Informal caregivers play a pivotal role in supporting patients approaching the end of life. The Carer Support Needs Assessment Tool (CSNAT) is designed to facilitate person-centred assessment and support through a process that is practitioner-facilitated, but carer-led. This study explored practitioners’ experiences of implementing the CSNAT in palliative homecare.We conducted qualitative interviews/focus groups with 20 practitioners in one UK hospice homecare service  before and after the implementation of the CSNAT. Thematic analysis of the data was underpinned by framework analysis.Not all practitioners appreciated that using the CSNAT required a shift towards a more person-centred approach to assessment; consequently they tagged the tool onto their existing practitioner-led practice. Practitioners who did use the CSNAT as intended were able to act as role models and support their colleagues in making this transition. Practitioners’ comments revealed a number of contradictions: 1) Most felt that they ‘already do’ identify carer support needs, but feared using the CSNAT could increase their workload; 2) some worried about introducing the CSNAT ‘too soon’, but recognised that it was ‘too late’ once patients were close to the end of life; 3) whilst practitioners stated ‘they were there for the family as well as the patient’, care provision was overtly centred around patients.This study provides vital insights into barriers and facilitators to implementing the CSNAT as part of a person-centred approach to assessment. The findings identified the training and support required to help practitioners make this transition to this new way of working. The pivotal role played by informal caregivers in supporting patients approaching the end of life (EOL) in a home care setting is recognised ,2. FolloHospice services play a key role in carer support, given that they are there for the family as well as the patient . HoweverWhilst carer assessment tools exist, they often consider the impact of long-term caregiving . Tools sThe Carer Support Needs Assessment Tool (CSNAT) was developed in response to the need for a comprehensive assessment tool that was suitable for use in practice by both carers and practitioners ,19. The The CSNAT provides a framework for a process of assessment which commences when the CSNAT is introduced to the carer. The carer is then given time to consider their support needs, using the domains as a prompt. A subsequent ‘assessment conversation’ with a practitioner enables the carer to prioritise their support needs and indicate the kind of support they would find helpful. This person-centred approach  facilitaThe central tenet of the CSNAT is that it should be used as part of an assessment process which is facilitated by the practitioner, but led by the carer. Fundamental to this process is that the carers’ perspectives take centre stage not only in identifying support needs, but also suitable solutions. Consequently, incorporating the CSNAT into practice requires a fundamental, albeit subtle, shift into how carers’ support needs are identified and responded to.We conducted a feasibility study to explore the potential role of the CSNAT in improving carer support in palliative home care. The aims were to:explore practitioners’ experiences of implementing the CSNATuse practitioners’ experiences to inform the development of a training guide to support implementation of the CSNATThe study was successful in identifying the key barriers and facilitators to individuals’ adoption of the CSNAT and the implications for practice and training.The views of family carers regarding the CSNAT were also sought; however these findings are beyond the focus of this paper.A qualitative study design was used. Data were collected via individual interviews and focus groups, and were supplemented by field notes.The study was conducted in one UK hospice home-care service .The CSNAT was implemented as part of service development, as such there was an organisational expectation that practitioners would incorporate the CSNAT into routine practice. Training was provided for all practitioners by the service manager and the authors. This one-hour session provided details about development of the CSNAT and guidance on how to use the tool in practice; it also provided practitioners with an opportunity to meet the researchers and gain an understanding of the purpose of the study.The qualitative research ran in parallel to implementation. Whilst all practitioners were eligible to take part in the study, participation was voluntary. Invitations were sent to all 22 qualified nurses, of which18 participated (82%). Two HCAs who used the CSNAT also volunteered to take part.Data were collected between February 2011 & January 2012. All authors were involved in data collection and field notes were made throughout the study (LA).Focus groups of between 1–1¼ hours were held at three stages  and guidPre-implementation focus groups explored existing approaches to identifying carer needs, initial responses to the CSNAT and how staff anticipated using the tool in practice. The second round, four months post implementation focused on practitioners’ experiences of using the tool and the implications for training and support. The final meeting, 11 months post implementation acted as a closing session for the study. A number of recurrent themes emerged throughout this process with ‘data saturation’ of key themes.Audio recordings were transcribed in full and thematic analysis conducted following the principles of framework analysis . Transcrth January 2011 (reference number 10308). Written consent was gained from participants when joining the study and reconfirmed at subsequent stages.University ethics approval was received from The University of Manchester Committee on the Ethics of Research on Human Beings on 6A unique alphanumeric code is used in the text to identify participants. We have adopted a generic term ‘P’ for all grades of staff to maintain participants’ anonymity.The principles of rigour, pertaining to qualitative studies, were observed throughout the study; the COREQ criteria  have guiThe 20 participants comprised 14 CNSs, three sisters, one staff nurse and two HCAs who, collectively, had worked for the service between 20 months and 12 years. They provided information on existing practice relating to carer support, and barriers and facilitators to implementing the CSNAT. Their comments provided invaluable help for development of a training guide for CSNAT implementation.Practitioners stressed that they ‘already do’ identify carers’ support needs as part of usual practice. However, further discussion established that current approaches were informal and shaped by practitioners’ perceptions of carers’ needs, with ‘joint assessment’ of patients and carers being common:P20 We don’t actually use a tool when we do an initial assessment…although you have an agenda of your own…In your head are the things you need to extract from them to do your initial assessment.Subsequent contacts with the family provided further opportunities for the carer’s needs to be raised. Practitioners noted the importance of developing relationships in order that carers would feel comfortable enough to discuss their concerns, allowing issues to emerge naturally:P24 I think as the patient’s journey goes on, and you’re building up a rapport with the patient and the carer, things crop up.As part of this on-going relationship, exploration of concerns could be initiated by the practitioner who looked for ‘cues’ as to when the carer wished to talk, enquiries commonly framed in general terms such as ‘How are you?’ Carers could also initiate discussions, the frequently cited ‘doorstep conversations’ being the archetypal example:P10 Sometimes you’re stood on the doorstep. These are the ‘I don’t want them to know I’m talking to you’ conversations…that might be the time when you say to them ‘And how are you?’Practitioners’ ‘initial response to the CSNAT was broadly favourable. The CSNAT domains reflected issues known to be of relevance to those in a caring role, as such, the CSNAT did not contain any ‘surprises’. However, comments indicated that the very resonance of the domains left some practitioners unclear as to how the tool could bring about tangible benefits:P21 In some ways I hope it doesn’t change our practice in that I hope we’re covering it all already, so if that is the case then it won’t hugely affect how we address things.The comment above illustrates that some practitioners had not fully appreciated that implementation of the CSNAT required a shift from existing practice  to the approach required to incorporate the CSNAT . This perhaps accounts for P21’s description of how she attempted to ‘tag’ the CSNAT onto her existing practice:P21 I tend to do my whole assessment with the patient and the carer…as before, and give them information, at that point, as I would have before… but then say, “—and this is the form.”The construction of the CSNAT as a ‘form’ or ‘paperwork’, as opposed to an opportunity for carers to express their support needs is of interest and has clear implications for training. This perception could inevitably shape how the CSNAT was introduced and subsequently interpreted by carers:P11 People don’t like forms, do they? They struggle with things, like the attendance allowance and if that was left to their own devices they wouldn’t fill that in half the time. So I think some people, they just get overwhelmed, don’t they, and it’s another form that somebody wants them to fill in.Comments by others suggested that they construed the CSNAT as a ‘tick-list’ to demonstrate that topics had been discussed, e.g. during ‘joint assessments’. The primary benefit of the CSNAT being that, if nothing else, it made existing practice visible to others both within and beyond the organisation:P20 I think it does make additional work but, I think, in this day and age [with] …commissioning, and things like that, then, we have to be able to be seen to be providing that service and, perhaps, it helps us in doing that.Tagging the CSNAT onto existing practice in order to document assessments was not uncommon during early stages of implementation. However, there was some recognition that the CSNAT required a shift in practice; most notably it required practitioners to make an overt distinction between the needs of the patient and the carer and ask about needs more directly. This led to some fears about the potential impact on workload due to the time required to undertake assessments and respond to support needs identified. One practitioner [P21] felt the overt assessment of carers could effectively double her caseload, others were concerned that extending the length of visits to accommodate separate assessment could be problematic for both the practitioner and the family:P10 I haven’t got negative feelings about [the CSNAT]. I worry a little bit that it might extend the period of time that we are there, which already can be quite a long time.Furthermore, there was a fear that presenting carers with a predefined ‘list’ could trigger identification of previously unmet needs. Practitioners expressed discomfort with this as it could lead to carers having ‘unrealistic expectations’ as to what the hospice service could provide:P12 I was going to say it could impact on time if the carers are given that focus that we’re there for them and…they may realise that they do need more and so we may actually end up giving more of our time to the carers, as well as the patients. So that could impact on the workload.The fear of ‘opening up a can worms’, in relation to needs identified, was related to discomfort felt by practitioners when presented with a concern that they may not be able to respond to or ‘fix’, the following practitioner giving a very open account of how she might feel:P10 I don’t know if it’s a nurse thing or what, but…if I’m going to be picking up things on here that I can’t fix that might make me feel uncomfortable…I know we can’t fix everything and I’m used to that day in/day out, but [the CSNAT] will make it very much in my face.Concerns were also expressed about appropriate timing of assessment both pragmatically and with regard to the impact on the carer and the practitioner-carer relationship. The first visit was commonly cited as ‘too soon’ as the patient would inevitably be the focus of attention for both practitioner and carer:P14 You would be more focused on the patient, possibly. Not saying that the carer isn’t important, but…getting the patient details is obviously more pertinent…To me that would be more a priority than filling out even more paperwork.Conversely, introducing