Despite the wealth of mental-health information available online to consumers, research has shown that the mental-health information needs of consumers are not being met. This study contributes to that research by soliciting consumer questions directly, categorizing them, analyzing their form, and assessing the extent to which they can be answered from a trusted and vetted source of online information, namely the website of the US National Institute of Mental Health (NIMH). As an alternative to surveys and analyses of online activity, this study shows how consumer questions provide new insight into what consumers do not know and how they express their information needs. The study crowdsourced 100 consumer questions through Amazon Inc.’s Mechanical Turk. Categorization of the questions shows broad agreement with earlier studies in terms of the content of consumer questions. It also suggests that consumers’ grasp of mental health issues may be low compared to other health topics. The majority of the questions (74%) were simple in form, with the remainder being multi-part, multifaceted or narrative. Even simple-form questions could, however, have complex interpretations. Fifty four questions were submitted to the search box at the NIMH website. For 32 questions, no answer could be found in the top one to three documents returned. Inadequacies in the search and retrieval technology deployed at websites account for some of the failure to find answers. The nature of consumer questions in mental health also plays a role. A question that has a false presupposition is less likely to have an answer in trusted and vetted sources of information. Consumer questions are also expressed with a degree of specificity that makes the retrieval of relevant information difficult. The significance of this study is that it shows what an analysis of consumer mental-health questions can tell us about consumer information needs and it provides new insight into the difficulties facing consumers looking for answers to their questions in online resources. Trusted community organizations such as NAMI  also provide comprehensive vetted information on mental health for consumers. Many other countries have similar online resources—the UK National Health Service (NHS) and Mental Health Foundation and the Mental Health Foundation of New Zealand, for example. With such rich resources, it might be expected that consumer mental-health information needs are being taken care of. But research shows otherwise. This article adds to that research, asking what can be learned about mental-health information needs by directly soliciting questions on mental health from consumers and assessing the extent to which those questions can be answered using a trusted and vetted source of online information. Included in our study is an analysis of the questions in terms of their surface form or structure and the categories they fall into.There is a wealth of vetted mental-health information specifically for consumers It has long been known that the Internet plays an important role in mental-health information seeking, with the lived experience of mental illness being of particular interest . KhazaalDespite the rich resources of the Internet, and other sources of patient information such as mental-health professionals, the mental-health information needs of patients and the larger community are not being met. In the Unmet information needs in mental health have been identified in two recent studies focused specifically on depression. In a large-scale survey, twenty one specific topics—organized under general information, lived experience, research and policies, and specific treatments—were confirmed as areas of need in the general population . In postAs an alternative to surveys that assess consumers’ knowledge about specific topics in mental health and as an alternative to analyses of online activity such as in mental-health forums, this study proposes that consumers be prompted to ask questions about mental health and that those questions be studied to better understand unmet information needs. No one has investigated what people want to know about mental health by asking them directly. The hypothesis behind this study is that a question-based analysis can provide important insight into what consumers do not know and, by trying to answer those questions using an existing trusted and vetted source of online information, provide an understanding of the extent to which the information needs represented by these questions can or cannot be met online. Directly solicited questions can also be categorized in terms of content and form to provide insight into the information needs they represent.Clinical information needs have been extensively studied by examining real clinical questions collected from physicians . The queA large-scale source of general consumer health questions is the US National Library of Medicine (NLM). Its customer services receive about 90,000 requests a year from around the world. To provide help in responding to these requests, it recently launched the Consumer Health Information and Question Answering project . The proWhy do people who use drugs experience more mental illnesses?  The general assumption behind this question is that drug use and mental illness commonly occur together, a fact research shows to be the case  What kinds of mental-health questions do people have? (2) What form do the questions take? and (3) How easily are answers to those questions found in trusted and vetted online sources of information? The aim is to develop new insight into what consumers do not know, how they express their information needs in language, and the extent to which those expressed needs can be met using trusted online information.To obtain consumer questions on mental health and illness, we used crowdsourcing; in particular, the platform provided by Amazon Inc.’s Mechanical Turk (AMT). AMT is a digital marketplace that allows workers to perform tasks online in exchange for specified fees. Workers are anonymous to the person requesting the task, as is the requester to the workers. A worker’s unique identifier allows the requester to control the number of times any worker responds to the task request. Results are provided in an Excel spreadsheet that can be downloaded for analysis. AMT has been shown to be a reliable source of participants for clinical studies in consumer health informatics . StudiesWhat do you want to know about depression, anxiety, schizophrenia, bi-polar disorder, suicide or ANY mental-health topic? Enter a full question, not just a phrase. For example: “Do people ever recover from schizophrenia?” NOT ”Schizophrenia recovery.” We do NOT want simple questions of the form “What is X?” where X is a commonly known mental illness. Your question does not have to have perfect grammar but it must be perfectly clear. For example: “Do viruses cause mental illness?” is acceptable. “Is there cause for mental illness?” is not. Spelling mistakes are alright as long as the question is still clear.One hundred questions were collected from 100 unique workers between April 26, 2014 5:47 PM PDT and April 26, 2014 9:46 PM PDT for a total cost of $40 and an effective hourly rate for workers of $16. Each worker was allowed to ask only one question to ensure our data represented the information needs of more than those few people who might respond to the AMT task first and provide many questions each, thereby excluding others from offering their questions.The questions were analyzed three different ways:1.Consumer health category analysis: Using categories adopted from the NLM consumer health question answering project, each question was labelled as to the kind of question it was.2.Analysis of question form: The structure or surface form of each question was investigated.3.Analysis of answer accessibility: An assessment was made as to whether or not each question had an answer that was accessible through the search box at the NIMH site http://www.nimh.nih.gov/.The questions were analyzed as asked, that is, without correction or editing. The number was limited to 100 to permit a thorough manual search of the trusted and vetted information resources of the NIMH. It is especially time-consuming to establish a negative, namely that a given question cannot be answered from a given document. We discuss at the end of the paper the kinds of automated text analysis that will make further studies of this kind possible with a larger collection of questions. The full set of 100 questions is given in Twenty question categories were defined, adapted from the 14 categories of the NLM study of consumer health questions. Adaptions were as follows. In the NLM study, the greatest number of questions (23%) fell in the Management category. This category was replaced by the four NLM subcategories of TREATMENT, CURE, PREVENTION, and OTHER MANAGEMENT to better understand the questions being asked. The Information category had the next highest number of questions (18.1%) in the NLM study. For our study on mental health, any question that appeared to be seeking information to understand mental illness in general or a specific disorder fell into this category. And if a question would require as part of an adequate answer some explanation of mental illness in general or of a specific disorder, it was considered an INFORMATION question. In the NLM study a combined category called Susceptibility had the next highest concentration of questions (14.4%). It was replaced by the three NLM subcategories of DISTRIBUTION of a DISEASE in a POPULATION, INHERITANCE PATTERNS, and TRANSMISSION PATTERNS for infectious diseases. Although the transmission category might not seem justified for mental health, consumers could ask questions that fit the category. Finally, one category was added that is important for mental-health, namely PSYCHOSOCIAL, which covers questions about the social-emotional ramifications of a mental-health disorder. Is Bi-Polar disorder curable at all or will people always be dependent on their medication? falls into four categories; namely, CURE, PROGNOSIS, TREATMENT and INFORMATION, the last because the question suggests the need for general information on bipolar disorder.A question could receive more than one category label. For example, the question All 100 questions were categorized by the first author (CEC) and a randomly selected subset of 20 questions was additionally categorized by the second author (JBK). Those 20 questions are indicated by starred rows in Is schizophrenia genetic? The mother of an adult child similarly diagnosed may say: My father had schizophrenia. Is that why my child has it? Different acts and different words used to express the question but, in important ways, the same question with the same content, namely an inquiry into the heritability of schizophrenia. However, the first question act may have behind it a concern over whether or not the questioner is at risk. The second may have behind it a concern that she or her family heritage are to “blame” for her child’s illness. The reason for or motivation behind a question is an important part of understanding what is being asked and determining the most appropriate answer.It is a fundamental tenet of semantics that meaning does not exist solely within words and sentences but resides in the situations in which the words are uttered and the attitudes of those speaking and hearing the words . To undeIs X true? (Are anti-depressants dangerous?), or What causes X to occur? (What causes PTSD?). This simplifying assumption