto a 4–12% acrylamide gel, ran at a constant 70 V through stacking, and then increased to 200 V for resolving of the protein bands. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes at 400 mA for 1 h. The membranes were blocked for 1 h using Tris-buffered saline and Tween 20 /5% milk and washed in TBST for 5 min (six times). The membranes were then placed in TBST/5% milk containing primary antibody (β2-ART) , pβ2-AR , or pβ2-AR  and then incubated overnight at 4°C. The blots were washed in TBST for 5 min (six times) at room temperature and then placed into TBST/5% milk containing biotinylated anti-rabbit  secondary antibody for 1 h. Next, the blots were washed in TBST for 5 min (six times) and then incubated with Supersignal Chemi Substrate  for 5 min at room temperature then apposed to CL-Xposure Film . Antibodies were stripped from the blots by applying Restore Stripping Buffer  for 15 min at 37°C with gentle agitation. The blots were then washed twice in TBST for 10 min each and blocked for 1 h with TBST/5% milk. The blots were then reprobed for β-actin  as a loading control. Analysis of blots was completed using MCID 7.0 software , and GraphPad PRISM software was used for data analyses. Spleen and DLN cell pellets containing 5 × 106 cells/mL were plated into 24-well plates  and then placed in an incubator set at 7% CO2 and 37°C for 24 h. After 24 h, the supernatants were harvested and stored in the freezer at −80°C. ELISAs for IFN-γ were completed with standards run on each plate and according to the manufacturer's directions. Plates were read at 450 nm within 30 min with λ correction 570 nm using an ELISA plate reader  and cytokine levels determined by comparing optical densities from a standard curve created using known concentrations of IFN-γ present on each plate. Spleen and DLN cells suspended at a final concentration of 2 × 10P < 0.05) values were subjected to Bonferroni posthoc testing. The sum of the radiographic scores for the hind limbs was averaged within the treatment groups and expressed as a group mean ± SEM and subjected to Kruskal-Wallis statistical analysis  followed by Dunn posthoc testing.  The average of the right and left foot pad measurements was obtained for each animal, and then these individual means were averaged within each group, expressed as a mean ± standard error of the mean (SEM), and analyzed using a two-way analysis of variance (ANOVA) with repeated measures. Significant ANOVA  followed by Bonferroni post hoc analysis. Differences between treatment groups for production of IFN-γ were determined using a Student's t-test (P < 0.05). Only data where significant differences (P < 0.1) between treatment groups were observed will be described. Replicated individual optical densities (OD) from P < 0.0001) between D12–14 compared with Saline-, SMB-, or MO-treated rats (P < 0.0001 at each time point). Dorsoplantar foot pad widths of CFA-challenged rats continued to increase through D21–24 (severe disease) and then stabilized through D28 (chronic disease). Radiographic analysis of the ankle joints (P < 0.0004). CFA-challenged rats had increased soft tissue swelling, bone loss, periosteal bone formation, narrowing of their joint spaces, and reduced bone density by D28 after CFA challenge compared with the Saline- (P < 0.01), MO- (P < 0.001), or SMB- (P < 0.01) challenged rats. Animals challenged with saline, SMB, or MO developed no clinical signs of arthritis throughout the duration of the experiment . Erythemted rats  . Similarly, isoproterenol-stimulated cAMP production rose 40% (P < 0.05) higher than in unstimulated spleen cells from nonarthritic rats. In contrast, adding isoproterenol to the spleen cell suspension from arthritic rats had no effect on cAMP production. On D21, unstimulated splenocytes from Saline- and CFA-challenged rats contained similar baseline levels of cAMP. Treatment with forskolin  significBmax between treatment groups (P < 0.0002). Mean Bmax . The mean KD differed significantly between treatment groups (P < 0.0009). KD was significantly higher (P < 0.01) for MO- or CFA-treated compared with SMB-treated rats. There was a trend (P < 0.1) for an increased mean KD in splenocytes from MO- and CFA- compared with Saline-treated rats. On D21 after treatment with Saline, MO, SMB, or CFA, saturation isotherms obtained from binding experiments with splenocytes are shown in Figures n Bmax  was signβ2-AR expression is altered in animals with established AA, splenocyte β2-AR expression was assessed by western blot  in splenocyte β2-AR density between treatment groups (P < 0.01) or SMB (P < 0.05) significantly elevated the splenocyte β2-AR density compared with Saline or MO treatment. There also was a significant difference (P < 0.005) in splenocyte β2-AR density between treatment groups on D28 , MO , or SMB  treatment groups. A representative immunoblot depicting β2-AR expression in splenocytes from each treatment group on D28 after CFA challenge is shown in β2-AR expression in DLN cells was observed between any of the treatment groups at either time point after CFA challenge  were observed  by 43, 47, and 42% compared with Saline-, MO-, and SMB-challenged rats, respectively  by 52, 52, and 49% compared with Saline-, MO-, and SMB-challenged rats, respectively  and D28 (P < 0.0042), when expressed