the CSNAT to carers of patients close to the end of life was frequently viewed as inappropriate, given the need to assess and respond to the patient’s needs in the short space of time available:P11 I’ve felt more comfortable leaving [the CSNAT] at earlier stages…I’ve struggled, perhaps, more when I’ve gone in to meet someone who is, actually, very much approaching end-of-life…I’ve always felt leaving the form gives [the carer] something else to worry about.Finding a suitable point to introduce the CSNAT was dependent, nominally, on how long the family was likely to be in contact with the service and frequency of visits. However, some practitioners were concerned that, if introduced at the ‘wrong’ time, the CSNAT could distress carers by ‘forcing’ them to think about issues before they were ready, or even cause them to worry about things that they hadn’t anticipated:P14 It might bring things up as well that maybe at that particular time the carer’s not prepared to, or doesn’t want to, identify…So, I don’t know, by asking that are you bringing things out that they are not ready to discuss?This ‘gatekeeping’ role with regard to when to introduce the CSNAT was driven by both practicalities, such as visiting patterns, and a desire to be sensitive to the perceived readiness of the carer to discuss some issues. It was feared that introducing the CSNAT at the wrong time would not only fail to elicit carers’ support needs, it could also distress the carer and potentially damage a fledgling relationship:P11 You’ve almost got to try and maintain that relationship…and keep that foot in the door to try and be there throughout and I think, sometimes, if you try and push too much or encourage too much, it can be to the detriment of that relationship so…sometimes, then, filling forms in doesn’t feel appropriate…I think it’s that gut instinct…and our experience that actually helps you make that decision as to whether it’s actually going to be helpful or not.As seen, barriers to implementation related, largely, to the extent to which practitioners felt the CSNAT could enhance existing practice. Consequently, practitioners frequently used their judgement as to whether the tool would be beneficial in a particular circumstance, as opposed to using routinely for all carers. However, a number of practitioners did perceive the CSNAT as an enhancement to existing practice and were able to incorporate the tool in a meaningful way. The following practitioner gave a clear example of how using the CSNAT overcame the limitations of existing practice:P21 on one occasion, a patient said to me, oh, she wants to go through the [CSNAT] with you…the relative had never previously made any move to talk to me, or sit with me, or be part of the conversation. So there are conversations in which it has been really helpful in just giving that opportunity, which I might not otherwise have had’Similarly, some respondents felt the CSNAT had improved their existing practice by acting as a tool to facilitate communication:P14 Now that we are going through this…the carers are having the questions put to them rather than the CNS picking it up in conversations, more of a direct [other agreeing] you know… ‘do you need more support with -?’ It’s more of a direct contact than just picking it up in little snippets.Those who felt positive about the CSNAT appeared to have understood the underlying principles of the approach which distinguished carers’ and patients’ needs and allowed the carer to prioritise their support needs and identify potential solutions:P03 I think it's good in a sense that it gets them, not us—because we can go in and say you need this, this and this, but this isn't designed for the patient's needs, it's designed for the carer's needs. So I think it's good that it gets them to prioritise what they feel their needs are, certainly not what we think.These natural role models were able to champion this person-centred approach to assessment. For example, the focus groups provided an opportunity for these natural ‘champions’ to share examples of how they had integrated to CSNAT into their way of working and enhance carer support. These champions also used the focus groups to help problem-solve issues or challenge perceptions. This can be seen in the following exchange where practitioner P11 responds to a colleague’s concerns:P20 When they’re looking at the [CSNAT] questions that say “Do you need more advice on help with your personal care?” and things like that, they don’t want to look at what might be happening in the future…it’s the here and now. And that actually sometimes makes them think about; “well, does this mean that…I’m not going to be able to stay on my own at night?” and things like that.P11 But I guess turning it around, it may well be reassuring for some families to actually talk about; “well actually, if I got to that point and I did need help, [I’d] know who to ring, or what support might be available.”These informal exchanges took place on other occasions, including where practitioners shared office space or attended team meetings. These conversations enabled colleagues to reflect on how they were using the CSNAT, share examples of good practice and support each other during the transition to this new way of working.Participants provided invaluable insights into their existing practice regarding identification of carers’ support needs, their perceptions of the CSNAT, and their feelings about using the tool. Collectively, the factors which facilitated or acted as barriers to implementation fed directly into the training developments.Many practitioners recounted occasions on which the tool proved beneficial . HoweverUse of the CSNAT was also affected by practical issues such as workload, visiting patterns, sensitivities around potential discomfort for carers, and the extent to which carers’ needs could be accommodated separately from those of the patient. Consequently, for a number of practitioners, practice in relation to carer assessment remained largely unchanged; the CSNAT simply acting as a ‘form’ for recording ‘existing practice’.Understandably, it could take practitioners time to become fully acquainted with how to use the tool. However, some practitioners appreciated the potential benefits of the CSNAT at an early stage. These role models were willing to engage with their colleagues in group settings to help them overcome perceived problems in relation to the implementation of the CSNAT.Interestingly, discussions with practitioners revealed what appeared to be a number of contradictory comments; these related to identifying carers’ needs, when to introduce the CSNAT, and the carer as a ‘client’ of the service. The following sections explore three of these apparent contradictions alongside the literature which helps to illuminate them.The CSNAT did not, of course, arrive in a vacuum, however, existing practice was informal and practitioner-led, reflecting practice in hospice services more widely . Given tThe concerns suggest that there were some misconceptions about the tool. For example, the perception that the CSNAT will ‘open up a can of worms’ and identify issues that the practitioner would then need to ‘fix’ runs counter to the idea of a carer-led, practitioner-facilitated approach in which the carer prioritises their needs and helps to identify potential solutions.The desire for practitioners to ‘fix’ problems and feelings of guilt if they are unable to is a recognised phenomenon ,28. WhilFor some, the fear that the CSNAT could increase workload was enough to limit use of the tool. However, even practitioners who planned to introduce the CSNAT found it difficult to find a suitable ‘window of opportunity’. Whilst practical issues such as visiting patterns came into play, the overriding factor seemed to be sensitivities around when it was appropriate to broach potentially distressing topics. There was a misconception that the CSNAT might force people to consider issues before they were ready, as opposed to the domains legitimising concerns and providing the opportunity for carers to open up a conversation about topics important to them. Conversely, if the patient was very close to the end of life it was felt to be ‘too late’ to discuss the carer’s needs given the speed at which events were occurring.Determining the appropriate time to open up ‘difficult’ conversations is not unique to carer assessment. Boot and Wilson  found thDeveloping relationships was valued as practitioners believed that carers’ needs would come to the fore as the relationship progressed. Hill et al  challengWhilst services were nominally ‘there for the family as well as the patient’, it was clear that this was subject to a number of provisos. Although ‘joint assessments’ took place, patient’s needs were clearly prioritised. Likewise, patient assessment formed part of routine practice, whereas carer ‘assessment’ was less overt and subject to the practitioners’ perception of whether it was appropriate. Explicit provision for carers was largely viewed as ‘extra’, as revealed by the comment that the caseload would ‘double’ if carers’ needs were to be assessed separately from those of the patient. This leads to the question as to whether existing practice constitutes ‘assessment’.Whilst carer assessment has been much discussed in policy and professional literature, guidance on how this can be achieved is relatively sparse . In contThe apparent contradictions raised in this discussion can be understood when viewed in the light of how ‘existing’ practice enables practitioners to contain some of the demands placed on them. Whilst practitioners ‘already do’ consider carers’ needs, the extent to which these are explored is limited by time constraints, fear of causing distress, and the conflation of patients’ and carers’ needs. That is, practitioners exert some control over what is discussed, who it is discussed with and when. Consequently, practitioners’ sensitivity to carers’ support needs did not necessarily translate into an opportunity for carers to express these needs in full. In view of these findings it is perhaps not surprising that practitioners may have had reservations about incorporating the CSNAT into routine practice. When used as intended, the CSNAT clearly shifts the locus of control to the carer, allowing them to shape the assessment process and determine which support needs they would like to discuss and when.In summary, adoption of the CSNAT was not straightforward for many practitioners as they had an established working pattern which helped them manage demands placed on them whilst being sensitive to the carers’ needs. Implementing change is not straightforward, a prerequisite to success being that the change desired is viewed as beneficial .The CSNAThe misconceptions of some practitioners have implications for practice as it is clear that practitioners require time to make the transition to this new way of working. Consequently, the means by which the underlying tenets of the CSNAT are conveyed to practitioners requires further consideration if successful implementation of the tool is to enhance practice. As a result of this study the training and on-going facilitation relating to implementation have been substantially revised . In partThe study provides key information relating to the adoption of the CSNAT by individual practitioners working within hospice homecare. Whilst some information was gained on the contextual factors practitioners work within, this was not extensive. Context can clearly play a key role in the implementation of a new way of working. Hospices show wide variation in both the size and nature of services they provide and the composition of teams. Further work has now been undertaken with a larger group of services which will provide more detailed information on key contextual factors which impact on the successful integration of the CSNAT.The study highlights a number of factors influencing uptake of the CSNAT into routine practice. The findings revealed that, as with any tool, the CSNAT it is not a ‘quick fix’ solution to assessing carers’ needs. In order for the tool to be meaningful it needs to be used as part of a person-centred approach to assessment. Practitioners need training and support to make a transition from existing practitioner-led practice to one which incorporates a carer-led approach. Whilst this new approach builds on existing skills it differs from what practitioners ‘already do’. Transition to this new way of working needs to be underpinned by training in the underlying principles of this approach and requires on-going facilitation until this new way of working is embedded into routine practice.