may have been necessary for early work on question taxonomy development, but questions real people pose are seldom that simple. The NLM questions project recognizes this reality in its work on decomposing complex questions into their simpler parts  was chosen to conduct a review of the extent to which answers are accessible to consumers. Questions were entered into the search box at the NIMH site as they were asked, without any editing or rephrasing. To make the assessment as reasonable as possible, long questions were omitted, thereby precluding many questions of complex form which are naturally more difficult to find answers for. There were 54 short questions (fewer than 255 characters) and these were taken as the test questions. The NIMH website was accessed by CEC on October 17, 2014 and the top three URLs returned for each search were retrieved. A URL (Uniform Resource Locator) is an Internet address that points to a document and it was these documents that were examined for answers to the given questions.It is important not only to know what questions consumers have but also whether or not they can find answers to those questions at comprehensive trusted and vetted sources of online information for consumers. The NIMH site  examined the documents that the top three URLs pointed to and made one of the following three judgments for each question:An answer could not be found in the top three documents returned, or no results were returned.An answer could be found but it was in a long or complex document that required extensive reading and some interpretation.An answer could be found quite easily in the documents returned, starting with the first, moving on to the second or third as needed.In the second assessment, AMT workers were used. For each question an AMT task was set in which the question was presented along with the top URL retrieved by CEC earlier. The worker was asked to indicate if an answer could be found in the document the URL pointed to by following the link and copying and pasting an excerpt containing the answer in a text box or, if no answer could be found, to enter NO ANSWER, or if no document was returned to enter NO DOC. Each task was performed by two workers on February 5, 2015, for a total cost of $76.20. Workers spent longer than expected on each task—on average 13 minutes compared to the 4 minutes expected—giving a lower than intended effective hourly rate of $3.79.Given this average time of 13 minutes, we limited the AMT worker assessment to just the top search result. The length of time it took to ascertain whether or not there was an answer in a document is itself a result that establishes the difficulty of the task for the consumer. We discuss this further in the Results section.For the categorization of all 100 questions by CEC, k = (Na − Nc)/(Np − Nc), where Na = number of agreements overall, Nc = number of agreements that could be expected due to chance, and Np = maximum number of agreements possible. An agreement for a question is an instance in which a given category is either assigned by both raters to that question or by neither. A disagreement for a question is an instance in which a given category is assigned by one rater to the question but not by the other. The number of agreements overall is the sum of the agreements for all 20 questions. With 20 questions and 20 categories, the maximum number of agreements possible is 20 × 20. Chance assignment of a category to a given question is 0.5 for each of the 20 categories, which sums to 0.5 × 20 = 10 for each question. For 20 questions, the number of agreements due to chance is therefore 10 × 20 = 200.For the 20 randomly selected questions that were additionally categorized by JBK, we computed inter-rater agreement using the chance-adjusted measure according to the formula The agreements and disagreements are shown in k = 0.9. If we omit the INFORMATION category, which had a disproportionally high number of agreements, the score stays the same. If we omit the categories shown by shaded row labels in the Table that were not relevant to the subset of 20 questions and so also contributed a high number of agreements, we again get a score of 0.9.Taking all 20 questions and categories, we obtained Is a predisposition for bi-polar disorder hereditary or can it be caused by environmental factors?—may have an exclusive-or or an inclusive-or interpretation and so is not merely two simple questions. That is, the question may have any of the following three summarized interpretations: Is there an inherited predisposition and no environmental effect? Are environmental factors the cause and a disposition is not inherited? Do inherited and environmental factors both play a causal role?The majority of the questions (74 out of 100) are simple in form. Twenty six are complex, with 11 being multi-part, 9 multifaceted and 6 narrative, as shown in Complex questions present another challenge, not so much for their interpretation but for finding answers. Complex questions often include information that is very specific, such as being unfaithful or disappointed, experiencing failure, experiencing a two-week episode, or having a timid father. Although it is often possible to formulate general answers that take specifics into account, search and retrieval technology behind search boxes at websites cannot make the leap from the specific to the general. Nor can the consumer always make the necessary generalization; to know that timidity, for instance, is not a personality trait that generally shows up in discussions of mental health.The first assessment of answer accessibility (by CEC) had the results shown in http://www.nimh.nih.gov/health/topics/depression/index.shtml (accessed 6 February 2015). Some but not all yielded answers to the specific question posed. Sixteen URLs pointed to news items on the NIMH site, such as the following: http://www.nimh.nih.gov/news/science-news/science-news-about-depression.shtml (accessed 6 February 2015). Although an answer might lie within such a document, it was typically difficult to find among all the news article links given, which had to be followed individually. Some URLs pointed to documents such as the following 2011 Director’s Report, which is not consumer-oriented and did not contain an answer: http://www.nimh.nih.gov/about/advisory-boards-and-groups/namhc/2011/september/directors-report.shtml (accessed 6 February 2015). Some URLs pointed to a long pdf (portable document format) document, such as the following budget report, which did not provide an answer: http://www.nimh.nih.gov/about/budget/cj2007_33873.pdf (accessed 6 February 2015).Twenty of the fifty five top URLs that were returned pointed to consumer-oriented material organized under Health Topics at the NIMH website—such as the following, for example: On average, how much time does a person suffering from bi-polar disorder spend in a manic state compared to a depressive state? The underlying assumption is that it is possible to predict the relative lengths of manic and depressive states in general. The question Suicide is most common with which mental illnesses? assumes enough is known about suicide to associate its occurrence with specific mental illnesses in a quantifiable way. Such presuppositions can be identified for 11 of the 32 questions without an answer and these are given at the top of What is the relationship between circadian rhythms and Bipolar Disorder? assumes there is a relation between circadian rhythms and mental illness and that it has been studied. In fact, the NIMH search returns a report on the topic from an NIMH workshop that does provide an answer to the question.A number of the questions entailed presuppositions that are not necessarily true and which may have precluded an answer being found at any trusted and vetted information site. Take, for example, the question The second assessment of answer accessibility, conducted by AMT workers, had similar results, with answers found by both or one of the AMT workers for 20 of the questions. However, the AMT workers failed to find answers to questions numbered 36, 38, 39 and 53 in Question 7: Stress management techniques and meditation can help people with anxiety disorders …, aerobic exercise may have a calming effect. …, certain illicit drugs, and even some over-the-counter cold medications can aggravate ….Question 11: Children who develop depression often continue to have episodes as they enter adulthood. Children who have depression also are more likely to have other more severe illnesses in adulthood.In two instances of answers not being found, the URL pointed to a pdf file which produced the following standard warning before it was opened:Opening <filename>.pdf. Some files can contain viruses or be otherwise harmful to your computer. It is important to be certain that this file is from a trustworthy source. Would you like to open this file?The AMT workers most likely declined to open such a file, which accounts for their reporting the result of NO DOC (no document returned).k = 0.8 between the first and second assessment was calculated using Cohen’s kappa, computed as k = (Pa − Pe)/(1 − Pe) on the results in Inter-rater agreement of The length of time it took for the AMT workers on average to ascertain whether or not there was an answer in a document—13 minutes—made us decide to restrict the second assessment to just the top document. If it takes that long to check one document it seems unlikely that consumers would be prepared to search one document after another for an answer. Despite the difference in the number of documents used in the first and second assessments, there was still high inter-rater agreement. This suggests that much of the time if an answer exists, it can be found in the first, second and/or third documents, just in different forms.http://www.nimh.nih.gov/health/statistics/index.shtml, accessed 20 January 2015). Nonetheless, any follow-up study collecting questions should mention all disorders that are of interest to the study or none, and should explicitly include or exclude substance abuse disorders. Other disorders specifically named in the collected questions were ADD, DID , narcissistic personality disorder, “Neurosis and Psychosis,” borderline personality disorder, drug use, OCD, Erotomania, and PTSD (twice).The high percentage of mental-health questions categorized under INFORMATION—81% compared to 18.1% for the general consumer health questions from the NLM—suggests that consumer grasp of mental illness is low compared to other health topics. Questions sought both general information and information on specific disorders, notably depression (29 questions), bipolar disorder (16), anxiety (14) and schizophrenia (12). These four disorders were named in the AMT task description, which may have increased the likelihood of their being specifically asked about. However, in the US anxiety and depression have the highest 12-month prevalence figures for adults and the four named disorders are among the top six contributors to disability-adjusted life years for mental and behavioral disorders, with drug and alcohol use disorders being the other two  in our study were questions about the lived experience of mental illness for the patient and other affected individuals. The category MANIFESTATION (first cluster) is largely about symptoms and these questions were common, as were questions labeled TREATMENT (first cluster). There were some but not many (nine) questions labeled DIAGNOSIS, which was not in the top two category clusters. There were many questions labeled