as a pβ2-ARPKA/β2-ART  towards lower pβ2-ARPKA/β2-ART expression for SMB- compared with Saline- or MO-treated on D21 (β2-ARPKA/β2-ART were significantly reduced by 58 (P < 0.01), 61 (P < 0.01), and 48% (P < 0.05) in arthritic CFA- compared with Saline-, MO-, and SMB-treated rats on D21, respectively. Similarly, pβ2-ARPKA/β2-ART were significantly decreased on D28 by 26 (P < 0.05), 34 (P < 0.01), and 28% (P < 0.05) in arthritic rats compared with the Saline, MO, and SMB treatment groups, respectively  compared with Saline-treated rats ). On day 28, pβ2-ARGRK trended towards being reduced (P < 0.1) in SMB- compared with Saline-treated rats (β2-ARGRK in splenocytes from arthritic rats were significantly increased compared with MO (28%) and SMB . A representative immunoblot depicting pβ2-ARGRK expression in splenocytes from each treatment group on D28 after CFA challenge is shown in Analysis of western blots revealed significant treatment effects on expression of pted rats . Only pββ2-ARGRK were also expressed as pβ2-ARGRK/β2-ART  and D28 (P < 0.0001)  (P < 0.1). pβ2-ARGRK/β2-ART in CFA-treated rats significantly increased (P < 0.001) compared with Saline- (44%), MO- (46%) or SMB- (52%) treated rats on D28 after immune challenge  . On D21, < 0.05) . In MO-thallenge .β2-ARPKA was also assessed in DLN cells from arthritic and nonarthritic treatment groups  and D28 (P < 0.0136). On day 21, pβ2-ARPKA/β2-ART in DLN cells increased in SMB-treated rats compared with Saline or MO treatment (P < 0.05) and in CFA-challenged rats compared with Saline- or MO-treated rats   on D28 compared with Saline-treated rats  . In contted rats . β2-ARGRK/β2-ART for both D21 and D28 after immune challenge  in CFA-challenged rats compared with either the Saline- or MO-treated rats  increased in SMB- compared with MO-treated rats. There was a trend (P < 0.1) for a higher ratio in SMB- compared with Saline-treated rats. Analysis of western blots revealed significant treatment effects on expression of pted rats . pβ2-ARex vivo secretion of IFN-γ in spleen (a) and DLN (b). IFN-γ production did not differ significantly between spleen cells obtained from arthritic rats treated with terbutaline compared with arthritic rats treated with the vehicle (γ production compared with vehicle treatment (P < 0.05). vehicle . In contβ2-AR expression and signaling in immunocytes from lymphoid organs that mediate disease processes for RA. We report that in AA, during severe disease, β-AR agonists fail to induce cAMP production in splenocytes. Loss of β-AR-induced cAMP is accompanied by reduced affinity and expression of β-AR, indicative of receptor desensitization and downregulation. pβ2-ARPKA and pβ2-ARGRK are strikingly different in splenocytes and DLN cells from AA rats. In splenocytes, pβ2-ARPKA is reduced regardless of the time point examined, while in DLN cells, pβ2-ARPKA is increased during severe AA and reduced during chronic disease. Similarly, in splenocytes, pβ2-ARGRK is unchanged and increased with severe and chronic disease stages, respectively, but increased in DLN cells during both disease stages. Differential pβ2-AR support disparate regulation of spleen and DLN cell functions by the SNS in AA rats. These findings are consistent with previous findings from our lab [in vivo terbutaline treatment showing increased IFN-γ production by DLN cells, but no change in splenocyte IFN-γ production ex vivo.In the present study, we have determined the effects of CFA, and its components, on  our lab  and in tβ-AR stimulation failed to alter cAMP levels in splenocytes from AA rats during peak disease severity (D21) compared with controls. Synthesis of cAMP was not impaired, because forskolin induced intracellular cAMP production in AA rats. Since β2-AR-Gs protein coupling is required for adenylyl cyclase-induced cAMP production, these findings support an uncoupling of β-ARs to Gs proteins in splenocytes from AA rats. Loss of catecholamine-induced cAMP production indicates altered receptor function and is supported by findings from the receptor binding studies. Receptor affinity (1/KD) was reduced in splenocytes from AA rats by D21 after CFA challenge compared with controls . Receptor affinity was also decreased in MO-treated controls, indicating that the MO component of CFA induced this effect. Bmax values were reduced in splenocytes from SMB and AA compared with Saline- and MO-treated rats. The effects of SMB treatment on β-AR density support a role for the bacterial cell wall component of CFA in this response. Collectively, our data indicate that splenocyte β2-ARs are desensitized and downregulated in severe AA, effects that are mediated via MO and SMB, respectively. In the present study, basal levels of cAMP were similar in AA and nonarthritic rats, but β2-ART expression (membrane and intracellular) in splenocytes obtained from AA rats on D21, an effect also observed in SMB-treated controls. In contrast, our binding data show reduced cell surface expression of β2-ARs, suggesting increased internalization of β2-ARs. By D28, β2-ARs were decreased in AA rats compared with all controls, indicating a disease-specific internalization and degradation of receptors between D21 and D28. In contrast, β2-ARs levels returned to baseline in SMB-treated rats at D28, supporting recycling of