Clinical evidence supports the use of second-generation dopamine D2 receptor antagonists (D2RAs) as adjunctive therapy or in some cases monotherapy in patients with depression. However, the mechanism for the clinical antidepressant effect of D2RAs remains unclear. Specifically, given accumulating evidence for decreased ventral tegmental area (VTA) dopamine system function in depression, an antidepressant effect of a medication that is expected to further reduce dopamine system activity seems paradoxical. In the present paper we used electrophysiological single unit recordings of identified VTA dopamine neurons to characterize the impact of acute and repeated administration of the D2RA quetiapine at antidepressant doses in non-stressed rats and those exposed to the chronic mild stress (CMS) rodent depression model, the latter modeling the hypodopaminergic state observed in patients with depression. We found that acute quetiapine increased dopamine neuron population activity in non-stressed rats, but not in CMS-exposed rats. Conversely, repeated quetiapine increased VTA dopamine neuron population activity to normal levels in CMS-exposed rats, but had no persisting effects in non-stressed rats. These data suggest that D2RAs may exert their antidepressant actions via differential effects on the dopamine system in a normal vs. hypoactive state. This explanation is supported by prior studies showing that D2RAs differentially impact the dopamine system in animal models of schizophrenia and normal rats; the present results extend this phenomenon to an animal model of depression. These data highlight the importance of studying medications in the context of animal models of psychiatric disorders as well as normal conditions. However, the therapeutic mechanism of these agents in depression is largely unknown. For quetiapine specifically, existing theories highlight the role of a drug metabolite N-desalkyl quetiapine, which functions as a potent norepinephrine reuptake inhibitor2. However, this model does not incorporate the D2RA properties of quetiapine.Quetiapine and other second-generation dopamine D2 receptor antagonists (D2RA), e.g., lurasidone, are increasingly popular monotherapy and as an adjunct to other antidepressant drugs in patients with inadequate responses to traditional antidepressants alone6. Thus, it is important to reconcile how a compound that is thought to block dopamine transmission may actually help a condition that results from already reduced dopamine system function7. Importantly, existing theories are based on data from administering quetiapine to normal rats, whereas the effect of quetiapine on the dopamine system in rodent depression models has not been examined. However, as has become clear in other settings such as animal models of schizophrenia, the effects of D2RAs on a normally functioning dopamine system often differ substantially from their effects on a dopamine system functioning in a pathological state8. It is therefore critical to examine the impact of quetiapine on animal models of depression that exhibit clinically relevant pathological functioning of the dopamine system.Mounting evidence suggests that in depression the dopamine system is underactive9. Most dopamine neurons alternate between these two firing patterns, however in order to burst fire a neuron must already be in a spontaneously active state. Using single unit electrophysiological recordings of identified dopamine neurons, one can quantify the relative number of spontaneously active dopamine neurons, as well as the proportion of their firing that occurs during a burst. We have recently reported that across multiple animal models of depression, including chronic mild stress (CMS)10 and learned helplessness11, the number of spontaneously active dopamine neurons is markedly reduced without changes to their firing rate or amount of burst firing. In the CMS model specifically, we have further elucidated that this decrease in dopamine population activity following exposure to chronic stress is due to enhanced afferent GABA-ergic input from the ventral pallidum driving dopamine neurons into a silent state, as inactivation of this region restores dopamine population activity to normal levels12.Dopamine neurons fire in two patterns: a slow, irregular discharge pattern of tonic activity, and a rapid sequential discharge of burst firing13 and human patients with depression14 suggest that restoring normal dopamine system function is a valuable approach to treating depression, particularly in cases with prominent dopamine related symptoms such as anhedonia. Thus, if quetiapine were to increase the population activity of dopamine neurons, this would be expected to contribute to its antidepressant effect. While the effects of quetiapine on the dopamine system have been extensively studied in normal rodents, the effects of quetiapine on the dopamine system in animal models of depression have not been examined. Specifically, acute administration of quetiapine at antidepressant doses has been shown to increase dopamine population activity15, whereas after repeated administration dopamine neuron population activity is at baseline or lower levels16. However, behavioral studies using the CMS model suggest that quetiapine may have the ability to reverse deficits in behavioral assays relevant to depression, specifically when administered at doses of 10 mg/kg for long durations17. This dose is comparable to 150–300 mg daily in humans, which has been supported to have antidepressant effects and is substantially lower than the doses used for schizophrenia18. This suggests that the effects of repeated quetiapine may differ in the presence of factors driving a hypodopaminergic state, e.g., chronic stress.Multiple studies across animal modelsIn the present study, we evaluated the acute and repeated treatment effects of antidepressant dosage of quetiapine in both non-stressed and CMS-exposed rats. We find that acute and repeated quetiapine administration produces strikingly different effects in non-stressed vs. stressed rodents.All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.Adult male Sprague-Dawley rats  weighing 300–340 g at baseline were used for all experiments. They were acclimated to the facilities for >7 days, housed in pairs in a temperature (22 °C) and humidity (47%) controlled colony room (lights ON 7:00 a.m.–7:00 p.m.). Food and water were available ad libitum. Sample size was determined by calculating the group size needed to achieve power ≥ 80%. Rats were randomly assigned to CMS or control and drug or vehicle groups.12. CMS exposed rats were single-housed in individual cages within an isolated colony room. Stressors included periodic restricted access to food and water, cage tilt, damp bedding, continuous overnight illumination, intermittent paired housing with an unfamiliar cage-mate, white noise (80–90 dB), stroboscopic lighting, and predator odor, all delivered in the home cage. Each week rats were exposed to 3–4 stressors in a pre-determined order consistent with our prior published studies19.CMS was performed as reported previously15 and behavioral17 effects at this dose. Vehicle treated rats received 1 ml/kg injections of the same solution without the addition of quetiapine; the two solutions were equivalent in pH and temperature. Compared to humans, rats produce markedly lower levels of the norquetiapine metabolite from quetiapine, allowing us to partially dissociate the D2RA effects of quetiapine from the norepinephrine effects of the norquetiapine metabolite20.Quetiapine 10 mg/mL was dissolved in 0.3% tartaric acid in physiological saline and neutralized with sodium hydroxide to a final pH of 5-6. This dose (10 mg/kg) was selected based on prior studies showing electrophysiologicalElectrophysiological recordings were performed counterbalanced for light phase and experimenter across groups. Rats anesthetized with chloral hydrate  were placed in a stereotaxic frame  and maintained at 37 °C using a thermocouple-controlled heating pad . To access the VTA, the skull was cleared of skin and fascia, and a partial craniectomy of the skull was performed 5.3–5.7 mm posterior and + 0.6–1.0 mm lateral to bregma.Single barrel electrodes were constructed with a vertical electrode puller  using 2 mm diameter borosilicate capillary tubes , and then broken to a target of 6–10 MΩ under microscopic control and filled with a 2 M saline solution with 2% Chicago Sky Blue (Sigma Aldrich). Signals were fed through silver wire to an amplifier  operated with open filter settings , displayed on an oscilloscope , and stored on a computer running Lab Chart 7 . Units were recorded when signal-to-noise ratio exceeded 3:1. Electrode integrity was ensured by replacing electrodes and repeating tracks when either (1) no cells with biphasic waveform (either dopamine or non-dopamine) were identified on two consecutive tracks, or (2) an acute obvious change in recorded neuron shape or background noise level suggested damage to the electrode.10. Electrodes were lowered into the VTA using a manual hydraulic microdrive (Kopf Instruments) and dopamine neurons were identified using well-established criteria including location, slow (<12 Hz) and irregular firing pattern, and long duration action potential  with variable shape and biphasic waveform22. Identified dopamine neurons were recorded for 3 min (1 min minimum), and three parameters of dopamine neuron firing were calculated: (1) the number of spontaneously firing dopamine neurons identified in each track , (2) firing rate, and (3) proportion of spikes occurring as burst firing (%SIB) in which the burst onset was defined as two spikes with ≤80 msec interspike interval and termination by >160 msec interspike interval23. Additional bursting characteristics historically examined in studies of dopamine neurons were also calculated including burst duration (sec), bursting rate (Hz), number of spikes per burst, intra-burst inter-spike interval, and coefficient of variation. No significant group differences in these parameters were found  was then delivered intravenously at autoreceptor-selective dosages via the lateral tail vein until a decrease in firing rate of >20% was observed, with most rats showing response with 40–80 µg/kg, consistent with prior studies8. The VTA was then resampled for population activity. These recordings were counterbalanced across animals for anteroposterior starting location within the VTA.Final electrode placement at the conclusion of recording was marked by electrophoretic ejection of Chicago Sky Blue. Rats were