CAUSE (first cluster). The prevalence of these questions suggests a strong need to understand the origins of mental illness. The CAUSE questions overlap with those categorized under INHERITANCE PATTERNS (second cluster). Taken together the questions in these two categories were twice as frequent as those in any other category besides INFORMATION. There were seven questions about stigma, four asking if something is “normal,” one specifically mentioning stigma, one asking about violence, and one questioning the safety of marriage to a person with a mental illness. There weren’t any questions on disclosure in our study. The many questions combined under PROGNOSIS (first cluster), COMPLICATION (second cluster), and CURE (second cluster) would be aligned with the topics of coping and comorbid health problems identified in earlier studies. Despite the fact that the topics of earlier studies and the NLM categories are not fully aligned, there seems to be broad agreement on the content of consumer questions, but with a lesser emphasis on diagnosis and disclosure in our question collection.What internal coping mechanisms can you use to help deal with bi-polar disorder?, CEC assumed that by “internal coping mechanisms” the questioner meant things she could do herself without help from health-care professionals and so categorized it as a PSYCHOSOCIAL question whereas JBK linked “internal coping mechanisms” with cognitive behavioral therapy and so categorized the question as a TREATMENT question. For question 22, How does poor nutrition during childhood affect one’s susceptibility to develop bipolar or some other mood disorder like depression?, JKB assumed the questioner had in mind that good nutrition might confer a preventive advantage and so assigned the category PREVENTION. For question 5, What causes mental illness to occur in people? JKB determined that an adequate answer would entail some discussion of genetic factors and so assigned the category INHERITANCE PATTERNS.There was strong agreement between the two raters on the categorization of the questions. Differences that did exist revealed something about the process of categorization. Interpreting a question to determine which categories applied to it entailed making assumptions about what the questioner may have had in mind and what an adequate answer to the question would entail. For example, for the question numbered 89 in Why is there a stigma if you have Bipolar Disorder that all of those who have it must be crazy? A grammatically correct version of this question would be Why do people hold the stigmatizing belief that a person with bipolar disorder is “crazy”? This is in the first place a question about stigma, about trying to understand the origins of or reasons behind the stigma of mental illness. But it is also about particular attitudes towards a particular disorder, namely that the person with bipolar disorder is a different, stranger, lesser person, as suggested by the colloquial term crazy.In this initial study, question form analysis was restricted to the top-level characterization of simple, multi-part, multifaceted or narrative. Despite the fact that the majority of questions were simple in form, categorization was not simple and finding answers was not simple. Questions that are simple in form can still have complex meaning. An example is question number 1 in Are X and Y related? implies a different level of knowledge from How are X and Y related? The first question shows uncertainty as to whether a relation exists while the second presumes a relation and seeks to know more about it. The form of the question therefore constrains the kind of answer that is appropriate and relevant to the information need. For these reasons, further study of consumer questions on mental health needs to include a deeper analysis of the question form, one that can profit from the large body of research in education.The form and content of questions have long been studied from the perspective of education. The aim has been to understand what questions to ask students that will best guide their learning. A further aim has been to glean from student questions what they need to learn. An early and oft-cited work is that by http://www.nimh.nih.gov/health/topics/index.shtml, accessed 20 January 2015). However, answers to more than half of the questions were not readily available. Unanswered questions came from any category and included questions that were both simple and complex in form. Admittedly, answers satisfactory to the AMT workers may have been found in the second or third document returned, which research shows is examined around 24–32% of the time. If answers were to be found in the fourth and later documents not checked by CEC, it is less likely these would be found by consumers. There is the possibility that by adding the online resources of trusted consumer organizations such as NAMI and The Mental Health Foundation of New Zealand, more answers would be found. But at the same time a higher number of irrelevant documents would likely be returned as well, the core problem of the online searching experience. Nevertheless, the restriction to the top documents is an acknowledged limitation of the current study.The NIMH provides extensive patient-oriented material and supports research on almost all the topics addressed by the collected questions , that are not used in professional medicine. Here however, we have the problem of precision. The distinction between susceptibility and prevalence important to question 33 is not one that can be elided and carefully written material will not confuse the two. But if the act of asking the question is properly understood, that is, if the questioner’s purpose in asking the question is taken into account, the conceptual overlap between susceptibility and prevalence might be acknowledged and a suitable answer given about the prevalence of anxiety in different age groups.The second reason a question may not find an answer is related to language. There is information at the NIMH site about the prevalence of anxiety in different age groups and it may very well be that the questioner would be satisfied with information about prevalence. It was the word What is the difference between bi-polar 1 and 2? Taking the three top documents returned at the NIMH site for this question, two were about an NIMH Strategic Plan and one was a report from a workshop on mental health and mass violence. The phrase difference between and the word bi-polar were matched to the content in these documents and nowhere, it appears, did the words bi-polar 1 and 2 appear in a document that used the word difference.A third reason for answers not being found has to do with the surface form of questions and the sensitivity of the search process to the surface form. Take the question At what age do symptoms of schizophrenia usually begin to show in those affected? But the second question did not: How early do individuals with schizophrenia begin to manifest symptoms? The most likely reason for the difference is that there are direct discussions of age of onset but these do not use the word early.To appreciate how important the actual phrasing of the question is to the likelihood of an answer being found through the search box, consider the following two questions, both asking about the onset of schizophrenia symptoms. The first question did have an easily found answer in the top document: fourth reason consumer questions may not readily find answers lies in their specificity. “Being more positive,” “children under 10,” “detoxing heavy metals”—these specific references are unlikely to find an exact match in consumer information materials even if there are discussions of patient attitudes and children and toxic metals and illness. The narrative questions all include details that are specific to someone’s circumstances. Such questions will require individualized answers. Is it worth the effort to provide them? That remains to be seen but it is likely that the stories they tell will provide insight for others in similar circumstances. The studies by A In looking at the high number of questions for which answers could not be found, it might be argued that consumers ask the “wrong” questions. That view, however, does not respect the right of consumers to have information needs of any nature and to express those needs in whatever way they are able.This study aimed to contribute to the research on consumer mental-health information by soliciting consumer questions directly, categorizing them, analyzing their form, and assessing the extent to which they can be answered from a trusted and vetted source of online information. The Internet is a much-used resource for health information by consumers. According to a recent Pew report, 59% of U.S. adults, and 72% of Internet users, looked online for health information in the previous year . The dowGiven the need for more research, what is the significance of the study reported in this paper? What do we know about mental-health information and consumers that we did not know before?http://trec.nist.gov). Second, the nature of consumer questions in mental health mitigates against the easy provision of answers. There are presuppositions that consumer questions harbor; a question that has a false presupposition is less likely to have an answer in trusted and vetted sources of information. Consumer questions are also expressed with a degree of specificity that makes the retrieval of relevant information difficult.We have shown that even highly trusted and well-organized sources of information such as the NIMH may not readily provide answers to consumer questions online. Two kinds of explanations presented themselves. First, the clear inadequacies in the search and information retrieval technology deployed at Internet resources such as those of the NIMH. There is no easy fix for this. The problem of matching a question expressed in ordinary language to the text in documents is a long-standing and active area of research on a non-trivial problem, as demonstrated by the 24-year history of the Text Retrieval Conference (TREC) workshop series, which encourages research in information retrieval by providing large test collections for tasks such as the retrieval of documents on the Internet and question answering  words that occur together more often than would be expected by chance. The only four-word n-gram of note in our 100 questions is what is the best and, apart from the phrases mental illness(es), bi(-)polar disorder, and the brain, the other noteworthy bi-grams are the best, symptoms of, and recover from. It appears to be a common theme that consumers want answers that provide comparative results, as indicated by the best. An interest in diagnosis may be suggested by symptoms of, but it may also suggest attempts to understand the experience of someone with a mental illness, wanting to know if observed behavior is a manifestation of a disorder or not. An intuitive reading of the questions as a whole supports the idea that the bi-gram recover from gives voice to a hope that mental illness can be recovered from, as opposed to being managed as a chronic condition. These observations are tentative given the relatively small collection of questions but they point to the potential of large-scale question collection for robust quantitative analysis. Question should be collected in a number of different contexts, including online at a consumer organization such as NAMI, through crowdsourcing, and at locations used in other studies—psychiatrists’ offices and hospitals, for example.With a large enough collection of consumer questions, quantitative analytic methods can be deployed. We end with a brief discussion of what these methods offer. Quantitative text analysis has been used to do text data mining in the scientific literature  and deteHow does depression harm the brain? Because the presupposition—that depression does indeed harm the brain—does not have a consensus evidence-based opinion, an answer will not be found for consumers in trusted and vetted information sources. But using a general-purpose search engine, a consumer will find many “answers” to the question at less trusted and non-vetted online information sources. It is imperative that the most trusted online sources of mental-health information be the ones that consumers go to, because it is at those sources that their questions can be answered.With sufficient consumer questions, quantitative methods can also be deployed to identify common presuppositions in questions. Understanding common presuppositions could lead to a better understanding of the knowledge consumers may need but may not realize they need. Understanding common presuppositions also reveals the questions that must be addressed at trusted and vetted online sites, even if just to point out there is no definitive answer. Take the question 10.7717/peerj.867/supp-1Data S1Click here for additional data file.