β2-AR to the membrane or β2-AR synthesis keeps pace with degradation.Western blots revealed an increase in β-AR agonist-induced cAMP and β-AR densities in PBMC and B cells in RA patients that negatively correlate with systemic disease activity [β-AR agonist-induced cAMP do not differ in CD4+ and CD8+ T cells and are increased in PBMCs in RA patients compared with healthy controls in other studies [β2-AR stimulation, use of membranes versus whole cells, and/or reported increase in the incidence of polymorphisms in the β2-AR in RA patients. RA patients have a greater incidence of the Arg16Gly polymorphism in the β2-AR compared with healthy controls, which in association with HLA-DR alleles imparts a greater risk for developing RA [27Glu and Thr164Ile imparting an increased risk for developing arterial hypertension in RA patients [16Gly polymorphism imparts differential signaling of β2-ARs. Similar to Arg16 encoded receptors, the polymorphisms Arg16→Gly or Gln27→Glu is recruited to the cell membrane to a similar extent with similar ligand binding affinities. They also have similar activation and deactivation kinetics after a single stimulation with an β-AR agonist. However, receptors encoded by Arg16, Arg16Gly, and Gln27Glu β2-AR genes display different activation kinetics after repeat activation that result in differential efficacies to generate cAMP [16Gly polymorphism result in faster, more persistent, and more effective downstream signaling via cAMP regardless of its association with Gln27 or Glu27. In contrast, the Arg16Gln27 variant demonstrates slower activation kinetics and reduced cAMP production, an effect independent of receptor internalization, as this did not differ among the three β2-AR variants. Enhanced activation of the Arg16GLy is coupled with greater phosphorylation of the receptor by GRK-2 and a more rapid recruitment of β2-arrestin to β2-AR, which is required for enhanced activation kinetics. Once β2-AR is stimulated the altered activation kinetics is independent of activation by G proteins. Thus, reported inconsistencies in lymphocyte β2-AR-induced cAMP between RA and healthy controls may result from differences in duration and repetitiveness of receptor stimulation.Others have reported reduced activity , 43. In  studies , 43. Theoping RA . Furtherpatients . Ahles epatients  have demate cAMP  reviewedate cAMP . The Argβ2-AR. if they do, they could explain differences in strain susceptibility to induction of inflammatory arthritis. If so, these strains could provide models for understanding the contribution of β2-AR polymorphisms to disease onset and severity in RA patients. An understanding of the physiological and clinical relevance of β2-AR variants could substantially enhance our understanding of mechanisms for differences among experimental subjects and patients in their response to β2-AR agonists reviewed in [β2-AR polymorphisms impart different catecholamine-induced effects on lymphocyte functions in RA.It is not known whether different rodent strains also have variants in genes encoding iewed in . Collectβ-AR signaling in splenocytes of AA rats is expected to promote disease processes. However, previous studies from our group [β-AR agonists reduce disease severity if administered from disease onset through severe disease. Disruption of β-AR signaling in splenocytes after disease onset suggests that the attenuating effects of β-agonists on disease outcome are not due to regulation of splenocyte function via the β-AR-Gs-cAMP pathway. Alternatively, the attenuating effects of β-AR agonists could reflect signaling via cAMP in other secondary lymphoid organs and/or at the affected joints.Since the SNS dampens inflammation and cell-mediated responses, impaired ur group  and otheur group  have shoβ-agonist-induced cAMP response and β2-AR desensitization observed in this study is likely mediated, in part by, altered NE availability. While sympathetic activity is increased in RA (reviewed in [Loss of iewed in ), no stuiewed in  are consiewed in , 49 thatiewed in . Elevateiewed in . Furtheriewed in –53. β-AR are dependent on pβ2-AR by PKA and GRK, which occurs via site-specific phosphorylation at different serines/threonines [β2-AR in splenocytes are disease specific. In AA rats, pβ2-ARs at the PKA site are reduced on D21 and D28, while pβ2-ARs at the GRK site are unchanged on D21 and elevated on D28 compared with each control group. pβ2-AR by GRK2 uncouples β2-AR from Gs protein and facilitates the binding of β-arrestin to the receptor causing receptor downregulation by internalization [β2-AR by PKA and GRK provide a mechanism for β2-AR downregulation and alternative second messenger signaling observed in splenocytes from AA rats.Desensitization and downregulation of reonines –18. Pattβ2-AR protein and its pβ2-AR patterns in splenocytes and DLN cells are strikingly different in AA rats. On D21 and D28, the β2-ART protein levels in DLN cells are similar in all treatment groups, indicating no effect of AA induction on β2-AR expression. Unlike the spleen, pβ2-ARPKA are increased on D21 in DLN cells from CFA- and SMB-treated rats compared with the other control groups . These findings suggest that the cell wall component of the adjuvant is responsible for increased pβ2-ARPKA. By D28, pβ2-ARPKA site is reduced in CFA-, SMB-, and MO-treated rats compared with the Saline control group. In contrast, on D21 and D28, pβ2-ARGRK is greater in CFA- and SMB-treated rats than in rats treated with MO or Saline. These findings indicate a role for the mycobacterial cell wall component of CFA in driving pβ2-ARGRK and therefore implicate TLRs in regulating pβ2-ARGRK, particularly TLR2 and TLR4 [β2-ARGRK patterns in spleen and DLN cells [β2-AR by GRK5/6 shifts signaling into the ERK 1/2 cascade in HEK-293 cells treated with high physiological concentration of β-agonists [Total and TLR4 , 55. DifLN cells , 27, 28.agonists , 20, 26.