overdosed with additional chloral hydrate and decapitated. Brains were removed and fixed in 8% paraformaldehyde followed by 25% sucrose for cryoprotection. Sixty micrometer coronal sections were prepared using a cryostat , mounted onto gelatin-chromalum coated glass slides, and stained with cresyl violet and neutral red for microscopic confirmation of recording electrode location. Rats were excluded from the study if histological review showed recording electrode location outside of the VTA. Histological review was conducted with the investigator blinded to an animal’s experimental group.Spike time courses for individual neurons were exported from Lab Chart into Neuroexpolorer  for calculation of firing rate and burst firing. Within each rat, population activity was calculated by dividing the total number of dopamine neurons identified by the number of valid tracks performed, yielding the metric of Cells Per Track (CPT). The average firing rate and amount of burst activity for dopamine neurons recorded were averaged across rats in a group, considering each cell as an independent replicate. Data were analyzed in SPSS 23  and Prism 7 . For experiments 1 and 2, 2-way ANOVAs with post-hoc tests were performed to compare treatment groups. For experiment 3, paired t-tests were performed to compare data collected before and after apomorphine administration.F = 4.3, p = 0.049, Fig. n = 8; CON-QTP-Acute 1.4 ± 0.068 CPT, n = 11; 2-way ANOVA interaction F = 4.3, p = 0.049; post-hoc t25 = 3.3, p = 0.0054; Fig. 15. There was no change in the average firing rate of recorded dopamine neurons  2 h prior to assessing VTA dopamine neuron firing properties. This resulted in a significant interaction effect between treatment with acute quetiapine and exposure to CMS for dopamine population activity  = 72.6; p < 0.0001; post-hoc t25 = 4.2, p = 0.0006; Fig. n = 69; CMS-VEH-Acute 3.7 ± 0.30 Hz, n = 19; 2-way ANOVA p > 0.05 for main effects of stress or interaction; Fig. n = 69; CMS-VEH-Acute 29.9 ± 6.1%SIB, n = 19; 2-way ANOVA p > 0.05 for main effects of stress or interaction; Fig. In line with prior results, CMS-exposed rats treated acutely with vehicle had fewer spontaneously active dopamine neurons as compared to non-stressed rats treated acutely with vehicle  = 5.2, p = 0.028; Fig. n = 10; CON-QTP-Repeated 0.92 ± 0.10 CPT, n = 13; t42 = 0.53, p = 0.84; Table n = 78; CON-QTP-Repeated 3.4 ± 0.21 Hz, n = 92; 2-way ANOVA p > 0.05 for main effects of drug or interaction; Fig.  = 5.4; p = 0.020), a post-hoc test within non-stressed rats did not reveal a significant effect  = 2.0; p = 0.084; Fig. We next assessed whether repeated quetiapine administration differentially affected dopamine neuron activity in non-stressed and CMS-exposed rats. As with acute administration of quetiapine, we observed a significant interaction between CMS exposure and repeated treatment with quetiapine  = 5.4; p = 0.020). Histogram review suggests preserved firing rate distribution  was administered via the lateral tail vein (infusion verified by a decrease in firing rate of a single recorded dopamine neuron), and an additional 6–9 tracks of dopamine neuron recording was performed, allowing for pre- and post-apomorphine assessments of dopamine neuron population activity within the same animal. We observed no change in dopamine neuron population activity following apomorphine administration , suggesting that depolarization block did not occur following repeated administration of 10 mg/kg quetiapine.Repeated administration of antipsychotic drugs, including high antipsychotic doses of second generation agents such as quetiapine, have been shown to induce depolarization block following > 21 days of administration to normal ratsIn this study, we found divergent effects of acute and repeated administration of quetiapine in non-stressed and CMS-exposed rats. While acute quetiapine increased dopamine population activity in non-stressed rats, it had no effect on CMS-exposed rats. Conversely, quetiapine administered repeatedly was uniquely capable of restoring normal levels of dopamine neuron population activity in CMS-exposed rats, but had no persisting effects on population activity in non-stressed rats. Furthermore, we show that the return to baseline levels of dopamine neuron population activity in non-stressed rats administered quetiapine for 21 days is unlikely to be due to the induction of depolarization block in VTA dopamine neurons.17. However, as far as we are aware, this is the first study to examine the electrophysiological impact of quetiapine on the dopamine system in rats subjected to an animal model of depression. Our results suggest that quetiapine has significantly different effects on the dopamine system in CMS-exposed rats as compared to its effects in non-stressed rats. These data add to existing literature on the dopamine system impact of acute and repeated quetiapine treatment in normal rats. These studies found that acute administration of quetiapine at doses of 10 mg/kg or greater will induce acute increases in population activity of the A10  but not A9  dopamine neurons15. Moreover, the impact of repeated administration of quetiapine was examined at increasing dosages, and found to induce depolarization inactivation only when administered at doses of 40 mg/kg or greater16. This higher dose is more in line with the doses used clinically in the treatment of psychosis or acute mania , whereas the dose used in the treatment of depression  is more in line with the 10 mg/kg dose used in the present study, as measured by D2 receptor occupancy18.Prior studies have administered quetiapine to CMS-exposed rats and observed behavioral effects consistent with an antidepressant action17. However, whether quetiapine can rescue an existing depressive phenotype remains to be examined.Although behavioral confirmation of the anhedonic state would have been useful to corroborate the electrophysiological studies, no universally accepted behavioral assays are available. Thus, future studies should assess whether stress pre-exposure alters the impact of a one-time dose or repeated doses of quetiapine. Of note, prior studies have demonstrated the ability of quetiapine to prevent stress induced depressive behaviors25. Similarly, the induction of depolarization block by high-dose antipsychotic drugs is also believed to occur via hyperactivation of these pathways, resulting in over-depolarization of the dopamine neuron membrane potential to the point of spike inactivation24. One plausible mechanism by which quetiapine or other D2RA medications could be effective as antidepressants is that, at the low doses used to treat depression, the drugs produce sufficient activation of these feedback pathways to restore dopamine population activity to normal but not sufficient to induce depolarization block. In contrast, the low level of striatal D2 receptor blockade produced by low doses of quetiapine is likely to be compensated homeostatically by increased D2 receptor number, increased dopamine synthesis, increased release, etc. As a result, the normalization of dopamine neuron population activity in the depressed state produced by quetiapine results in a greater restoration of phasic dopamine system responsivity to afferent drive3 compared to the low tonic level of D2 receptor blockade.The mechanism through which quetiapine, like other D2RAs, increases dopamine neuron population activity is believed to occur via activation of striatal-midbrain feedback circuits, given that lesions of these feedback pathways block the antipsychotic drug-induced increase in dopamine population activity26. Thus, D2RAs are hypothesized to enhance accumbal inhibitory drive of the VP, releasing dopamine neurons from VP inhibition and subsequently increasing the number that are spontaneously active. We have shown previously that the VP is also a critical node in the pathway driving the CMS-induced hypodopaminergic state12. Therefore, these collective data lead to the intriguing possibility that convergent and counteracting effects on the VP is the mechanism of action of these D2RA medications’ antidepressant effects, with CMS increasing and quetiapine decreasing VP inhibitory tone over VTA dopamine neurons.The principle brain region believed to impact VTA dopamine neuron population activity is the ventral pallidum (VP), as pre-infusion of the GABA-A receptor antagonist into the VP blocks the increase in dopamine population activity observed following acute administration of sertindole or haloperidol27. Moreover, the binding profile of quetiapine is much different than other D2RA antipsychotic drugs, even within the second generation  class28. Thus, quetiapine demonstrates a unique receptor occupancy curve wherein it binds fleetingly with high potency and rapid dissociation, rather than a prolonged occupancy seen with other compounds29. It is possible that these additional collective qualities may account for the unique ability of quetiapine to act as an antidepressant medication while haloperidol and other more traditional antipsychotic drugs are not effective as antidepressants. Of note, recent clinical data supports a dopamine enhancing mechanism for D2RA treatment in MDD30. It is worth mentioning, however, that quetiapine and its metabolites do have a number of effects on other receptors such as 5-HT1A, which likely also contribute to a major portion of its antidepressant actions31.The approach of using subsets of D2RA medications for an antidepressant effect is a promising therapeutic mechanism. The present study focused only on quetiapine rather than other D2RAs such as haloperidol or clozapine because quetiapine has been shown to possess antidepressant effects clinicallyIn conclusion, the present data support unique and divergent effects of acute and repeated administration of the D2RA quetiapine in non-stressed and chronically stress-exposed rodents. These data offer compelling evidence for further investigating the dopaminergic component of the therapeutic mechanism of select D2RA medications, and highlight the importance of conducting these studies using an animal model of depression in addition to normal rats.Figure S1Table S1Table S2