Nicotiana benthamiana) leaves transfected with the magnICON deconstructed viral vector expression system. L1 proteins were extracted from agro-infiltrated leaves using a series of pH and salt mediated buffers. Expression levels of L1 proteins and VLPs were verified by immunoblot and ELISA, which confirmed the presence of sequential and conformational epitopes, respectively. Among three constructs tested , TPL1F, containing a full-length L1 and chloroplast transit peptide, was best. Extraction of HPV16 L1 from leaf tissue was most efficient  with a low-salt phosphate buffer. VLPs were purified using both cesium chloride (CsCl) density gradient and size exclusion chromatography. Electron microscopy studies confirmed the presence of assembled forms of HPV16 L1 VLPs. Collectively; our results indicated that chloroplast-targeted transient expression in tobacco plants is promising for the production of a cheap, efficacious HPV16 L1 VLP vaccine. Studies are underway to develop plant VLPs for the production of a cervical cancer vaccine.Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an alternative to the currently available virus-like particle (VLP) vaccines, and would be much less expensive. We optimized methods to express HPV16 L1 protein and purify VLPs from tobacco ( Over 95% of cervical cancers are caused by human papillomaviruses (HPVs). Cervical cancer is the second most common HPV-associated cancer in women, with approximately one-half million new cervical cancer cases and almost 250,000 deaths each year worldwide . The grein vitro and in vivo with either T = 7 symmetry, identical to native virions, or T = 1 comprised of 12 capsomers [The major recombinant HPV vaccines, Gardasil and Cervarix, are composed of major capsid proteins of HPV which self-assemble into highly immunogenic VLPs apsomers –5. Threeapsomers . AlthougPlants have emerged as an alternative biotechnological means of producing pharmaceutical  and therAnother challenge is the chromatographic purification of plant-based proteins. It has been demonstrated that different physiological conditions, together with extraction and purification processes, play a crucial role in denaturation of recombinant protein and determination of yield, stability, and biological activity . CsCl anNicotiana benthamiana) use a high-efficiency binary vector system with a chloroplast signal sequence to assemble the HPV L1 proteins in chloroplasts. The yield of purified plant VLPs was high and antigenically similar to VLPs produced in the baculovirus expression system (Cervarix). Using simple purification steps, this is possibly the first report to present high yield of intact HPV16 L1 VLPs from tobacco plants. We anticipate that our isolated VLPs are safe and can be used as an affordable vaccine worldwide.In this study, to express and purify HPV16 L1 VLPs from tobacco plants, we re-designed our expression construct and addressed major shortcomings from previous plant research, including the low yield and less than optimized purification procedures of earlier plant-based technologies. Our improved conditions for HPV16 L1 production in tobacco plants (BamHI site was obtained by PCR with the end-tailoring primer 16L1d22-Bam-R (5’–GGGGATCCTTATCCTAATGTGAACTTTGGCTT). For chloroplast targeting, a 59 amino acid plastid peptide (TP) coding sequence from rbcS was fused to the initiation codon of the L1 gene using overlap extension PCR. The plastid targeting sequence was amplified from pICH12190  using the primers TP-Xho-F (5’-ccctcgagaacaatggcttcttctatgctttcttc) and TPL1-OL-R (5’-GAAGGGAGCCAAAGAGACATGCATTGGACTCTTCCTCCGT), while the 5’ end of the L1 gene was amplified using primers TPL1-OL-F (5’- acggaggaagagtccaatgcatgtctctttggctcccttc) and 16L1-447-R (5’- cacaactgtgtttgcttgtaatcc). The two overlapping fragments were mixed and amplified using the terminal primers TP-Xho-F and 16L1-447-R, and the resulting product was digested with XhoI and NcoI and ligated with pIBT210.1 [NcoI site. The L1 genes were end-tailored by PCR with BsaI sites to allow insertion into pICH26212 (Icon Genetics), yielding p26212-16L1d22 , p26212-TPL1d22 , and p26212-TPL1F .We designed a plant codon-optimized coding sequence for HPV 16 L1 (505 amino acids) based on a strain isolated at the University of Rochester Medical Center (Robert Rose), which is nearly identical to Genbank accession ACV53978.1, except for the occurrence of Ser in place of Thr at position 389. Codons were chosen to optimize expression in tobacco, and avoiding spurious mRNA processing signals . The C-tIBT210.1 , digesteE. coli and Agrobacterium transformed plasmid DNA was confirmed for integration using BamHI and XhoI restriction digestion analysis and the PCR amplification method. The set of primers used were MZ-1L (5'-GCAGGTGTGGTTGACGAAT-3') and MZ-1R (5'-CAAACGTGCGTTAACAGGTG-3')  for amplification of N- and C- termini of the target gene, respectively.The A. tumefaciens (GV3101) cells were made electro-competent [-1) and incubated with shaking for 1 h and then plated onto YenB media containing appropriate antibiotics.Initially, ompetent , and 50 Agrobacterium cultures containing integrated HPV16 L1 genes. Cultures were grown from starter cultures with shaking at 28°C to attain the exponential phase  in YenB broth containing the appropriate antibiotics. The bacteria were pelleted down by centrifugation at 3000×g for 10 min, suspended in infiltration buffer . Final OD600 after centrifugation and resuspension was 0.1. The vacuum infiltration technique was employed to infiltrate 2–4 week old leaves of N. benthamiana plants. Following infiltration, the plants were grown for 1–9 days post infiltration (DPI) under conditions of 16 h light, 8 h dark at 23°C.Agro-infiltration of tobacco leaves was completed with transformed g) for 10 min at 4°C. The supernatant collected after clarification was used for further analysis.In the preliminary analysis, 200 mg of tobacco leaves were harvested at 6 DPI and proteins were extracted using HEPES buffer as described . For timThe extracted soluble protein samples (supernatant) collected from homogenized and clarified tobacco leaves were mixed with sodium dodecyl sulfate (SDS) loading buffer , and heated at 95°C for 5 min. These proteins were separated on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gels . Gels were either stained with coomassie blue  or transferred to a PVDF membrane  and saturated with 5% milk in Tris-buffered saline and Tween (TBST) for 1 h. The membrane was then incubated overnight at 4°C with HPV16 L1 specific monoclonal  or polyclonal  antibodies with 3% milk/TBST (Tris and NaCl containing 0.1% Tween 20). The membrane was washed three times with TBST and finally incubated with 1:5000 diluted secondary antibody . Proteins were detected by chemiluminescence  and bands were visualized on x-ray film  using an SRX-101A processor . Baculovirus-infected insect cell VLPs made in-house was used as a positive control. The purified fractions from CsCl, ammonium sulfate (AMS), size exclusion chromatography (SEC), and ion-exchange chromatography (IEC) fractions were also analyzed for protein expression, as described above.16A and C are murine monoclonal antibodies made in the laboratory against intact virus-like particles and characterized  whereas The ELISA was performed on all homogenized, purified leaf extracts and sera collected from immunizing mice as described in sections 2.4, 2.5 and 2.14. The 96-well ELISA plates  were coated overnight at 4°C with 100 μL of plant extracts or 500 ng per well of purified plant VLPs re-suspended in 1X Dulbecco's phosphate-buffered saline (DPBS). 500 ng of baculovirus-infected insect cell VLPs were loaded as a positive control. The plates were washed three times with washing buffer (PBS-T) and blocked with 5% BSA in 1XPBS (PBSA) for 1 h at 37°C. After washing three times, plant extracts coated plates were incubated with monoclonal or polyclonal antibodies against HPV16 L1 conformational or sequential epitopes, at a dilution of 1:1000 in 1% PBSA for 1 h at 37°C. Similarly, the purified plant VLPs coated plates were reacted with collected mouse sera, at a dilution of 1:100 in 1% PBSA for 1 h at 37°C. Moreover, to test the immunogenicity of plant derived VLPs, pooled mice sera were diluted 4-fold in 1% PBSA (1:50–1:51200) and titrated against 500 ng of insect cell derived VLPs. Thereafter, plates were washed thrice with PBS-T, and incubated with respective alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit IgG (H + L)  at a dilution of 1:5000 in 1% PBSA for 1 h at 37°C. Unbound secondary antibodies were removed by washing and bound antibodies were stained with an alkaline phosphatase chromogenic substrate , and absorption was measured at 405 nm. One percent PBSA and sera collected from unimmunized mice were used as negative controls for the background reaction.2 and homogenized with extraction buffer . The lysate was centrifuged at  for 30 min at 4°C. The cleared supernatant was collected and mixed with cesium chloride (CsCl) solution to a concentration of 1.31 g mL-1 as determined using a refractometer  and centrifuged at  for 20 h. The bands on CsCl gradients were collected, their densities measured, and the CsCl solution was replaced with 1XDPBS containing CaCl2 and MgCl2 (Life Technologies) by dialysis. The dialyzed sample was again centrifuged for 2 h at the same speed to pellet down the VLPs, which were then suspended in a minimal amount of 1XDPBS for storage at -80°C until analysis.Freshly harvested tobacco leaves (5 g) were grounded in liquid Ng) for 10 min at 4°C. The pelleted protein was re-suspended with PBS+ 0.01% Tween 20, and the re-suspended solution was stored at 4°C.To remove contaminants from L1 proteins, ammonium sulfate precipitation was conducted as previously described . The preg) for 10 min. The obtained supernatant was used for the purification process.To remove further contaminants, the ammonium sulfate precipitate was dialyzed against PBS + 0.01% Tween 20 . Using t-1, followed by the elution buffer PBS containing 2.0 M NaCl  and 0.01% Tween 20. The total run lasted 1.5 h. The fractions corresponding to peaks were pooled and dialyzed against binding buffer