β2-ARGRK, as IFN-γ and IL-6 or TNF-α, IL-1, and IL-12 induce different expression of GRKs and pβ2-AR in PBMC from healthy controls [β2-AR by PKA and GRKs and their consequences for immune function in AA is required to understand disease-specific SNS-immune crossregulation in RA. Proinflammatory cytokines elevated in inflammatory arthritis may also contribute to the altered pcontrols –58. Giveγ production in AA CD4+ TH cells, cells that are prevalent in the arthritic joints and drive disease processes [β2-AR can increase IFN-γ by activating ERK 1/2 under conditions of chronically elevated NE release. If so, then it would provide a mechanism to explain the increased IFN-γ production after β2-AR stimulation in activated T cells [β2-AR, including CD4+TH1, CD8+ T cells, NK cells and B cells. Recently, CD4+FoxP3+ T regulatory (Treg) cells have been demonstrated to express β2-AR, and when stimulated, enhances their suppressive activity in a cAMP-PKA-dependent manner [β2-ARs on Treg cells enhances the ability of Treg cells to convert from CD4+CD62L+FoxP3+ T cells to FoxP3+ inducible Treg (iTreg) cells, an effect mediated by cAMP-PKA pathways. These cells are essential for maintaining tolerance and regulating adaptive immune responses [β2-AR changes observed in this study. Given that multiple T lymphocytes express β2-AR and the importance of interactions between various T cell subtypes, it will be necessary to determine the T cell subtypes responsible for the altered β2-AR expression, signaling and phosphorylation reported here. Future studies to understand the mechanism for altered β2-AR signaling in different lymphoid compartments in specific immune cell subtypes in our model and in RA are required. An understanding of β2-AR signaling changes that occur in RA also will be required before the potential to use β2-AR agonists are realized for treatment of RA. Understanding the mechanisms for altered β2-AR signaling may also lead to the improvement of current clinical use of β2-AR agonists.Of significance for RA, the ERK 1/2 signaling pathway increases IFN-rocesses –61. It i T cells , in sple T cells , 63 and  T cells . Many lyt manner . Furtheresponses , 65. A lesponses –70. Consesponses . Cytokin


Resveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol./KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins  induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by resveratrol.Resveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin) and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27These data suggest that FOXO transcription factors mediate anti-proliferative and pro-apoptotic effects of resveratrol, in part due to activation of extrinsic apoptosis pathway. Prostate cancer, one of the more common neoplasms in the western world, arises through the progressive development of one or more pre neoplastic lesions into adenocarcinoma, and subsequently to metastatic disease Epidemiological data support the concept that naturally occurring compounds in the human diet may be devoid of toxicity and have long lasting beneficial effects on human health. Resveratrol has been shown to exhibit several potential chemoprotective activities in cell and animal models PI3K signaling plays a pivotal role in intracellular signal transduction pathways involved in cellular transformation, cell growth, and tumorigenesis. Inactivation of AKT results in dephosphorylation and activation of FOXO transcription factors, reported to mediate cell cycle arrest, DNA repair, and apoptosis /KIP1 and cyclin D1). Inhibition of PI3K/AKT pathway or overexpression of FKHR, FKHRL1 and AFX enhanced resveratrol-induced FOXO transcriptional activity. In contrast, inhibition of FKHR, FKHRL1 or AFX blocked resveratrol-induced expression of TRAIL, DR4, DR5, Bim and p27, and inhibited the expression of cyclin D1. These data suggest that resveratrol induces apoptosis and growth arrest through activation of FOXO transcription factors, and inhibition of PI3K/AKT pathway activates FOXO transcription factors and further enhances antiproliferative and proapoptotic effects of resveratrol.The objectives of our study were to examine the effects PI3K/AKT pathway on the regulation of FOXO transcription factors and their target gene products, and assess whether PI3K/AKT/FOXO pathway regulates anti-proliferative effects of resveratrol in prostate cancer cells. Our data demonstrated that inhibition of PI3K/AKT pathway enhanced FOXO DNA-binding and transcriptional activity, resulting in the regulation of its gene products , constitutively active AKT (CA-AKT) and dominant negative AKT (DN-AKT). LNCaP cells were transiently transfected with empty vector, WT-AKT, CA-AKT or DN-AKT and treated with or