Using the NMR data, we roughly estimate apparent rate constants for association, dissociation, and translation of α-CD during the pseudo-rotaxane formation based on a simplified kinetic model. These rate constants are indicative of face-selective and ratchet-like translation of α-CD on the axis because of the 2-methylpyridinium residues in the axis. We also evaluate apparent first-order rate constants for the deuteration. Comparison of these rate constants indicates that the face-selective translation of α-CD somehow couples with the deuteration. On the basis of this study, it is concluded that a translational molecular ratchet can be constructed using a large energy gradient with appropriate energy barriers and an enthalpically-driven coupled reaction.In the molecular world, molecular ratchets can realize the unidirectional movement in molecular machines. However, construction of artificial molecular ratchets has been still a great challenge. In this study, we investigate the formation of pseudo-rotaxane of a newly designed two-station axis molecule with α-cyclodextrin (α-CD) and the deuteration of acidic protons in the axis in D These biological molecular motors are based on Brownian ratchet, which withdraws the direction from thermal fluctuations on a sawtooth-shape potential surface7, or on coupling with a chemical reaction driven enthalpically2. In the real world, the ratchet mechanism regulates the direction of movement. Similarly, in the molecular world, molecular ratchets can realize the unidirectional movement in molecular machines11. However, construction of artificial molecular ratchets has been still a great challenge. Some groups have published pioneering works on artificial molecular ratchets. Kelly et al.14 devoted their efforts toward rotational molecular ratchets using triptycene and pawl moieties. Leigh et al.15, Arduini et al.19, and Chen et al.20 have reported translational molecular ratchet based on a rotaxane or pseudo-rotaxane, in which the direction of rotor component is regulated by attaching and detaching a pawl moiety on the axis component. Stoddart et al.21 have elaborated an artificial molecular pump based on a pseudo-rotaxane of cyclobis(paraquat-p-phenylene) and an axis molecule with an isopropyl group as energy barrier, in which redox and thermal stimuli cause unidirectional translation of the rotor component. Leigh et al.24 have realized a molecular information ratchet based on a rotaxane possessing a stilbene moiety, of which the Z–E ratio in the photostationary state depends on the photosensitizer. We have reported face-selective translation of the rotor, i.e., α-cyclodextrin (α-CD), in pseudo-rotaxanes composed of decamethylene stations and methyl-substituted pyridinium residues28, in which the 2-methyl group on pyridinium acts like a barb of fish hook for α-CD. We have also investigated deuteration of acidic protons in a two-station axis molecule catalyzed by α-CD in deuterium oxide (D2O) during the formation of pseudo-rotaxane29. Here we show a pseudo-rotaxane that realizes the face-selective translation of α-CD coincident with the α-CD-catalyzed deuteration in D2O, aiming at a translational molecular ratchet.Living organisms utilize translational and rotational molecular motors, e.g., kinesin, dynein, and ATP synthase, when they have to move or operate29. Figure 1H NMR spectra of a binary mixture of the two-station axis molecule and α-CD measured in D2O at 70 °C. The lower spectrum of Fig. 25, signals at 9.11 and 9.08 ppm are ascribed to the first station included by α-CD from the primary and secondary hydroxy sides, respectively, and signals at 2.90 and 2.88 ppm are assigned to the second station included by α-CD from the primary and secondary hydroxy sides, respectively −1 (black circles). The molality of first station included by α-CD from the secondary hydroxy side increases rapidly corresponding to the rapid decrease in the molality of free first station, reaches a maximum at ca. 1 day, and then decreases gradually (green triangles). On the other hand, the molality of first station included by α-CD from the primary hydroxy side increases gradually to ca. 0.7 mmol kg−1 over 7 days (red squares). As can be seen in the lower figure of Fig. Using the spectra measured at 70 °C and different times, the molalities of free and two complexed first and second stations were determined by deconvolution, and were plotted in Fig. xy, where x and y denote the inclusion states of the first and second stations, respectively. Values of x and y are 0, 1, or 2; 0 means the free state, and 1 and 2 denote the complexed states, in which the station is included by α-CD from the primary and secondary hydroxy sites, respectively. The two-station axis molecule can thus take one of nine states, i.e., 00, 10, 20, 01, 02, 11, 21, 12, or 22. The association and dissociation rate constants between the 00 and 10 states are defined as k1′ and k−1′, respectively. Similarly, the association and dissociation rate constants between the 00 and 20 states are defined as k1″ and k−1″, respectively. The rate constants for forward and backward reactions between 10 and 01 are defined as k2′ and k−2′, and those between 20 and 02 are defined as k2″ and k−2″, respectively. For simplicity, it is assumed that the association and dissociation rate constants for the first station are independent of the state of second station. As described in Supplementary information, we roughly estimated the eight rate constants by fitting the calculated molalities, i.e., [0 y] (=[00] + [01] + [02]), [1 y] (=[10] + [11] + [12]), [2 y] (=[20] + [21] + [22]), [x0] (=[00] + [10] + [20]), [x1] (=[01] + [11] + [21]), and [x2] (=[02] + [12] + [22]), to the experimental data in Figs xy] is the molality of xy state. As can be seen in these figures, the calculated molalities are in fairly good agreement with the experimental data using an appropriate set of values for the rate constants. It should be noted that it was almost impossible to determine sets of the rate constants unambiguously, but it was feasible to obtain the magnitude relationship of the rate constants at each temperature. Table k1″ > k1′, k−1″ ≥ k−1′, k2′ > k2″, and k−2′ ≥ k−2″. This may be because the numbers of α-D-glucopyranose units of unstable conformation in α-CD are different upon passing the 2-methylpyridinium units from its primary and secondary hydroxy sides30. It is noteworthy that k2′ > k−2′ and k2″ > k−2″, indicating that α-CD moves preferably from the first to second stations presumably because of the thermodynamic stability, which may be ratchet-like movement. From the intercepts and slopes in the Arrhenius plot  and activation energies (Eapp) were roughly estimated although we would not discuss these values in detail  before and after heating in D2O at 70 °C in the presence of α-CD . On the other hand, the MS chart for the axis molecule heated in D2O at 70 °C for 2 days in the presence of α-CD exhibits a series of signals centered at m/z = 192.84, which is assignable to a trivalent molecular ion peak of m = 578.52 , dextrin, and methyl α-D-glucopyranoside (MGlc). The deuteration ratios were quantified using the ratio of area intensities of signals due to the eight protons of methyl and methylene to that of the phenyl proton on the 4-position of the 3,5-dimethylpyridinium residue, which did not undergo deuteration. The deuteration ratios were then plotted in Fig. kapp) were determined from slopes of the first-order-plots. Figure kapp values. The deuteration reactions in the presence of α-CD and β-CD exhibit higher kapp ((7.7 ± 0.2) × 10−6 and (3.1 ± 0.1) × 10−6 s−1 for α-CD and β-CD, respectively). On the other hand, kapp values were much smaller under other conditions (~10−7 s−1). 1H NMR spectra indicated that α-CD and β-CD included the axis molecule to yield a rather stable pseudo-rotaxane whereas the others did not. It is thus concluded that pseudo-rotaxane structure is critical for the deuteration of two-station axis molecule.To study the effect of α-CD on the deuteration, deuteration experiments of the two-station axis molecule were also carried out in D2O was examined at different temperatures. Figure kapp were evaluated from the slopes of first-order plots at different temperatures.  Table kapp values estimated. The Arrhenius plot does not obey a single straight line  calculations for model systems, as can be seen in Fig. kapp may be underestimated because once deuteration occurs exchange of deuterium between a D atom on the methyl or methylene and D in D2O becomes non-detectable. It is important which hydroxy side of α-CD is more effective for the deuteration. We looked closely at the signals ascribed to the methyl and methylene protons which underwent deuteration, and observed that the corresponding signals due to the complexed ones become weaker and overlapped, as can be seen in Fig. The α-CD-catalyzed deuteration of the two-station axis molecule in Dine Fig. ; the slok−1′, k−1″, k2′, k2″, k−2′, and k−2″, range 10−15–10−9 s−1, whereas kapp ranges 10−15–10−11 s–1. These data indicate that the time scales of translation and deuteration significantly overlap. On the other hand, the Arrhenius plot for kapp . Using the eight rate constants evaluated, the molalities of all the nine states of the two-station axis molecule can be calculated. If we choose the average molality of complexed axis molecule in the period of 0–5 days as a representative value for c, we can roughly estimate kapp′ values at 50, 60, 70, 80, and 90 °C. The Arrhenius plot for kapp′ indicates the slope is almost zero in the temperature range of 60–90 °C, indicative of the apparent activation energy of almost zero for the deuteration . An Agilent INOVA 600 spectrometer was also employed to obtain spectra of two dimensional J-correlation spectroscopy with gradient coherence selection (gCOSY), two dimensional total J-correlation spectroscopy for scalar coupled spin systems with a zero-quantum filter for artifact suppression (zTOCSY), and two dimensional heteronuclear single-quantum 1-bond J-correlation spectroscopy with adiabatic 180° X-nuclei pulses and gradient coherence selection (gHSQCAD). Deconvolution of 1H NMR spectra was carried out using a MestReNova software (version 10.0.2). Electrospray ionization mass spectroscopy (ESI-MS) data were recorded in a positive mode on a Thermo Fisher Scientific LTQ-Orbitrap-XL, controlled by the XCARIBUR 2.1 software package. The condition of ionization was set to the following parameters; ion spray voltage at 3.5 kV, ion spray temperature at 100 °C, and ion transfer tube temperature at 275 °C. Internal calibration of ESI-MS was carried out using the monoisotopic peaks of sodium adducted ion of diethylphthalate (m/z 314.1410), protonated ion of di-2-ethylhexylphthalate (m/z 391.2843), and sodium adducted ion of di-2-ethylhexyl-phthalate (m/z 413.2662).N-decyl-2-methylpyridinium or 4-decyl-2,N-dimethylpyridinium) using the Gaussian 09 program32. In all the calculations, DFT with PBE0 functional was used, and 6–31 + G(d) basis sets were applied for the hydrogen, carbon, nitrogen, and oxygen atoms. All the geometries of the model systems were fully optimized.To investigate the structures of metastable complexed states of α-CD and the two-station axis molecule, DFT calculations were carried out for model systems composed of α-CD and a station model (i.e., Supplementary information


