Very few studies have examined the risk of short-term adverse hemorrhage of low-dose aspirin use in primary prevention. This case-crossover study examined the transient effect of low-dose aspirin use on major hemorrhagic risks.A representative database of 1,000,000 patients randomly sampled from the Taiwan's National Health Insurance Research Database in 2000 was analyzed. The study cohort consisted of a total of 501,946 individuals, aged 30–95 years old, at risk of a major bleeding event in 2000. A case-crossover study was used to retrieve data on 10,905 incident patients with major hemorrhagic complications  and prescribed low-dose aspirin (≤300 mg/day) from 2000–2008. A 56-day time window (∼2 months) was used as the case period for which the odds ratio (OR) was estimated using the ratio of patients exposed during the 56-day case period only (1–56 days before the index date) compared to its corresponding 56-day control period only (57–112 days before the index date).Four hundred eighty-nine (4.5%) of the 10,905 hemorrhagic patients had used low-dose aspirin during the 56-day case only period; 294 (2.7%) of the same patients had used low-dose aspirin during control only period. Low-dose aspirin use increase the risk of developing a major hemorrhage 1.33-fold . Significance was found prominent in 4,453 non-hypertensive and non-diabetic subjects .Transient low-dose aspirin use increases risk for major hemorrhagic events in Han Chinese. Long-term antiplatelet treatment has been estimated to reduce ∼25% of non-fatal myocardial infarction, non-fatal stroke, and vascular death et al., the clinical guidelines of aspirin use in the primary prevention of cardiovascular events in Western countries may not be appropriately applied to Asian populations due to different disease preference in this population Because a more recent meta-analyses has revealed that aspirin has a chemopreventive effect on cancer incidence and mortality et al., analyzing a population-based cohort of 4.1 million Italian citizens, reported that low-dose daily aspirin use (≤300 mg) significantly increased the risk of both gastrointestinal and cerebral hemorrhages Recently, De Berardis Using Taiwan's nationwide population-based insurance claims dataset, we performed a case-crossover study to investigate the impact of short-term (56 days) low-dose aspirin use on major bleeding events. This timeframe should make it possible to elucidate the transient effect of aspirin on the risk of acute events 12,13]. The use of case-crossover design may provide more reliable data because the same subjects, who are investigated at adjacent time points, serve as their own controls, minimizing the confounding of both known and unknown time-invariant variables between the study patients.This study tapped Taiwan's single-payer National Health Insurance (NHI), promulgated by the Taiwan government on March 01 1995 International Classification of Diseases, Ninth revision, clinical Modification (ICD-9-CM)This study used a sampling cohort dataset obtained from NHIRD. National Health Research Institutes use a systematic sampling approach to randomly select a representative database of 1,000,000 patients from the year 2000 registry of all NHI enrollees (NHI 2000) ICD-9-CM codes 140.xx-208.xx), acute myocardial infarction (AMI) , ischemic stroke , major gastrointestinal hemorrhage  or cerebral hemorrhage (ICD-9-CM codes 430-432) between January 1, 1997 and December 31, 1999. Patients expiring or leaving the NHI program for unknown reasons in 2000 were also excluded.This case-crossover study recruited patients aged 30–95 years old in 2000 as potential study subjects . We exclWe prospectively followed the remaining relatively healthy patients starting in January 1 2000 until first primary diagnosis of AMI, ischemic stroke, major gastrointestinal hemorrhage, or cerebral hemorrhage during a hospital stay or death from other causes, withdrawal from the NHI program for unknown reasons, or the end of study period (December 31 2008). From those first diagnosed with major hemorrhagic complication, we excluded patients who were prescribed aspirin dosages >300 mg/day on any day during the study time period window (1–112 days before the date of that diagnosis), leaving us with only those prescribed low-dose aspirin .ICD-9-CM code 250) or hypertension (ICD-9-CM codes 401–405) was defined in a patient if he or she was diagnosed for the diseases in at least two outpatient claims or in one inpatient claim a year before the major hemorrhagic complication was first diagnosed. General health status was assessed by the Charlson co-morbidity index, which is the sum of the weighted score of 17 co-morbid conditions and is widely used to control confounding in epidemiological studies , and urbanization level . Diabetes . We estimated the odds ratio (OR) as ratio of patients exposed during the 56-day case period only , defined as 1–56 days before the first diagnosis of major hemorrhagic complication, to the same patients exposed during the same 56-day control period only, defined as 57–112 days before first diagnosis of major hemorrhagic complication. The advantage of this case-crossover design approach is that the transient exposure effect on acute outcome can be examined without between-patient confounders, such as smoking, etc 2 agonist, and vasodilators, as another time-variant variable. Exposure to these drugs was defined as having a prescription of one of them at least one day during the case period (56 days before first diagnosis of major