without resveratrol (20 µM). Transfection of LNCaP cells with empty vector, WT-AKT or CA-AKT had no effect on apoptosis . ResveraSince PI3K/AKT pathway inhibits resveratrol-induced apoptosis, we sought to examine the effects of resveratrol on the phosphorylation of PI3K and AKT . LNCaP cSince mTOR is a down-stream of AKT, we next sought to examine whether resveratrol also regulates phosphorylation of mTOR. As shown in PI3K/AKT pathway has been shown to regulate the phosphorylation of FOXO proteins We next examined the nuclear translocation of FKHR by resveratrol using wild type and mutant GFP-FKHR constructs. LNCaP cells were transfected with either GFP-FKHR or GFP-FKHR-TM (triple mutant) and treated with resveratrol for 24 h . TransloWe next examined the FOXO-DNA interaction by electrophoretic mobility shift assay (EMSA). An electrophoretic mobility shift assay (EMSA) is a common affinity electrophoresis technique used to study protein-DNA interaction. Treatment of LNCaP cells with resveratrol resulted in enhanced FOXO-DNA interaction . The incWe next examined whether inhibition of PI3K/AKT pathway enhances resveratrol-induced FOXO transcriptional activity in a luciferase reporter gene assay which measures FOXO function . We haveWe next examined the effects of AKT on resveratrol-induced FOXO activity in LNCaP cells . The AKTSince LNCaP cells express mutant PTEN resulting in constitutive activation of AKT, we sought to examine the role of PTEN on resveratrol-induced FOXO transcriptional activity . LNCaP cWe next examined whether resveratrol induces transcriptional activation of FOXO in the presence or absence of FOXO1-TM, FOXO3a-TM, or FOXO4-TM (phosphorylation deficient triple mutant) . LNCaP c/Kip1, cyclin D1, TRAIL, TRAIL-R1/DR4 and TRAIL-R2/DR5. These genes are direct targets of FOXO transcription factor. LNCaP cells were pretreated with LY-294002 or AKT inhibitor IV followed by treatment with resveratrol for 24 h, and the expression of Bim, p27, cyclin D1, TRAIL, DR4 and DR5 was measured by Western blotting , TRAIL, DR4, DR5 and p27/Kip1, and inhibited the expression of cyclin D1 in LNCaP/scrambled cells. Inhibition of FKHR, FKHRL1 or AFX by shRNA attenuated resveratrol-induced Bim, TRAIL, DR4, DR5 and p27/Kip1 expression. By comparison, inhibition of FKHR, FKHRL1 or AFX by shRNA slightly attenuated the inhibitory effects of resveratrol on cyclin D1 expression. These data suggest that resveratrol can regulate apoptosis  and cell cycle-related genes  through FOXO transcription factors. Interestingly, induction of TRAIL and its receptors DR4 and DR5 by resveratrol will result in activation of extrinsic pathway of apoptosis.FOXO transcription factors have been shown to regulate apoptosis and cell cycle-related genes by shRNA . Resvera/Kip1 and apoptosis, and resveratrol-induced inhibition of cyclin D1. Our data suggest that resveratrol induces cell cycle arrest and apoptosis through regulation of FOXO transcription factors in prostate cancer cells. Overall, these properties of resveratrol strongly suggest that it could be used as a cancer chemopreventive agent.In the present study, we have shown that resveratrol induced apoptosis in prostate cancer cells through inhibition of PI3K/AKT and mTOR pathway, and activation of FOXO transcription factors. Furthermore, downregulation of PI3K/AKT pathway increased resveratrol-induced apoptosis. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27FOXO transcription factors are tumor suppressors that are inactivated in the majority of human cancers, owing to the overactivation of the PI3K/AKT pathway 2O2-stimulated cells, and was blocked by co-transfection of dominant-negative type FOXO3a mutant. These findings indicate that FOXO is a key regulator of ROS-induced apoptosis in mammalian cells.ROS are thought to play multiple roles including tumor initiation, progression and maintenance, and ROS production is highly increased in cancer cells . High levels of ROS under stress conditions have been shown to inhibit phosphorylation and acetylation of FOXO proteins, resulting in enhanced FOXO-DNA binding activity. In the cell, ROS levels can be sensed by virtue of stimulatory and inhibitory oxidative modification of cysteine residues within proteins that control various signaling cascades. Recently it was shown that cysteines in FOXO can also act as sensors of the local redox state. The cysteine-dependent redox switch can regulate ROS signaling pathway upstream of FOXO. Furthermore, FOXO can also control ROS levels by transcriptional regulation of a multilayered antioxidant system. Thus, cysteine based redox signaling to FOXO could play a role in fine-tuning the optimal cellular response to ROS to control organismal lifespan. In our study, phosphorylation deficient mutants of FOXO enhanced resveratrol-induced FOXO transcriptional activity and apoptosis. This may be because resveratrol induces ROS leading to induction of SIRT1 which in turn causes deacetylation of FOXO proteins and enhances DNA binding activity /kip1 and apoptosis. Furthermore, the inhibition of FKHR, FKHRL1 or AFX by shRNA blocked the inhibitory effects of resveratrol on cyclin D1. Similar to our studies, FOXO silencing has been shown to decrease expression levels of Bim, TRAIL, FasL, p27/Kip1, which are FOXO target genes controlling cell