The WHO Classification of Tumours of Endocrine Organs considers the inmunohistochemical characterization of pituitary adenomas (PA) as mandatory for patient diagnosis. Recent advances in the knowledge of the molecular patterns of these tumours could complement this classification with gene expression profiling.Within the context of the Spanish Molecular Registry of Pituitary Adenomas (REMAH), a multicentre clinical-basic research project, we analysed the molecular phenotype of 142 PAs with complete IHC and clinical information. Gene expression levels of all pituitary hormones, type 1 corticotrophin-releasing hormone receptor, dopamine receptors and arginine vasopressin receptor 1b were measured by quantitative real-time polymerase chain reaction. In addition, we used three housekeeping genes for normalization and a pool of nine healthy pituitary glands from autopsies as calibration reference standard.Based on the clinically functioning PA , we established the interquartile range of relative expression for all genes studied in each PA subtype. That allowed molecularly the different PA subtypes, including the clinically non-functioning PA (NFPA). Afterwards, we estimated the concordance of the molecular and immunohistochemical classification with clinical diagnosis in FPA and between them in NFPA. The kappa values were higher in molecular than in immunohistochemical classification in FPA and showed a bad concordance in all NFPA subtypes.According to these results, the molecular characterization of the PA complements the IHC analysis, allowing a better typification of the NFPA. Pituitary adenomas (PA) constitute 10%–15% of intracranial neoplasms. They are currently classified according to their clinical, biochemical, radiological and inmunohistochemical (IHC) characteristics as well as their tumour behaviour , 3.Clinically, PAs are divided into two main types: functioning pituitary adenomas (FPAs) and non-functioning pituitary adenomas (NFPAs). FPAs are accompanied by a clinical syndrome related to hormone hypersecretion: Cushing’s disease, acromegaly, galactorrhea-hypogonadism or hyperthyroidism. NFPAs include a cluster of pituitary tumours without endocrine manifestations of hormone overproduction, which are diagnosed incidentally or due to neuro-ophthalmological symptoms. They comprise approximately 30%–35% of pituitary tumours , 5.Radiologically, two classification systems are currently used. The Hardy classification divides PAs into four grades based on their size and the invasiveness in the sella turcica . The KnoPathologically, different proposals have aimed to classify pituitary tumours over the years, ranging from the cellular staining properties to the latest World Health Organization (WHO) classification based on IHC and ultrastructural criteria . This clCurrently, translational research is based mainly on genetic and epigenetic characteristics. As experience accumulates in this field, molecular studies will gradually become integrated into the standardised clinical management of PA, similarly to other tumours ,11.Silent adenomas have been attributed to the release of pituitary hormone isoforms without biological activity or to the reduced or absent secretion of the normal pituitary hormones . Null ceThe aim of this study was to characterize molecularly the silent and immunohistochemically null cell adenomas in a large series of pituitary adenomas and to study the concordance between the IHC and molecular typification of PA.CEIC Hospital General Universitario de Alicante). None of the donors was from a vulnerable population and all donors or next of kin provided written informed consent that was freely given. One of the patients was 14 years old boy and his parents signed his informed consent.The study was performed within the context of the Spanish Molecular Registry of Pituitary Adenomas (REMAH), a Spanish Multicentre Clinical-Basic Project . The stuWe collected 142 PA specimens with complete clinical and IHC data and sufficient quantity and quality of RNA for molecular analysis. Additionally, we obtained nine normal pituitary glands from autopsies and used them as calibration reference samples.The tumours came from four university referral hospitals for pituitary surgery. Clinical, pathological, and radiological data were collected anonymously for each sample from the REMAH database. The information retrieved included sex; age; endocrine syndrome ; hormonal data; previous medical, surgical or radiotherapy treatment; tumour maximum diameter (TMD); radiological MRI extension of the tumour, and IHC results. Tumours were classified as invasive (Hardy’s grade IV) or non-invasive (Hardy’s grades I–III). We present the clinical baseline characteristics in All cases presenting clinical and biochemical characteristics of a recognised endocrine syndrome were classified as FPA. We considered participants to have NFPA when they did not present any clinical or biochemical characteristics of a recognised endocrine syndrome and were diagnosed according to neuro-ophthalmological or radiological signs.According to the IHC data, we defined the following subtypes of NFPA: 1) IHC gonadotroph adenomas (IHC-GT): tumours with positive immunostaining for LH, FSH or SU; 2) IHC silent corticotroph adenomas (IHC-sCT): tumours with positive immunostaining for ACTH without Cushing’s syndrome; 3) IHC silent somatotroph adenomas (IHC-sST): tumours with positive immunostaining for GH without acromegaly; 4) IHC silent thyrotroph adenomas (IHC-sTT): tumours with positive immunostaining for TSH without hyperthyroidism; 5) IHC silent lactotroph adenomas (IHC-sLT): tumours with positive immunostaining for PRL without clinical amenorrhea-hypogonadism; 6) IHC silent multihormonal adenomas (IHC-sMHA): tumours with positive immunostaining for more than one hormone without predominance of one of them, not associated to a clinical syndrome; 7) IHC null cell adenomas (IHC-NC): tumours without positive immunostaining for any pituitary hormone.th percentile of their corresponding FPA, as follows: 1) molecular gonadotroph adenomas (M-GT): tumours with a dominant expression of LH, FSH or S; 2) molecular silent corticotroph adenomas (M-sCT): tumours with a dominant gene expression of POMC, AVPR1b and CRH-R1 without Cushing syndrome [First, we defined the interquartile ranges (IQR) of gene expression for each clinically diagnosed FPA subtype Tables  and 3. Asyndrome ,14; 3) mAlthough we did not find any molecular sST, sLT or sMHA in our series, we considered these categories because they have been described by others authors . The expThe IHC studies were performed in the respective pathology departments of the four participating hospitals. The application of immunohistochemical techniques was performed by equal in the four hospital on complete sections of formalin-fixed tissue and embedded in paraffin in automated form with Autostainer Link48 (Dako-Agilent) equipment, with the Envision high sensitivity display system (Dako). The tissue sections were subjected to dewaxing by incubation in xylol and rehydration using decreasing solutions of ethanol and water. Antigenic recovery was performed with the PT-Link apparatus, treating the sections with target retrieval solution Envision Flex, with high pH (9.0). Primary antibodies were incubated in the Autostainer for 20 to 30 minutes, depending on the setting of the antibodies. Antibodies used and their dilutions in each centre are detailed in “All the molecular studies of the tumours were performed in the Research Unit of the General University Hospital of Alicante.® solution at 4°C for 24–48 h and then frozen at −80°C until molecular analysis. Molecular methods were adjusted and set up to obtain the highest possible quantity of material from the samples, preserving the quality of the nucleic acids.All specimens were preserved immediately after surgery in RNAlaterDR1, DR4, DR5, DR2 and its isoform DR2 long) were measured by qRT-PCR. The results of the dopamine receptors are detailed in “GAPDH, ACTB and HPRT-1) were analysed simultaneously and with the same protocol. The reactions were performed in 7500 Fast Real Time PCR System (Applied Biosystems), and results were analysed using SDS software (Applied Biosystems®), taking into account the three housekeeping genes. All samples and normal pituitary autopsy samples were analysed in duplicate and following the same protocol. Once normal pituitary autopsy samples were analysed separately and their concordance tested, we mixed them in equal parts into a pool that was analysed as another sample that served as calibrator. A relative quantification was established based on the ΔΔCt method.Before analysing the study samples, we performed efficiency analyses with two non-valuable samples and two commercial controls of total RNA from brain and total human RNA. The chosen method for extraction was the AllPrep DNA-RNA-Protein (Qiagen). We evaluated the quality and quantity of total RNA in the Nanodrop (Thermo Scientific) and the 2100 Bioanalyzer (Agilent Technologies). The protocol of retrotranscription, selection of primers and quantitative real-time-PCR (qRT-PCR) conditions were adjusted to the material and methods published by Luque et al . SpecifiStatistical analysis was performed with SPSS 15.0 software (IBM Software). Participant ages and tumour diameters were expressed as mean ± standard deviation (SD). Qualitative variables, including PA subtypes, were expressed as absolute and relative frequencies. Molecular variables showed a non-normal distribution (Kolmogorov-Smirnov test), therefore we chose percentiles as the measure of distributions and reported them as median plus interquartile range (IQR: p25-p75). Cohen's kappa coefficient was calculated to measure inter-classifications agreement (κ = 1 representing complete concordance and κ = 0 null concordance). P values < 0.05 were considered statistically significant.Clinical and biochemically, 70 patients (49.3%) in our series had a hormonal profile compatible with an endocrine syndrome and were considered as having FPAs, while 72 patients (50.7%) showed normal pituitary function or hypopituitarism and were considered as having NFPAs. The