and concentrated with an additional 2-h centrifugation step .The supernatant obtained after ammonium sulfate precipitation was dialyzed against the binding buffer  for 3 h at 4°C. The 5 mL cation exchange column  was used to purify the L1 protein. The column was equilibrated with binding buffer, and the dialyzed sample (5 mL) was loaded onto the column. The column was then washed with five column volumes of binding buffer with a flow rate of 0.4 mL min-1 for 4 h; their absorbance at 280 nm was measured with a spectrophotometer, and HPV16 L1 protein was analyzed by SDS–PAGE and immunoblotting, as described above.SEC was performed on dialyzed plant VLPs purified by the CsCl density gradient ultracentrifugation method and ammonium sulfate precipitation. Dialyzed samples were loaded on (16/600) Superdex column . The running buffer for this column was PBS, pH 7.2, 0.01% Tween 20. Fractions were collected from the column at 0.5 mL minTotal protein concentration of each VLP preparation and ammonium sulfate precipitated samples was determined using a Nanodrop ND-8000  at 280 nm and the Bio-Rad Bradford protein assay reagent  with bovine serum albumin  as a standard. The yield of L1 protein was quantified using SDS-PAGE analysis. Serially diluted HPV16 L1 VLPs from leaves and insect cells were loaded on an SDS-PAGE gel, and their band densities were measured by Quantity one image analyzer .The dialyzed, concentrated and purified HPV16 L1 VLPs were absorbed onto carbon-coated grids  and negatively stained with 2% phosphotungstic acid (pH 6.8). The quality of VLPs was examined at a final magnification of 41,000× by transmission electron microscopy (Phillips CM-12).C57BL/6J mice were obtained from the Jackson Laboratory .2 floor area) at a maximum capacity of four mice per pen. Mice were allowed free access to autoclaved food  and acidified water (pH 2.8–3.2). Mice were euthanized using CO2 in a chamber fitted with an appropriate pressure-reducing regulator and flow meter or equivalent equipment to ensure a gradual displacement of 10–30% volume/minute. All studies were done with the University of Louisville, Institutional Animal Care and Use Committee  and Biosafety Committee  approvals.Only female mice were used to minimize fighting. Mice were maintained in humidity-, temperature-, and light cycle (12:12) controlled vivarium under specific pathogen-free conditions. Mice were housed in double-pen polycarbonate cages . Statistical differences were determined by Student's t-test and one was ANOVA. Differences were considered to be statistically significant if the p-value was 0.05.Three constructed vectors p26212-16L1d22, p26212-TPL1d22, and p26212-TPL1F for protein expression in plants were designated as V1, V2, and V3, respectively . RestricThree steps were followed to optimize the expression level of the L1 protein. At first, the expression level of the L1 protein in plants by different expression vectors was confirmed at 6 DPI. Extracted L1 proteins  were detected at target size of 51 or 56 kDa when compared with baculovirus-infected insect cells used as a positive control . AlthougSecondly, to optimize and characterize the purification condition for the L1 protein extracted from infiltrated plant leaves, different physiological buffers with various pH (2–8), ascorbic acid supplement, and salt (0 and 0.1 M) were used. Proteins were successfully detected in crude extracts homogenized with pH 4 to 8 buffers, and higher concentrations were found in the presence (0.1 M) rather than the absence of salt containing buffers . OverallThirdly, a time point study was conducted to determine the optimal duration of protein expression in V3 transformed tobacco plants. Interestingly, the expression levels of the L1s were high at 5 to 9 DPIs, whereas non-infiltrated control leaves were negative for all the tested antibodies . The proBased on obtained results, 6 DPI leaves transformed with V3 vector and extracted with neutral PBS buffer was chosen for further purification and quantification studies. Compared with purified VLPs from insect cells, >2.5% of TSP for L1 proteins (2.5 μg L1 / 100 μg TSP) was calculated and overall, 250 mg of L1 yield was obtained per kg of plant biomass.3 density in centrifuge tubes and were referred to as the top and lower bands. L1 proteins were detected in both the collected bands, however, a relatively stronger signal was identified from the top collected band than the lower one  column. Differently assembled forms of L1 VLPs were eluted in a peak with an elution volume of approximately 42 mL of running buffer between 2000 and 75 kDa when compared with different molecular weight protein standards . L1 protIn order to optimize the conditions and strategy for collecting the accumulated VLPs, alternative purification methods were tested. Using forty-five percent of AMS, most of the L1 proteins and VLPs were successfully recovered. Following each step of the AMS treatment, TSPs were precipitated and clarified, which drastically reduced the TSPs to one-fifth of 760 mg (152 mg).These results indicated that more than 75% of TSPs were effectively precipitated; most of the recovered L1 proteins were soluble in PBS buffer, as shown in Based on the isoelectric point (pI) of 8.5 for the L1 protein, crude extracts, CsCl, and AMS precipitated protein samples were also subjected to CM Sepharose Fast Flow, a weak cation exchanger. We found that plant protein samples showed low binding affinity with the cation column and started eluting early with only 0.3 M of NaCl. Unlike the plant protein, the insect cell-expressed L1 protein was eluted at ≥ 0.7 M NaCl (data not shown). These results were also verified by immunoblot assay, which showed that most of the plant L1 protein was eluted without being bound and lost during the wash .Our results confirmed that purified HPV16 L1 protein self-assembled into stable VLPs in plants. As shown in To test the immunogenicity of plant VLPs, antibody titers were evaluated in immunized mice , where 1The aim of our study is to successfully express and purify HPV16 L1 VLPs from tobacco plants using MagnICON expression system. The L1 protein was designed to be accumulated in chloroplasts, which lead to the assembly of predominantly 35–55 nm VLPs with maximal protein level (>2.5% of TSP).Low levels of recombinant HPV L1 protein expression lead to decreased formation of VLPs, which in turn reflects the total outcome yield of purified VLPs ,24. TherCompared to nuclear transformation, it has been documented that genetic engineering of plastids offers many advantages, which make the system ideal for applications involving plant-made pharmaceuticals . These iExpression of L1 proteins was shown in plants transfected with all three constructs upon extraction with an HEPES buffer , and theLow yield of L1 in extracts with HEPES buffer suggested that L1 protein might be degraded during the extraction and homogenization process. To overcome the problem of low yield, we optimized our extraction protocol using different buffers and salts, and an antioxidant supplement was added to reduce L1 oxidation. Our results indicated that extraction of HPV16 L1 from leaf tissue was most efficient (>2.5% of TSP) with a low-salt phosphate buffer (pH 7.0). Using this neutral pH phosphate buffer, we found that L1 expression reached the maximum between 6 and 7 DPI. A study had previously shown that neutral or lower pH aided in the assembly of T = 1 or T = 7 L1 VLPs . SimilarNotably, our improved purification protocol gave higher L1 VLP's recovery with the successful removal of major plant proteins such as Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from the purified product. After that, different purification strategies to intensively recover highly stable and purified VLPs were tested in this study. Therefore, combinations of CsCl density gradient analysis, ammonium sulfate precipitation, and different chromatographic purification techniques were employed to remove contaminated proteins and recover purified ones and eventually to standardize the best conditions for purification. Since VLPs were only partially purified when a single purification method was used, we used a combination of steps to remove contaminated proteins. The VLPs recovered after the purification steps were abundant and highly antigenic as evident by electron microscopic and ELISA analysis. Although the HPV-neutralization activity of plant VLP-induced antibodies was not assessed in the present study, their high binding titers to insect cell-produced VLPs, as shown in Another problem in previous studies was the aggregation of proteins. Therefore, purification conditions that maintain the intact conformation of VLPs are important because recombinant VLPs are inherently unstable and tend to denature and aggregate in solution , 28. To AMS was used as an alternative method to purify L1 proteins. In agreement with a previous study , our resIn conclusion, we demonstrated that a magnlCON plant virus vector with full-length L1 and a chloroplast transit peptide successfully produced HPV16 L1 VLPs in tobacco plants. Extraction of HPV16 L1 from leaf tissue was most efficient (>2.5% of TSP) with a low-salt phosphate buffer in the presence of HPV16 L1 VLPs, predominantly 35–55 nm in size, evident from electron microscopic studies. Although we used CsCl density gradient centrifugation as the preliminary step to remove plant proteins and collect VLPs, these data may be useful for the development of chloroplast-based vaccines. We anticipate that our new purification methods described herein will aid in recovering highly antigenic HPV16 VLPs from tobacco plants towards the goal of reducing time, cost, and labor while increasing the capacity for industrial production.S1 FigImmunoblot represents the detection profile of HPV16 L1 protein where NC; negative control corresponds to non-infiltrated tobacco leaves, V3; PBS extracted 6 DPI tobacco leaves with V3 construct and PC; positive control, VLPs derived from insect cells. MoAbs (16E and 1H8) and PoAb (disCPV2) were used to detect sequential epitopes of HPV16 L1 protein.(TIF)Click here for additional data file.


Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose.We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung.We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF.Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway.NCT01725139, pre-clinical. Is there a scientific rationale for the use of a clinically advanced pan-PI3 kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor in idiopathic pulmonary fibrosis (IPF)?We provide a comprehensive scientific and dosing framework for targeting PI3 kinase signalling in IPF with the pan-PI3 kinase/mTOR inhibitor, GSK2126458, based on the exploration of novel functional endpoints in patient-derived cells and precision cut lung slices.This study provides key data to support the progression of this agent to a proof-of-mechanism trial in patients with IPF and further proposes a roadmap for the repositioning of existing oncology agents as novel therapeutic agents in the IPF setting with minimal requirement for the additional use of experimental animals.Idiopathic pulmonary fibrosis (IPF) represents one of the most rapidly progressive fibrotic conditions with a median survival of 3.5 years from diagnosis,Fibrotic foci, the sentinel lesions in IPF, comprise fibroblasts and α smooth muscle actin positive (αSMA+) myofibroblasts embedded in a type I collagen-rich matrix. These lesions are thought to represent the leading edge of the relentless fibrotic response and arise as a result of a highly abnormal wound healing response characterised by dysregulated epithelial/mesenchymal crosstalk and a complex network of proliferation and differentiation signals received from a remodelled and homeostatically degenerate microenvironment1.in vitro and in tumour necrosis factor α induced fibrosis in vivo.20Class 1 PI3K form an important oncogenic signalling node, integrating signals from a variety of inputs including tyrosine kinase receptors, G-protein coupled receptors and activated Ras,https://clinicaltrials.gov/ct2/show/NCT00972686).GSK2126458 is a potent, highly selective pyridilsulfonamide inhibitor of class 1 isozymes of PI3K in addition to mammalian target of rapamycin (mTOR).in vitro pharmacological assessment of GSK2126458 and related development compounds in lung IPF and non-IPF control fibroblasts. We also assess inhibitor effects using novel functional endpoints in precision-cut IPF tissue slices, and further evaluate pAkt levels in IPF bronchoalveolar (BAL) cells as a potential pharmacodynamic (PD) biomarker to monitor target engagement in future clinical studies. Integration of these data with pharmacokinetic (PK) data for GSK2126458 from oncology trials enabled modelling of an optimal dosing regimen for IPF and the progression of GSK2126458 to an IPF proof-of-mechanism (PoM) study . As well as providing a rationale for progressing a novel therapeutic agent in IPF, these studies further provide a new paradigm for the future re-positioning of existing drugs as novel anti-fibrotic agents.The aim of the present study was to evaluate the scientific and dose rationale for re-positioning GSK2126458 as a potential novel therapeutic agent for IPF. We explore evidence for PI3K pro-fibrotic signalling in IPF lung tissue in combination with performing All human samples were obtained with informed signed consent and with research ethics committee approval . Patients with IPF were diagnosed in accordance with current international guidelines.Primary human lung fibroblasts (LFs) were grown from explant cultures of IPF (IPF-LF) or non-IPF control (C-LF) lung tissue, and cultured as previously described.10.1136/thoraxjnl-2015-207429.supp1Supplementary dataS473, pAktT308, αSMA and CD68 was conducted on 4 μm formalin-fixed paraffin-embedded serial sections of human lung biopsy material from n=12 patients with IPF, using the avidin–biotinylated HRP enzyme complex method (Vector Laboratories). Confocal dual-immunofluorescence microscopy for these markers was also performed on representative fibrotic foci. See Immunostaining for pAkt2/100% humidity) prior to incubation with the PI3K inhibitors Compound 1 or GSK2126458. The effect of short-term exposure (2 h) to Compound 1 on Akt phosphorylation was assessed by Milliplex Map (Millipore) bead assay of lung slice homogenates prepared in ice cold PhosphoSafe buffer (Millipore). In separate experiments, the effect of GSK2126458 on levels of the collagen formation marker P1NP in tissue supernatant was assessed by proprietary competitive ELISA (Nordic Bioscience) as previously described.Precision cut slices of IPF lung, 8 mm, were cultured for 24 h in DMEM supplemented with 0.4% FCS (5% COThe effect of GSK2126458 on FCS-induced Akt phosphorylation in C-LFs (n=2) and IPF-LFs (n=5) was assessed by Meso Scale Discovery (MSD) system. Phosphorylation events (pAkt and pSMAD2) in C-LFs were also assessed by Western blot. Densitometric analysis of Western blots was performed using ImageQuant TL v8.1 software . The intensity of bands in phospho-protein blots was divided by the intensity of the total protein following stripping and re-probing. Values were expressed as a ratio of the positive (stimulated) control. The effect of GSK2126458 on bronchoalveolar lavage fluid (BALF) cell pellet obtained from patients with IPF (n=6) undergoing routine diagnostic BAL was also assessed by MSD. See The effect of GSK2126458 on FCS-induced proliferation in control (n=2) and IPF-derived primary human LFs (n=5) was assessed by MTS assay. Fibroblast proliferation was also assessed by 5-ethynyl-2′deoxyuridine (EdU) incorporation into DNA of primary human LFs . Fibroblast and HBEC apoptosis was assessed by caspase 3,7 activation. See Col1A1 gene expression in confluent fibroblast monolayers following TGFβ stimulation was assessed by RT-PCR following TRIzol extraction as per the manufacturer's instructions (Thermo Fisher). RNA was DNAse treated using a DNAfree kit (Ambion) and real-time RT-PCR conducted using the MESA GREEN qPCR MasterMix Plus for SYBR Assay (Eurogentec). Detailed methods are provided in the S473 and AktT308 localised to classic spindle-shaped, αSMA+ myofibroblasts and epithelium overlying fibrotic foci and cells morphologically characteristic of capillary endothelium  to explore evidence of functional PI3K signalling in areas of active fibrosis in IPF lung biopsy material (representative sections from n=12 patients shown), assessing the phosphorylation state of the critical downstream kinase, Akt. lium see  S1A,C,E.T308 see  S1A,C,G.T308 see  S1B,D,F.T308 see . These spAktS473 A–F. In t located M–R. αSMApAktT308 G–L.Studies performed in live cultured precision-cut lung slices obtained from a patient with IPF confirmed prominent Akt phosphorylation in lung tissue and this signal was reduced in a concentration-dependent manner following incubation with a closely related analogue of GSK2126458, Compound 1  (see onl50=0.84 nM (range 0.43–1.64), n=2 donors) and IPF-LF (50=1.52 nM (95% CI 0.73 to 3.18), n=5 donors) in IPF-LF. S473 phosphorylation. 50=18.70 nM , IPF-LF geometric mean=23.64 nM (95% CI 13.61 to 41.07), n=5 donors) assessed by MTS assay. The inhibitory effects of GSK2126458 on FCS-induced fibroblast proliferation were confirmed using a second assay based on the incorporation of the modified thymidine analogue, EdU, into newly synthesised DNA (Click-iT EdU). The IC50 for GSK2126458 obtained in this assay system was comparable to that obtained in the MTS assay ). Investigation of the effect of GSK2126458 on fibroblast apoptosis , showed that apoptosis was not detectable until concentrations ≥3 μM (We next assessed the effect of GSK2126458 on Akt phosphorylation and proliferation of primary IPF and non-IPF control LFs (IPF-LF and C-LF). GSK2126458 inhibited serum-induced pAkt in C-LF  revealed that macrophages within IPF alveolar spaces were highly immunoreactive for pAkt. We therefore reasoned that since macrophages are a prominent cell type in IPF BALF, this observation could be exploited to develop a PD biosensor to monitor in vivo inhibition of PI3K signalling by GSK2126458.IHC studies (50=0.58 nM (95% CI 0.40 to 0.86)). The inhibitory profile of GSK2126458 was further found to be maintained in BALF cells re-suspended in ice-cold saline for up to 35 min following incubation with the drug (data not shown).BALF differential cell counts confirmed that macrophages formed a prominent cell type n=6, see  S5. Akt in vitro studies were integrated with human PK data for GSK2126458 from ongoing clinical trials in patients with advanced solid tumours , according to the scheme outlined in To develop a dose rationale for GSK2126458 for future IPF clinical trials, the data from these The observation that the epithelium also displays a prominent pAkt signal lead us to further investigate the potential effect of GSK2126458 on epithelial cell apoptosis. 1 implicated as a critical mediator of this process.1 signalling.1-induced fibroblast collagen production.Progressive, excessive and disorganised deposition of collagen and other extracellular matrix (ECM) proteins underlies the gross remodelling leading to loss of lung function in IPF, with TGFβ1 (1 ng/ml) stimulation. 1 induces rapid phosphorylation of SMAD2 peaking at 2 h, with pAktS473 exhibiting a slower kinetic peaking at 12–24 h (COL1A1) gene expression , and leads to a reduction in cell number with an IC50 of 1.66 μM (95% CI 0.51 to 5.43). Induction of caspase 3,7 in molecular crowded conditions is consistent with concentrations of GSK2126458 which cause reduction in cell count, suggesting apoptosis in crowded conditions may be responsible for the observed reduction in cell number  in IPF tissue slices using a competitive ELISA based on a proprietary monoclonal antibody.PI3K represents a key oncogenic signalling node, implicated in a range of cellular processes from inflammation to cancer. More recent evidence also suggests a role for this pathway in fibrosis, however to the best of our knowledge there are currently no PI3K agents in clinical development for IPF or any other fibrotic condition. The aim of the current study was to further explore the rationale for targeting PI3K signalling in IPF and to define the pharmacological profile of a potent and clinically advanced PI3K/mTOR inhibitor, GSK2126458, as a novel anti-fibrotic.T308) and serine 473 (pAktS473) to initiate downstream signalling. The initial focus for our PI3K pathway validation studies in IPF was therefore to perform detailed IHC studies to determine Akt phosphorylation in IPF biopsy tissue. These studies showed demonstrable immunoreactivity for pAktS473 associated with activated myofibroblasts within IPF fibrotic foci, as previously suggested.T308 immunostaining associated with these myofibroblasts and thus provide the first evidence of full Akt activation in situ in this disease context. Immunofluorescent confocal co-localisation studies further provide the first evidence of cell-specific differential phosphorylation of Akt: whereas pAktT308 immunostaining predominates in myofibroblasts and a thin band of cells located at the epithelial–mesenchymal border of fibrotic foci, pAktS473 immunostaining was evident in both the epithelium and myofibroblasts. Moreover, heterogeneous distribution and phosphorylation of Akt were observed in bronchial epithelium and macrophages in the IPF lung. Differential phosphorylation of Akt has not been extensively studied; however in cancer, it has been proposed to influence the interactions of Akt with its substrates and thereby influence cell motility and invasion.T308 relative to pAktS473 is associated with poor prognosis.PI3K mediates the conversion of phosphatidylinositol 4,5 bisphosphate (PIP2) to phosphatidylinositol 3,4,5 trisphosphate (PIP3) at the plasma membrane, inducing the recruitment and phosphorylation of Akt at threonine 308 , GSK2126458 would significantly impact fibroblast PI3K signalling and proliferation in the lungs of patients with IPF.1-induced myofibroblast collagen production, based on limited literature reports suggesting the involvement of PI3K in SMAD-independent TGFβ1 signalling.1-induced myofibroblast collagen deposition at concentrations around the 100 nM range.The decision to progress GSK2126458 to a PoM trial in IPF was predicated on an appropriate dose rationale for this compound as a fibroblast anti-proliferative agent, based on the widespread evidence linking this pathway to cell growth and proliferative responses in multiple cell types and disease settings.1 signalling at clinically attainable doses, while potentially sparing its essential homeostatic and tumour suppressor functions. This is a particularly important consideration in patients with IPF who are at a heightened risk of developing lung cancer.39Our final aim was to directly link PI3K/mTOR signalling to pro-fibrotic collagen formation in IPF lung tissue using an ex vivo human tissue model. Type I collagen is the most represented extracellular protein in the lungs and its dysregulated turnover is a hallmark of fibrosis. During maturation of type I collagen, soluble pro-collagen is processed to its insoluble form by carboxy and amino terminal endopeptidase cleavage, generating peptide fragments such as the N-terminal P1NP which can be monitored as a sensitive biomarker of collagen formation.1-induced collagen production in IPF fibroblasts. Population modelling and integration of preclinical biomarker data with human PK data has enabled us to develop a dosing framework which should safely and effectively engage PI3K signalling in the lungs of patients with IPF, providing support for progressing GSK2126458 to further clinical evaluation in IPF in a dose-finding, double-blind, placebo-controlled study . To the best of our knowledge, this represents the first IPF clinical study aimed at assessing target engagement and tolerability of a PI3K/mTOR inhibitor in this disease setting.In conclusion, we have considerably extended the scientific rationale for targeting PI3K signalling in IPF using patient-derived cells and lung tissue. We provide evidence for active PI3K signalling in the fibrotic foci of IPF patients together with data that GSK2126458 attenuates serum-induced proliferation and TGFβ