cycle and apoptosis FOXO transcription factors play important roles in regulation of cell cycle and apoptosis /KIP1. In addition to regulation of FOXO through dephosphorylation, resveratrol may also regulate cell survival and/or apoptosis through global modulation of gene expression via deacetylation of FOXO transcription factor. Further studies are needed to assess the physiological significance of phosphorylation vs acetylation of FOXO proteins. A thorough understanding of the mechanisms of resveratrol may lead to discovery and development of novel therapeutic molecules for the treatment and prevention of human diseases.In summary, our study demonstrate that dephosphorylation of FOXO may stabilize and enhance FOXO's DNA binding and transcriptional activity. Our findings demonstrate that resveratrol regulates the expression of FOXO's target genes such as Bim, TRAIL, DR4, DR5, cyclin D1 and p27/KIP1, cyclin D1 and β-actin were purchased from Santa Cruz Biotechnology Inc. . Enhanced chemiluminescence (ECL) Western blot detection reagents were from Amersham Life Sciences Inc. . Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay kit and caspase-3 activity kit were purchased from EMD Biosciences . Resveratrol was purchased from LKT Laboratories, Inc. . All other chemicals used were of analytical grade and were purchased from Fisher Scientific  and Sigma-Aldrich .Antibodies against Bim, TRAIL, DR4, DR5, p272.LNCaP cells were obtained from the American Type Culture Collection  and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic at 37°C in a humidified atmosphere of 95% air and 5% COExpression plasmids for silencing of FKHR, FKHRL1 and AFX were constructed using psiRNA-DUO-GFP-Zeo vector .6 cells/dish. The next day transfections were performed using LipofectAMINE . Cells were transfected with expression constructs encoding wild type PTEN (pSG5L-HA-PTENwt), mutant PTEN (pSG5L-HA-PTEN-G 129E and pSGL5-HA-PTEN-G 129R), wild type-AKT (pUSE-WT-AKT), constitutively active-AKT (pUSE-CA-AKT), dominant negative-AKT (pUSE-DN-AKT), or the corresponding empty vectors (pSG5L or pUSE) in the presence of an expression vector pCMV-LacZ (Invitrogen Life Technologies) expressing β-galactosidase. For each transfection, 2 µg of DNA was diluted into 50 µl of medium without serum. After the addition of 3 µl of LipofectAMINE into 50 µl Opti-MEM medium, the transfection mixture was incubated for 10 min at room temperature. Cells were washed with serum-free medium, the transfection mixture was added, and cultures were incubated for 24 hrs in the incubator. The next day, culture medium was replaced with fresh RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin mixture and resveratrol was added for desired times for the measurement of apoptosis and protein expression.Cells were plated in 60-mm dishes in RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin mixture at a density of 1×10Cells were treated with resveratrol and apoptosis was measured by TUNEL assay as per manufacturer instructions.4 per well) were seeded in a 96-well plate with 200 µl culture medium. Approximately 16 h later, cells were treated with various doses of resveratrol to induce apoptosis. Casapse-3 activity was measured as per manufacturer's instructions (EMD Biosciences) with a fluorometer.Cells . Cell lysates containing 40 mg of protein were analyzed using 12.5% SDSPAGE. Transferred membranes were blocked using 5% skim milk and incubated overnight with primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase at 1∶5,000 dilution in TBS-Tween 20 for 1 hour at room temperature. Membranes were developed using ECL Substrate. Protein bands were visualized on X-ray film using an enhanced chemiluminescence system.Cells were grown on fibronectin-coated coverslips , washed in PBS, and fixed for 15 min in 4% paraformaldehyde. Cells were permeabilized in 0.1% Triton X-100, washed and blocked in 10% normal goat serum. After blocking, cells were incubated with primary antibody (1∶100) for 18 h at 4°C, washed with PBS and incubated with fluorescently labeled secondary antibody (1∶200) along with DAPI (1 µg/ml) for 1 h at room temperature. Finally, coverslips were washed and mounted using Vectashield . Isotype-specific negative controls were included with each staining. Stained cells were mounted and visualized under a fluorescent microscope.LNCaP cells were transfected with empty vector, FOXO1-TM, FOXO3a-TM or FOXO4-TM along with reporter plasmids, p6xDBE-luc and pRL-TK Total RNA was extracted using the RNeasy Mini Kit  and first strand cDNA was synthesized with the Omniscript RT kit (Qiagen), using 1 µg of RNA per 20 µl reaction and oligo(dT) primer. cDNA was then utilized in PCR reactions for FKHR, FKHRL1, AFX and GAPDH.32P] dATP by incubating oligodeoxyribonucleotide strands with 5× reaction buffer and 10 U T4 polynucleotide kinase for 1 h at 37°C. Then labeled oligonucleotides were allowed to anneal at room temperature for 10 min and 20 µg protein from each sample was used in 25 µl binding reactions, which consisted of 1 µg poly dI-dC, in 5× binding buffer  and 1 mM DTT). To determine specificity of DNA binding, samples were incubated with or without 20 ng of unlabeled competitor DNA for 10 min at room temperature. Then 0.1 ng of labeled probe was added and samples were further incubated for 20 min at room temperature. Samples were separated on a 5% non-denaturing polyacrylamide gel in 0.5% TBE and visualized by autoradiography.FOXO probes were end-labeled with [γ-The mean and SD were calculated for each experimental group. Differences between groups were analyzed by one or two way ANOVA using PRISM statistical analysis software . Significant differences among groups were calculated at P<0.05.