prevalence of the different subtypes according to the IHC or molecular identification is shown in Once we molecularly identified the different subtypes of PA, we assessed the contribution of the molecular study to the identification of the cellular origin of PA, comparing it with participants’ biochemical and IHC profiles. We contrasted these techniques against the established clinical diagnosis on an individual basis ., the concordance between the three techniques was generally high in all subtypes, with the biochemical study showing the highest kappa values, followed by the molecular analysis . The con(When we considered only NFPAs (n = 74), the concordance between IHC and molecular profiles was generally low, with the highest level of concordance (κ = 0.519) shown for the sCT subtype and the lowest one (κ = 0.259) for the NC subtype . It was In this study, we show the molecular typification of a large series of PA. The main strength of the article is the complete clinical, biochemical and immunohistochemical studies of all patients. Moreover, all molecular studies were performed in the same molecular laboratory. Conversely, the main limitation of the study is that the immunohistochemical studies were performed locally in the four University hospitals participants. As immunohistochemistry is a semi-quantitative technique and depends largely of the antibodies chosen, this fact biases significantly the inmunohistochemical results. Indeed, we observed an important variability in the IHC results between the four participating centresmeanwhile in the Lyon’s pathological series, using sensitive IHC techniques, the percentage of NC adenomas were reduced from 10% in 1992 to 1% in 2012 . The var were subclassified as M-sCT, but only 7 (9.5%) of these tumours were confirmed by IHC. The remaining 10 were classified as MHA (n = 4), NC (n = 1), GT (n = 1) and sST adenomas (n = 4). Previous reports in surgical series showed prevalences of sCT between 2.9–5.7% ,22 simil% were suIn the same way, gene expression identified five NFPAs as M-sTT, only one of which was confirmed by IHC. Three cases were identified as MHA . The last one is a discordant case, which we will discuss later.Eleven sST were identified by the IHC method but not by the molecular analysis. The primer chosen for GH had specificity for all four transcripts of GH, so it is unlikely that the absence of M-sST represented a false negative of the molecular study. Rather, the IHC has a low specificity, attributed to the dilution and the time of incubation with the antibody and to the immunoperoxidase reaction. Authors have highlighted the high number of false positive obtained with this technique . Indeed,Similarly, four sLT were identified by the IHC method but not by the molecular analysis. Of these, one tumour was molecularly identified as M-CT, and indeed, the patient had Cushing’s syndrome. This case represents, as above, a false positive from the IHC analysis. The other three IHC-sLT were molecularly identified as M-GT., which was attributed to a lesion of the dopaminergic pathways. Therefore, we consider again that all three of these cases are probably false positives of the IHC technique.One patient with clinical, biochemical and IHC diagnosis of LT did not express the PRL gene. However, the expression of dopamine receptors DR2 and DR4 in this tumour resembled that of the LT adenomas. Moreover, the tumour overexpressed the receptors DR1 and DR5 (50 and 21 FC respectively). This patient had been previously treated with high doses of cabergoline (3.5 mg/week) for four years. The most plausible explanation was that the long-term treatment with cabergoline downregulated the PRL gene and increased the DR1 and DR5 gene expression, as reported elsewhere ,32.One patient with clinical and biochemical diagnosis of LT expressed neither PRL protein nor the PRL gene and was erroneously identified via IHC and molecularly as having NC adenoma. Once again, the patient had been treated with cabergoline for a long time (more than four years). It is possible that different degrees of downregulation have sequential effects on the gene and the protein expression. In fact, other authors had shown that treatment with dopamine agonists can decrease PRL and its receptor mRNA levels . BecauseOne patient molecularly classified as M-ST had a clinical LT and showed positive immunostaining for all pituitary hormones. He was a 14-year-old patient with macroprolactinoma, in treatment with cabergoline for four years. Despite the normalisation of the PRL levels, the boy had a growth and puberty delay. The tumour expressed the GH gene at high levels (more than 3 FC) without gene expression of PRL. The most plausible explanation is that the sample analysed was normal pituitary, with a high growth hormone expression corresponding to adolescence and a lack of PRL gene expression due to the cabergoline treatment. This case highlights the need for a pathologist to select the tissue for analysis before performing any molecular study.In this large series, molecular profiling showed higher accuracy than the immunohistochemical study in the identification of pituitary adenomas subtypes. This finding entails changes in the known prevalence of these tumours with possible clinical repercussions. Indeed, the prevalence of silent CT, a tumour PA subtype that often behaves aggressively, increases significantly with the molecular compared to IHC techniques. Moreover, gene expression allows reclassification of a large part of the IHC null cell adenomas. Therefore, the molecular profiling complements the pathological information of the IHC in the study of PA, although previous medical treatments may act as confounders by modifying the gene expression. Even though the not centralized performance of IHC analysis in this study could bias the IHC results, this fact increases the value of the molecular typification of PA as complement of the IHQ information in NFPA. However, it is worth highlighting the importance that an expert pathologist carefully identifies the used tissue for molecular analysis.S1 Table(DOC)Click here for additional data file.S2 TableThis is the S2 Table legend: PA: pituitary adenomas, DR: dopamine receptors, NC: null cell adenomas, GT: gonadotroph adenomas, CT: corticotroph adenomas, sCT: silent corticotroph adenomas, ST: somatotroph adenomas, LT: lactotroph adenomas, TT: thyrotroph adenomas, sTT: silent thyrotroph adenomas.(DOC)Click here for additional data file.S3 TableThis is the S3 Table legend: Values show Cohen's kappa coefficient (κ = 1 represents complete concordance and κ = 0 the null concordance). All p-values were 0,000. IHC: Immunohistochemistry; H: Hospital; NFPA: Nonfunctioning Pituitary Adenomas; NC: non-calculable.(DOCX)Click here for additional data file.


AIDS-associated Kaposi's sarcoma (AIDS-KS) caused by human herpes virus 8 (HHV-8) is the most severe and resistant form of KS tumor. Our aim was to verify whether there is an association between HHV-8 variability and development of AIDS-KS in Brazil by comparing the HHV-8 variability between individuals without and with KS. Saliva samples and blood, when available, were analyzed by polymerase chain reaction (PCR) techniques for detection of the fragments of ORF K1 of HHV-8, which were then genotyped and analyzed regarding the genetic variability. Our study described 106 positive cases for HHV-8 in the saliva from 751 AIDS patients without previous KS. In addition, we performed a phylogenetic analysis of HHV-8 in 34 of the 106 AIDS patients without KS and in 33 of the 37 patients with active KS. The distribution of HHV-8 genotypes A, B, C, and F in AIDS individuals was indistinguishable by comparing non-KS and KS groups, as well as regarding ethnicity. Considering the KS group, genotype B was associated with better prognosis of KS tumor. Interestingly, we found a particular profile of diversity within clade C and 2 recombinant patterns of HHV-8 in the saliva of AIDS individuals without KS. We emphasize the need to achieve standard genotyping protocol for ORF K1 amplification, thus allowing for substantial detection of HHV-8 variants. Our findings can shed light on the role of HHV-8 variability in the pathogenesis of AIDS-KS. All patients have been treated with cart under routine program.After the informed consent form was signed, samples of saliva or peripheral blood mononuclear cells (PBMCs) and serum were obtained from patients enrolled at the outpatient clinic of the HIV/AIDS Patient Care Extension Service (SEAP HIV/AIDS) of the Clinical Division of Infectious and Parasitic Diseases of the University of São Paulo School of Medicine Clinical Hospital , localized cutaneous (CUT), and disseminated cutaneous (DISS). The clinical progression of KS was assessed based on the extension and number of KS lesions interpreted according to criteria set by Krown et al. Participants who had worsening of disease  were grouped into subgroup W and those with no progression or improvement of KS were grouped into subgroup S.Samples were collected from 2 different previous studies in 2 different situations that involved distinct groups. The first situation was characterized by the presence of HHV-8 among patients living with AIDS and without previous or concurrent KS manifestation. The period of HIV/AIDS diagnosis in this group was between 1987 and 2003. The second situation was characterized by the presence of AIDS-defining diagnosis of KS among patients who had anatomopathological and clinical diagnosis of active KS during the period from 1993 to 1998. Records on clinical progression of KS were based on medical consultations performed during 1998 and 1999, with a median of 4.41 consultations 2.1.1In a previous cross-section study conducted from January 2007 through December 2008, saliva samples were collected from 751 individuals living with HIV/AIDS without previous KS. In the present study, we selected those samples with detectable HHV-8 DNA load to study the HHV-8 sequences.In addition, we analyzed matched blood and saliva samples for 1 patient in this group taken at different times.2.1.2In another previous cross-section study conducted from January 1998 through December 1999, PBMC samples were collected from 37 HIV/AIDS individuals with active KS. In the present study, we included these samples for comparative evaluation of HHV-8 sequences.In addition, we investigated available serial blood samples or blood and saliva matched for these patients.2.22.2.1 and cryopreserved at –70°C until analysis.Saliva was collected according to the protocol described by Beyari et