The neurotransmitter dopamine (DA) inhibits the fibrillisation of α-syn into amyloid, and promotes α-syn aggregation into SDS-stable soluble oligomers. While this inhibition of amyloid formation requires the oxidation of both DA and the methionines in α-syn, the molecular basis for these processes is still unclear. This study sought to define the protein sequences required for the generation of oligomers. We tested N- (α-syn residues 43–140) and C-terminally (1–95) truncated α-syn, and found that similar to full-length protein both truncated species formed soluble DA:α-syn oligomers, albeit 1–95 had a different profile. Using nuclear magnetic resonance (NMR), and the N-terminally truncated α-syn 43–140 protein, we analysed the structural characteristics of the DA:α-syn 43–140 dimer and α-syn 43–140 monomer and found the dimerisation interface encompassed residues 43 to 60. Narrowing the interface to this small region will help define the mechanism by which DA mediates the formation of SDS-stable soluble DA:α-syn oligomers. Parkinson's disease (PD) is a progressive neurodegenerative disorder characterised by severe motor dysfunction, muscular rigidity and resting tremor.  PathologThe loss of dopaminergic neurons in the substantia nigra, and the subsequent depletion of dopamine (DA) in the striatum is a key feature of PD. α-syn expression can regulate DA metabolism since α-syn knockout mice have reduced striatal DA levels and an attenuated DA-dependent locomotor response to amphetamine.  There isThere is a clear difference in behaviour between α-syn and the DA:α-syn oligomer since the DA:α-syn oligomer cannot interact with lipid vesicles or cause membrane permeability while α-syn (in the absence of DA) does.  While DAStructural analysis using atomic force microscopy indicated the DA:α-syn oligomers possessed non-fibrillar morphologies. , 24 ElecFull-length (1–140) human α-syn cDNA sequence was cloned into the vector pRSETB  and expressed and purified as previously described.  PurifiedThe α-syn 1–60, 1–95, 43–140, H50N, T75P constructs were made using the QuikChange Site Directed Mutagenesis system . α-syn 1–60 was also produced synthetically using FMOC peptide synthesis . The α-syn 43–140 construct has a Met as the first residue. All truncated and mutant α-syn proteins except α-syn 1–60 and 1–95 were expressed and purified as for wildtype α-syn above. The α-syn 1–60 and 1–95 proteins were purified by ammonium sulfate (50%) precipitation, whereby solid ammonium sulphate was added to the lysed cell mixture (v/v) and stirred overnight at 4°C. Following centrifugation at 3200 × g for 1 hour at 4°C, the supernatant discarded and the pellet resuspended in 10 mM sodium phosphate buffer pH 7.5. After boiling for 20 mins and centrifugation at 3200 × g for 1 hour at 4°C the supernatant was collected. The supernatant was desalted using a PD-10 desalting column  and then loaded on a HiTrap SP Sepharose FF cation exchange column. Bound α-syn 1–60 and 1–95 were eluted using a linear salt gradient from 0 to 1M NaCl in 20 mM Tris HCl at pH 8.5. Fractions containing α-syn 1–60 and 1–95 were pooled and lyophilised. The sample was resuspended in 100 mM ammonium bicarbonate pH 7.5 and loaded onto a Sephacryl 300 SEC column  and eluted isocratically with 100 mM ammonium bicarbonate (pH 7.5) at a flow rate of 1mL/min. α-syn 1–60 containing fractions were pooled, dialysed and lyophilised. Protein identities were confirmed by mass spectrometry. The α-syn 43–60 peptide was synthesised using FMOC peptide synthesis 15N) or doubly  for NMR studies. [600 of ~0.6 was achieved, the cells were pelleted by centrifugation at 3200 × g for 15 minutes at 4°C. Cells were washed twice, centrifuging after each wash with M9 minimal media to remove contaminating LB media. Cells were resuspended in M9 minimal media containing either 15NH4Cl or 15NH4Cl and 13C glucose and allowed to equilibrate at 37°C shaking at 220 rpm for 2 hours. Expression was induced with 1 mM IPTG overnight at 37°C. Cells were harvested by centrifugation at 3200 × g, 1 hour at 4°C. Cell pellets were stored at -20°C until lysis and purification were performed according to the method outlined above. Different batches of the protein being expressed and purified during this study.α-syn was isotopically labelled either singly . The concentration of stock solutions were determined by UV absorbance measurements using an extinction coefficient of 5120MSamples were prepared in SDS sample buffer  (v/v) and heated at 100°C for 10 minutes. The samples, unless otherwise stated, were separated on a 12% Bis-tris SDS-PAGE gel (Life Technologies) and the proteins visualised by silver staining according to manufacturer’s instructions (Bio Rad). Some samples were analysed on a Novex 16% Tricine SDS-PAGE gel (Life Technologies) according to the manufacturer’s instructions.Oxidised DA was generated by incubating 2mM DA in PBS pH 7.4 at 30°C and shaking for 28 days. The reaction was then centrifuged at 200,000 × rpm for 60 min to obtain a pellet (insoluble oxidised DA corresponding to polymerised oxidised DA) and a supernatant (soluble oxidised DA) fraction. The pellet fraction was washed 3× with water and resuspended in the original volume with water. The supernatant fractions were analysed by ESI-MS and an UV-visible absorbance spectrum acquired between 200–700nm confirmed the absence of un-oxidised DA in the sample.DA mediated α-syn oligomerisation was performed as previously described  using 20Oligomerisation of α-syn for NMR analysis was performed by incubating 15mg of α-syn 43–140 at a concentration of 200 μM with 2 mM DA at 37°C on a heat block for 7 days to ensure maximal oligomerisation had occurred. After 7 days, the mixture was centrifuged 100,000 × rpm for 1 hour at 4°C and the supernatant collected. The supernatant of the crude reaction mixture was loaded onto a Superdex 200 16/60 GL SEC column . Fractions were eluted in 10 mM sodium phosphate pH7.5. Monomer and dimer-containing fractions were pooled and concentrated using an Amicon centrifugal unit with a molecular cut-off (MWCO) of 10 kDa. The monomer was concentrated using a unit with a 3 kDa MWCO. Different batches of the oligomers were generated and purified during this study.Circular dichroism (CD) measurements were performed on a JASCO 815 CD spectrometer , using the Jasco software. Far UV-CD spectra were collected between 180–250 nm at 37°C. Measurements were recorded at 50 nm/min with a 1 nm bandwidth and a 2s response time, averaging 3 accumulation scans per measurement. All spectra were background subtracted, and smoothed with the default algorithm in the Jasco software. Experiments were performed in triplicate.2O/90% H2O or 100% D2O as appropriate. 1H,13C-HSQC spectra were acquired on α-syn, at natural abundance. All double-resonance spectra were acquired with 2048 complex data points in the directly detected dimension and 512 complex data points in the indirect dimension.NMR spectra were acquired on a Bruker-Biospin Avance 800MHz spectrometer equipped with a cryogenically cooled probe. All experiments were performed at 5°C, and samples were typically 0.5mM protein in 0.5mL 10mM phosphate buffer pH 5.5 with either 10% D1H, 15N NOESY-HSQC with a mixing time of 100 ms, 1H, 1H NOESY with a mixing time of 300 ms, 1H TOCSY with a mixing time of 50 ms, 1H, 15N-HSQC, 1H, 13C-HSQC, HNCO, HN(CA)CO, HN(CO)CA, HNCA, HNCACB, CC(CO)NH, HNHA, and HNHB. [A series of standard two-dimensional (2D) and three-dimensional (3D) spectra were recorded to obtain sequence specific assignments, these included nd HNHB. Spectra wnd HNHB. 1H,15N X-filtered NOESY-HSQC experiment were performed using sample prepared by incubating α-syn in a reaction containing 50% unlabelled α-syn and 50% 13C, 15N labelled α-syn with DA in a 1:10 protein to DA ratio. This reaction was mixed and incubated at 37°C for 7 days. The solution was then centrifuged 100,000 × rpm, 1 hour at 4°C and the supernatant was then separated by SEC on a Superdex 200 16/60 column  to isolate the dimer. The dimer fractions were eluted in 10 mM sodium phosphate buffer at pH 7.5 and concentrated using an Amicon centrifugal filter unit  with a MWCO of 10 kDa to a final volume of ~500μl.A 120 ms Sequencing grade trypsin  was added to a solution containing either α-syn 43–140 monomer or α-syn 43–140 dimer in 50 mM Tris-HCl pH 7.5 at a 1:50 trypsin to protein ratio. Typically, 1mg of protein and 20 μg of trypsin was used in each experiment. The reaction was incubated at 37°C for 18 hours and analysed using reverse phase HPLC and ESI-TOF-MS.ESI-IMS-MS was performed as described.  All reagAggregation and spectroscopy experiments were repeated at least three separate times and were performed with different batches of expressed and purified protein and different batches of purified oligomers.To define which regions of α-syn are required for the formation of the DA-mediated soluble oligomers, we tested N- and C-terminal truncated proteins . α-syn 4The generation of DA mediated α-syn oligomerisation requires oxidation of DA to occur  and it iTo study the biophysical properties of the DA:α-syn 43–140 species we reacted α-syn 43–140 with DA and the dimer and monomer were purified using SEC and their identity confirmed by SDS-PAGE  and ESI-The DA:α-syn 43–140 dimer was characterised by far-UV CD spectroscopy and gave a single minima at λ = 200 nm that is indicative of a predominately random coil structure. A similar spectrum was observed for the DA:α-syn 43–140 monomer . TherefoTo investigate the stability of the DA:α-syn 43–140 dimer, we exposed it to trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP), which are known to dissociate oligomers to their monomeric species. , 36 The To further analyse the conformational properties of the DA:α-syn 43–140 protein we employed electrospray ionisation—ion mobility—mass spectrometry (ESI-IMS-MS). This method allows the gross conformation state and non-covalent interactions of a protein to be determined. , 38 The To analyse the behaviour and structure of the DA:α-syn 43–140 oligomers in solution we studied DA:α-syn 43–140 using NMR. This protein construct was chosen to reduce the spectral complexity arising from the presence of multiple repeat sequence motifs in α-syn and the resultant chemical shift redundancy.15N) or doubly  labelled protein (refer Methods). A proton nitrogen correlation spectrum  of the DA:α-syn 43–140 monomer was recorded at pH 5.5 to ensure favourable amide proton exchange rates. The 1H, 15N-HSQC spectrum of the DA:α-syn 43–140 monomer displayed the characteristic narrow chemical shift dispersion in the amide 1H resonances for an IDP [1H, 15N HSQC spectrum of DA:α-syn 43–140 dimer also showed the characteristic lack of chemical shift dispersion for the amide 1H resonances expected for an IDP  abuts 43–60 and is required for α-syn fibrillisation. A proline scan of this region identified residues that inhibited fibril formation, such as threonine 75.  