Chronic Obstructive Pulmonary Disease (COPD) is a complex pathological condition associated with an important reduction in physical activity and psychological problems that contribute to the patient's disability and poor health-related quality of life. Pulmonary rehabilitation is aimed to eliminate or at least attenuate these difficulties, mainly by promoting muscular reconditioning. The scope of this paper has been the analysis of the literature on pulmonary rehabilitation in COPD patients has appeared in the last five years, focusing on the principal outcomes obtained. The results demonstrate that pulmonary rehabilitation has a beneficial effect on dyspnoea relief, improving muscle strength and endurance. Moreover, pulmonary rehabilitation appears to be a highly effective and safe treatment for reducing hospital admissions mortality and improving health-related quality of life in COPD patients. It represents, therefore, a very important therapeutic option that, along with standard pharmachological therapy, can be used to obtain the best patient management. The favourable results obtained with pulmonary rehabilitation programs should stimulate researchers to improve our understanding of the mechanisms that form the basis of the beneficial effects of this therapeutic intervention. This would in turn increase the effectiveness of pulmonary rehabilitation in COPD patients. Pulmonary rehabilitation is defined by the American Thoracic Society and the European Respiratory Society as an “evidence-based, multidisciplinary, and comprehensive intervention for patients with chronic respiratory diseases who are symptomatic and often have decreased daily life activities.” As such it is an integral part of the clinical management and health maintenance of those patients with chronic respiratory disease who remain symptomatic or continue to have decreased lung function despite standard medical treatment. Integrated into the individualised treatment of the patient, pulmonary rehabilitation is designed to reduce symptoms, optimise functional status, increase participation, and reduce health care costs by stabilising or reversing systemic manifestations of the disease . All togCOPD is characterised by complex and diverse pathophysiologic manifestations. The inflammatory pulmonary process, principally triggered by cigarette smoke, induces a series of molecular and cellular reactions with detrimental effects on lung tissue . The maiIn spite of the patient's attempt to adopt more convenient breathing patterns, these adaptations are generally overwhelmed during exercise, when there is an acute increase in the ventilatory demand. Acute and chronic hyperinflation have been shown to contribute to exertional dyspnoea, reduced ventilatory capacity, and worsened exercise performance in COPD , 9. WastAlthough the initial pathology of COPD is confined to the lung, the reduction in physical activity and psychological problems associated to the progress of the disease increasingly contribute to the patient's disability and poor health-related quality of life. This forms the basis of the most important clinical manifestations of COPD, such as muscle dysfunction, cardiac impairment, skeletal and sensory deficits, malnutrition, and steroid-related myopathy , besidesExercise training is an important aspect of pulmonary rehabilitation, as it represents the best available means of improving muscle performance, with remarkable favourable impact on exertional dyspnoea, exercise tolerance, and improvement of daily activities . TraditiEducation of the patient is a core component of a complete rehabilitation program, together with the prevention and early treatment of respiratory exacerbations, implementation of breathing strategies, and bronchial cleaning. The combination of postural drainage, percussion, and forced expiration improve airway clearance, while the use of a positive expiratory pressure mask and assisted coughing have proven to be more effective than assisted coughing alone in COPD patients during an exacerbation . In factPulmonary rehabilitation programs should also address body composition abnormalities, which are frequently present but underrecognised in chronic lung diseases. Interventions relating to these aspects may be in the form of caloric supplementation, physiological interventions, pharmacological strategies, or combination therapy in order to induce weight gain without an overall fat mass increase. All of these interventions have resulted in an improvement in quality of life and justify the decision taken by official organisations to recommend pulmonary rehabilitation as an integral part of the long-term management of COPD –17. While the utility of pulmonary rehabilitation is undisputed, no general consensus exists regarding the parameters that should best represent the improvements achieved with pulmonary rehabilitation. Indeed, the