al2.2.26 cells/mL were cryopreserved in liquid nitrogen at –192°C until analysis.The PBMC samples were obtained from 5 mL of total blood with EDTA by means of density gradient centrifugation using the Ficoll–Hypaque method . A total of 1.0 × 102.2.3 whereas total DNA was quantified by using the NanoDrop spectrophotometer. All samples were suitable for viral DNA amplification. HHV-8 DNA was amplified with oligonucleotides directed to ORF-K1, recovering fragments from ∼300 to ∼860 bp,,36 as shown in Table per reaction was used whenever possible. The input of samples with low concentrations was adjusted at the maximum volume of 10 μL.DNA was extracted from both KS and non-KS samples by using the Genomic DNA extraction kit . DNA quality was monitored with PCR directed to beta-globin gene as internal control,2.3 The limit of detection based on a standard curve was 4 copies of pGEX-5X plasmid containing the ORF 73 insert .In order to compose a group of HHV-8-infected individuals without KS disease, salivary DNA samples were first submitted to real-time PCR (q-PCR) to detect and quantify a 68-bp fragment of the HHV-8 genome which encodes for latent nuclear protein (ORF 73), as described by Krishnan et al.2.4 and Poole et al. In parallel, the samples were also submitted to nested PCR to amplify shorter fragments of the ORF K1 hypervariable regions VR1 (386 bp) and VR2 (375 bp), with primers and cycling parameters being adapted according to Stebbing et al and Nascimento et al  to obtain a complete ORF K1 sequence (867 bp), with primers and cycling parameters being adapted according to Lacoste et al2.5PCR products were purified by using the QIAquick PCR Purification Kit (QIAGEN) and quantified with low DNA Mass ladder (Gibco). Approximately 20 ng of purified PCR were sequenced with Big Dye™ Terminator v 3.1 Cycle Sequencing Ready Reaction kit  according to the manufacturer's instructions and using the inner primers of the nested PCR described in Table 2.6http://www.ncbi.nlm.nih.gov/) and used as references for analysis. References and complete K1 sequences were aligned by using MUSCLE software and manually edited with Se-Al (available at http://tree.bio.ed.ac.uk/software/sea/). Phylogenetic inference was performed under maximum likelihood (ML) by using RAxML v. 7.4.8 with general time reversible (GTR)+Γ model of rate heterogeneity and 500 bootstrap replicates.Fifty-two HHV-8 K1 sequences collected worldwide were obtained from GenBank  and lytic-phase antigen was investigated by indirect immunofluorescence assay (IFA) according to the method described by Lennette et al2.8P values were <0.05.Data were analyzed with GraphPad Prism 5.0  and SigmaStat Analysis System Software 3.5 . The Mann–Whitney rank sum test was used for comparisons of 2 groups of continuous variables, whereas Yates’ chi-square correlation and 2-tailed Fisher's exact test, when appropriate, were used for comparisons of categorical data. The Kruskal–Wallis test was used for comparisons of 3 groups of continuous variables. Differences were considered statistically significant when 33.16 copies/μg of DNA). By using nested PCR, complete or partial ORFK1 were successfully amplified from saliva samples of 34/106 (32.1%) non-KS individuals and from PBMC samples of 33/37 (89.2%) KS individuals. The genotyping of HHV-8 was successfully performed in 100% of the samples where the HHV-8/DNA was amplified by nested PCR, totalizing 67 AIDS individuals screened. The clinical and epidemiological characteristics of 67 AIDS individuals of the non-KS and KS groups are presented in Table By using q-PCR, the fragment of ORF73 of HHV-8 was detected in the saliva of 106/751 (14.1%) individuals of the non-KS group (from 4 copies to 6.0 × 10 34/106 3.1% non-K 34/106 3.1% non-K3.2P = 0.6111). However, there was a statistical difference in relation to the distribution of genotypes A, B, and C within the non-KS group . In the KS group, we did not find any statistical difference in distribution of genotypes. One sample from each group had virus from genotype F.Figure Matched saliva and blood samples from the same patient presented HHV8 DNA with close similarity to each other, as showed in the taxa BR38_b and BR38_s as well as BR08_KS_b and BR08_KS_s. In the KS group, 5 patients with serial blood samples available for analysis presented HHV-8 sequences with close similarity to their own earlier samples  as well as KS  groups.There was no statistical difference in the genotypic distribution of HHV-8 among patients tested positive for anti-HHV-8 antibody against latency-associated nuclear antigen and lytic antigen by IFA in non-KS  as well as in KS  groups. There was neither difference in the genotypic distribution of HHV-8 between visceral and non-visceral forms of disease in the KS group , nor HHV-8 viral load among HHH-8 genotypes in the non-KS group .No statistical difference was observed in the genotypic distribution of HHV-8 between African and non-African ethnicities in non-KS . VR2 assay did not amplify 16 of the 26 genotype B samples (61.5%) (P = 0.0005), whereas VR1 assay did not amplify 14 of the 26 genotype C samples (53.9%) (P = 0.0125). It was noticeable that certain primers preferentially amplify one genotype over another.Most AIDS patients n = 65) were distributed among the 3 different genotypes of HHV-8 ,  and  according to the genotyping assays by using the 3 sets of primes together , as showed in Tables  were dis3.4P = 0.0182). Genotype A in comparison to genotype non-A  and genotype C in comparison to genotype non-C  did not show any statistical difference regarding subgroups W and S.The distribution of HHV-8 genotypes A, B, and C between subgroups W and S of the AIDS-KS group is presented in Table 4We analyzed genetic variation of the HHV-8 K1 gene of a series of 67 viral isolates obtained from Brazilian individuals with AIDS. We also provide basic epidemiological information on the dissemination of HHV-8 genotypes, subgenotypes and recombinant forms for individuals with AIDS without KS and those with AIDS-KS.,27,45,46 Nevertheless, prevalence of detectable HHV-8 salivary shedding in the group without KS was 14.1% , which is close to the prevalence rates reported in previous studies, that is, ranging from 9.5 to 11%.,46 In the saliva samples from those non-KS individuals, our protocol using 3 sets of primers together  allowed us to genotype 32.1% of the samples, at even low viral load. In the PBMC samples from individuals with KS disease, our genotyping protocol reached 89.2% (33/37). The sensitivity reported in previous studies was lower compared to the present one, with estimates varying from 26% to 74% in the analysis of 1 or both HHV-8 ORF-K1 partial targets in biopsy specimens of AIDS-KS lesions.,47Saliva proved to be a particularly convenient source for virus detection in asymptomatic HHV-8 infection, despite intermittent viral salivary shedding and low HHV-8 load in absence of the KS tumor.,20 The discordant data on the prevalence of genotype B among febrile children in Africa was also observed.,22 Discrepancies among genotype B frequencies partly reflect the need to achieve a standard method for ORF K1 amplification in genotyping studies, thus allowing for substantial detection of HHV-8 variants. Analysis of the alignment containing reference sequences of HHV-8 revealed important mismatches within the sites of the primers set for VR1 and VR2 (data not shown), which ultimately may lead to the biased genotyping. Based on our genotyping protocol, we could reduce biased HHV-8 genotyping by using the 3 PCR assays together .Our comparative data on the genotyping performance show that VR2 assay resulted in underestimation of HHV-8 genotype B, but worked very well to genotype C. This might be attributed to the fact that certain primers preferentially amplify one genotype over another. In previous studies conducted in South America, HHV-8 genotype B prevalence ranged from 2% to 21%. Unlike previous studies demonstrating a high prevalence of HHV-8 genotype A among individuals with AIDS-KS in the United States, Latin America, Northern Europe and Asia,,23,24,48 we have observed a lower prevalence of genotype A in relation to genotypes B and C in both non-KS and KS groups in AIDS individuals, reaching statistical significance particularly in the former group. In fact, our data regarding the predominance of genotypes B and C in Brazilian HHV-8 isolates agree with previous report by Caterino which was based on a small fragment of the ORF-26 gene.We have found genotypes, sub-genotypes and recombinant forms of HHV-8 similar to those described worldwide Fig. . It is n including slowly progressing classic KS and intra-familial transmission in the Mediterranean countries.,14,50,51 On the other hand, there was a lack of diversity within clade C in the KS group, which was composed of 100% of HHV-8 subgenotype C3,  from non-KS patients was similar to that previously reported in a HHV-8 endemic African country,, or C7 f,46,50–52 thus supporting that the currently detected recombinant forms do not originate from new events, but they represent previous circulating recombinant forms.,49Although recombinant forms of HHV-8 were found in non-KS group, we did not find co-infection with different HHV-8 genotypes in any patient analyzed, including 7 cases for which serial blood or matched blood and saliva were sampled in time interval up to 8 years   Table . AlthougOur study, however, has some limitations. First, it was not possible to obtain serially CD4 count and HIV viral load, so it would be very useful to verify some additional relationship with clinical progression of KS. Second, the limited number of samples analyzed precludes detailed investigation on the impact of ethnicity on HHV-8 genotypes.5In conclusion, the distribution of HHV-8 genotypes A, B, C, and F in AIDS individuals was indistinguishable between the groups with and without KS. However, it was possible to suggest that genotype B might be associated with better tumor prognosis in the KS group. Interestingly, salivary shedding of HHV-8 among AIDS individuals without KS presented a particular diversity of strains within clade C and 2 already described recombinant patterns. Moreover, we emphasize the need to achieve a standard method for ORF K1 amplification in genotyping studies, thus allowing for substantial detection of HHV-8 variants.FAPESP (State of São Paulo Research Support Foundation) for funding resources.The authors thank Dr. Vanda Akiko Ueda Fick de Souza  for both helpful insights and support to this study as well as LIM 52 (HCFM-USP) and CNPq .