To examiThe N-terminally truncated 43–140 α-syn dimer was chosen for structural characterisation, over the C-terminal truncation or full-length α-syn, as α-syn 43–140 formed DA-mediated soluble oligomers with a similar oligomeric pattern to full length α-syn but significantly simplified the NMR analysis by reducing the number of sequence repeats to minimize chemical shift redundancy in the spectra. Moreover, the ESI-IMS-MS analysis of 43–140 α-syn showed it behaved in a similar manner to full-length α-syn by occupying both extended and compact conformations under neutral conditions. In the presence of DA the highly extended state monomeric state bound three DA ligands without the need to oxidise the Met residues. These similarities between 43–140 and full-length α-syn support the utility of using the 43–140 construct to investigate which regions of α-syn are required for the formation of the DA-mediated oligomers. In this study we compared the monomeric and dimeric species in order to understand the mechanism by which DA mediates α-syn oligomerisation.1H, 15N HSQC spectrum showed amide resonances with limited chemical shift dispersion, characteristic of an IDP [1H,15N HSQC of α-syn 43–140 dimer were unshifted compared with those in the monomer, indicating that there is not a gross conformational change when compared to the monomer. Amide resonances of T43, V48, V49, H50, G51, T59, K60, E114, M116, V118, E126, M127, and S129 for the α-syn 43–140 dimer, are shifted or broadened compared to those in the monomer. These changes are centred around the N-terminus and the M127 residue at the C-terminus and indicates changes in chemical environment surrounding specific residues upon interaction with DA. Furthermore, the similarity in the 1H,15N-HSQC spectra between the dimer and monomer α-syn 43–140 suggests that, only a limited number of residues are modified by DA.CD and NMR spectra showed the DA:α-syn 43–140 dimer adopted a largely unstructured conformation in solution. The f an IDP , while Cf an IDP  The majoWhile CD, NMR and mass spectrometry data identified very few differences between the DA:α-syn 43–140 dimer and monomer, the dimer displayed altered behaviour in a membrane-mimetic environment. The presence of SDS caused α-syn 43–140 monomer to adopt α-helical structures and similar behaviour has been observed for full length α-syn. , 54–56 IOur studies expand upon the NMR and biophysical analysis of full length α-syn modified by DA quinones  which shThis study has provided novel and important data on the structural characteristics of the DA:α-syn oligomer, and the contribution of distinct domains and residues to their formation. We have narrowed the interface to a small region and while the exact residues involved and the nature of the interaction with DA / DA-products remains unresolved, our new data further defines the mechanism by which DA mediates the formation of these SDS-stable soluble DA:α-syn oligomers.S1 FigSilver stained SDS-PAGE gel of the different α-syn proteins treated with either freshly prepared or pre-oxidised DA. No DA refers to the proteins minus the addition DA. Fresh DA refers to DA (200uM) that has been added to the α-syn soon after the DA has been dissolved, as per the other incubations performed in this study, i.e. (TIF)Click here for additional data file.


Sarcoidosis is an inflammatory disease with a wide range of clinical presentations. The manifestations and prognosis in sarcoidosis are dependent upon not only organ involvement but also age and sex. The purpose of this review is to describe the systemic and ocular manifestations of sarcoidosis with a specific focus on sex-dependent difference in presentation and management. Sarcoidosis is more common in women, particularly in patients who present after age of 50 years. Women with sarcoidosis are more likely to develop cystoid macular edema and the mortality rate is higher than that of men. Sarcoidosis is a systemic inflammatory disease of unknown etiology that can target almost any organ of the body. The most commonly involved organs include the lungs, lymph nodes, skin, and eyes . The cliPatients suspected of having sarcoidosis may undergo a battery of diagnostic tests that are not specific for sarcoidosis, but rather markers of granulomatous inflammation. Angiotensin converting enzyme (ACE) may be elevated in 60–70% of patients with sarcoidosis . The serWhile various diagnostic tests can support a diagnosis, none are confirmatory, and, thus, sarcoidosis is considered a diagnosis of exclusion. Experts agree that three criteria must be met prior to assigning a diagnosis of sarcoidosis: (1) clinical or radiologic findings consistent with sarcoidosis, such as pulmonary disease, uveitis, mediastinal hilar lymphadenopathy, or erythema nodosum; (2) tissue biopsy with histologic evidence of noncaseating granulomas; (3) absence of other causes of granulomatous disease . A diagnOcular involvement occurs in 11–83% of cases of sarcoidosis patients and can lead to significant morbidity –18. The Initial management of patients with systemic sarcoidosis is challenging because evidence exists that the use of systemic corticosteroids actually increases the likelihood of relapse . In patiUnderstanding the epidemiology of sarcoidosis is complicated by the variability in presentation and diagnostic criteria and the disease is certainly underdiagnosed. As an example, one recent study reported sarcoidosis in 22% of patients with mediastinal incidentalomas identified on chest CT .Sarcoidosis presents most often in young adults under the age of 50, with the highest incidence reported in patients between 20 and 39 years of age . CountriAs with many inflammatory diseases, sarcoidosis affects women more than men. Three population-based studies performed in three different countries have produced similar findings. In Japan, the rates were 1.2 versus 1.4 per 100,000 in males versus females  and, in The increased incidence of sarcoidosis in females has led some to hypothesize that hormones may be a compounding factor. The Black Women's Health Study (BWHS) followed black women between 1995 and 2009 and determined that factors not associated with an increase in incidence of sarcoidosis included age at menarche, age at menopause, and parity. A reduced incidence of sarcoidosis was noted with a later age at first birth, and a weak association was suggested between recent birth and reduced incidence of sarcoidosis , althougP = 0.003). Uveitis was more common in the elderly group versus the younger group , and 80% of the patients with uveitis in the study were female [A second trend is the recognized second peak of sarcoidosis diagnosed in patients over 50 years of age , 27, 32.e female .The relapse rate of systemic sarcoidosis may also be influenced by sex. White males reportedly have a higher relapse rate of sarcoidosis than white females and African Americans of either gender . In thisThe array of clinical manifestations associated with sarcoidosis is quite varied and includes both constitutional and organ-specific presentations. In any patient with suspected sarcoidosis, a complete review of systems is essential to help characterize the systemic disease and direct diagnostic testing and management. Generalized symptoms of sarcoidosis include fatigue, night sweats, weight loss, arthralgia, and exercise intolerance . Gender Organ-specific involvement is easier to characterize and several sex differences have been identified in this regard. Males tend to have higher rates of pulmonary and cardiac involvement, while females are more prone to peripheral lymph node, skin, eye, and liver disease , 38, 39.Endocrine abnormalities are common and possibly underreported in sarcoidosis. Hypercalciuria and hyperprolactinemia have been reported in up to 30% of patients with sarcoidosis , 42. AbnGenitourinary sarcoidosis will obviously differ between men and women. Among men, clinical signs include testicular swelling and a painless mass in the scrotum , while wSarcoidosis can involve almost any ocular structure, including the globe, orbit, and adnexa. As with systemic sarcoidosis, ocular manifestations of the disease are more common in women –48. In ASeveral studies have demonstrated a second peak of uveitis in patients over the age of 50, and the vast majority of patients in this subset are female , 49, 50.Patients with sarcoidosis have been successfully treated with numerous anti-inflammatory agents, ranging from topical and systemic corticosteroids to antimetabolites and monoclonal antibodies. Because sarcoidosis often occurs in patients of childbearing age, special considerations must be made in patients who may have planned or unplanned pregnancy. The pregnancy class of many medications used to treat sarcoidosis is listed in A recent international poll of sarcoidosis specialists found that 80% of physicians considered MTX their preferred second-line treatment option for sarcoidosis, after systemic corticosteroids . They alTopical and periocular corticosteroids are commonly used to treat uveitis during pregnancy. In the case of topical administration, patients should be educated on punctual occlusion after administering the medication to reduce systemic absorption. Intravitreal bevacizumab, a class C drug, has reportedly been used for choroidal neovascularization, a potential complication of posterior segment inflammation which can lead to significant and permanent vision loss, in pregnant patients. In a small series of 4 patients treated off-label with intraocular bevacizumab, vision improved and no adverse events were observed . The usePatients with sarcoidosis often report a diminished health-related quality of life , with a Extrapulmonary manifestations of sarcoidosis differ by race and sex. Black Americans have a higher prevalence of extrathoracic involvement . A recenDeath from sarcoidosis is closely associated with respiratory, cardiac, neurologic, and hepatic involvement . A recenWomen tend to have higher rates of many autoimmune diseases , includiGender differences may also be related to genetics. A recent Brazilian study found that certain HLA alleles were more likely to be found in mestizos and blacks with sarcoidosis . It is pAlmost every aspect of sarcoidosis, from epidemiology and clinical presentation to treatment options and prognosis, is influenced by patient sex. Men are diagnosed at a younger age than women, and the onset of sarcoidosis in women follows a bimodal distribution with a second peak of onset occurring after age of 50. In this older cohort of patients, chest X-ray may not be sensitive enough to identify pulmonary changes and computed tomography is recommended. Women tend to have more CME and a worse overall visual prognosis than men. Women are more likely to have cutaneous involvement than men, and the mortality rate is higher in women. Several medications used to treat sarcoidosis are contraindicated in pregnancy; therefore, discussions regarding birth control should be thorough and repeated at every visit.