various research groups have focused on different parameters, like exercise performance, endurance, dyspnoea, and quality of life, while little or no attention have been paid to a number of parameters concerned with respiratory mechanics and gas exchange. The absence of homogeneity regarding the study endpoints largely limits the comparison among the various studies, besides evaluation of their results. With this in mind, we have analysed the literature on pulmonary rehabilitation in COPD patients that has appeared in the last five years, focusing on the main outcomes used and their evaluation. We conducted a MEDLINE search using the keywords “pulmonary rehabilitation” and “COPD”: of the resulting 1294 articles, 574 had been published in the last five years, but only 398 had pulmonary rehabilitation as the relevant issue. These papers could be classified as follows: 121 clinical trials, 78 randomised clinical trials, 10 meta-analyses, 4 practical guide lines, 131 reviews, and 54 systematic reviews . Guide lIn all studies, the duration of the pulmonary rehabilitation programs was six-to-twelve weeks. Furthermore, all programs were based on a multidisciplinary approach: exercise training, patient education, psychosocial and behavioural interventions, and nutritional therapy to contrast weight loss and muscle wasting. It is important to underline that only three papers made the distinction between primary and secondary outcomes. Furthermore, two of them have evaluated the improvement of the quality of life after the Saint George Respiratory Questionnaire (SGRQ) score as the primary outcome , 21, wheThe conclusion common to all papers listed in Twelve studies have analysed the six-minute walking test distance (6 MWTD) as a functional parameter; all of them concluded that pulmonary rehabilitation of COPD patients leads to an increase of the covered distance. Nine of those papers also evaluated other variables such as the incremental shuttle test, leg strength, and the peak oxygen intake and found that they were correlated with the 6 MWTD. Moreover, in a retrospective analysis involving a cohort of 815 severe or very severe COPD patients undergoing a pulmonary rehabilitation program based on increasing exercise tolerance, transfers, and stair climbing, Enfield et al.  found thForced Expiratory Volume in the first second (FEV1) as a functional parameter. Stav et al. [1 decline or even a suppression of that decline after three years of pulmonary rehabilitation, while Ergün et al. [1 after 8 weeks of pulmonary rehabilitation. Only 3 studies have considered v et al.  reportedn et al.  and Chann et al.  found no1 were more likely to enjoy an attenuation of breathlessness, besides greater improvement of both subjective (SGRQ) and objective outcomes (6 MWD).Four studies evaluated dyspnoea using the MRC or Borg scale. Ergün et al.  demonstrFew data are available concerning the effectiveness of pulmonary rehabilitation on dyspnoea relief in less severe COPD patients. In a 2-year randomised controlled trial on patients with moderate airflow obstruction but impaired exercise capacity, it was found that a significantly greater decrease of MRC dyspnoea score from baseline occurred in the group of patients subjected to active treatment .Ten studies have evaluated the improvements in quality of life using either the SGRQ, CRQ, or Hospital Anxiety Depression (HADs) scale. Van Wetering et al.  have conIt should be stressed, however, that these studies were carried out on patients with a stable disease, while it is well known that exacerbations are an important and negative prognostic element in the natural history of the disease , becomin The purpose of this paper has been to analyse the outcomes used in studies on pulmonary rehabilitation in COPD patients published over the last five years, besides the efficacy of these treatments in improving the quality of life and the ability in carrying out daily life activities. Almost all studies have assumed as a primary outcome for the evaluation of the pulmonary rehabilitation programs the distance covered during the six-minute walking test and the peripheral muscle strength, with less attention paid to the impact on the quality of life.A growing amount of literature advocates home-based rehabilitation as a useful adjunct for COPD management , 43. HomOf particular interest is the hypothesis that pulmonary rehabilitation, through the exercise and nutritional intervention, could reduce the risk of cardiovascular accidents , becauseThe primary goal of pulmonary rehabilitation should be, however, the improvement of lung mechanics, in order to lower the work of breathing and restore ventilation-perfusion distribution, with enhanced gas exchange and exercise performance. These pathogenetic cornerstones of COPD should be treated both with pharmacological bronchodilation and pulmonary rehabilitation, in order to reduce respiratory symptoms in stable patients and during exacerbations , 53. TheCurrent literature supports the notion that pulmonary rehabilitation provides clinically relevant improvements in quality of life, breathlessness, exercise performance, and psychological status. Also the usefulness of the association of conventional pharmacological treatment and pulmonary rehabilitation has been repeatedly proven . However