hemorrhagic complication) or control period (57–112 days before first diagnosis of major hemorrhagic complication). In addition, the number of outpatient visits during these two time periods was counted.To control for confounding variables, drugs that could potentially and significantly accelerate or reduce bleeding tendency and changes in their prescriptions over time were included in the model. These drugs were anticoagulants, antilipemic agents such as statin, nonsteroidal anti-inflammatory agents (NSAID), proton-pump inhibitors (PPI), antidepressants such as tricyclic antidepressants, selective serotonin reuptake inhibitors, monoamine oxidase inhibitors, or other antidepressants, and corticosteroid, all of which have been considered by a previous study For the case-crossover analyses, we first used McNemar test to investigate the significance of aspirin use between case and control period, and conditional logistic regression to estimate the odds ratio (OR) and its 95% confident interval (CI) of gastrointestinal or cerebral hemorrhage or both. Then, subgroup analyses were performed by stratifying the different time-invariant and time-variant characteristics of the patients, including age, gender, place of insurance registry, urbanization, year and season of index date and other medical conditions diseases such as Charlson co-morbidity index, hypertension, and diabetes as well as the presence of other confounding drugs during the study time window.Conditional logistic regressions were performed to explore the effect of aspirin use on hemorrhagic events after adjusting for the time-variant confounding factors which were significant in the univariate analysis listed in In addition to our analysis of a 56-day case period, we also computed odds ratios for different case periods, including 28-, 84-, 112-, 140-, 168-, 252-, and 336-day durations  to test the robustness of the results. This kind of analysis was also applied to different sites of hemorrhagic complications. All statistical operations were performed using SAS 9.2 statistical software; two-sided P value <0.05 was considered significant.A total of 10,985 cohort patients were hospitalized for a primary diagnosis of incident hemorrhagic complication between 2000 and 2008 . We exclOf the 10,905 patients, 489 (4.5%) had used low-dose aspirin during the 56-day case only period and 294 (2.7%) during the control only period . User raAfter adjusting for other time-variant medication variables and number of outpatient visits, we found that low-dose aspirin use conferred a 1.33-fold risk  of major hemorrhage during the case period, compared to the control period. Even after excluding the 99 study patients whose averaged daily dosage was >100 mg/day, the result remained similar, with an adjusted odds ratio (AOR) of 1.36 . Replacing history of hypertension by anti-hypertensive agents and adjusting this time-variant variable in the regression model, we found that low-dose aspirin use conferred a 1.18-fold risk  of major hemorrhage during the case period, compared to the control period. Similar and significantly increased risks related to low-dose aspirin use were also found in almost all other sub-categories of the time-invariant variables, including age, gender, urbanization level, hypertension, diabetes, Charlson index score, and year of event . BecauseThe AOR risk of bleeding events associated with aspirin use was 1.35  in gastrointestinal hemorrhage and 1.28  in cerebral hemorrhage . Figure As can be seen in In this case-crossover study using a nationwide representative sampling cohort, we found that short-time low-dose aspirin use increased the risk of major bleeding events among relatively healthy patients. Its adverse health effect was found to be particularly prominent in gastrointestinal hemorrhages and was consistently present when it was prescribed for two months or more. While increased risk was found in patients with and without diabetes as well as patients with and without hypertension, the risk was found to be higher in patients without these diseases. The case-crossover design of the study eliminated confounding by between-subject time-invariant factors and reduced the likelihood of “reverse causation” often associated with retrospective cohort designs et al., who reported a ∼55% increased risk The 33% increased risk we found was slightly lower than that reported by De Berardis vs. 33% increase, respectively). This finding was slightly different from De Berardis et al., who reported a large and significant increase in such risk in patients who did not have diabetes but only small and insignificant increase in patients with diabetes , again probably due to different study designs et al. also included case patients with previous hospitalization for cardiovascular events. We found increased risk of bleeding in both non-hypertensive (93%) and hypertensive subjects (23%). Together, our findings raise concern regarding the use of low-dose aspirin for primary prevention of cardiovascular diseases in the Taiwanese population with diabetes or hypertension.This study found the increased risk of major bleeding to be similar for patients with and without diabetes  than in the user group of NSAID (AOR = 1.20), suggesting whether use of NSAID in the observational period can modify the effect of low-dose aspirin use on the hemorrhagic event . The simet al., using existing published data on the rates of coronary heart disease, hemorrhagic stroke, and major gastrointestinal bleeding from the Japanese population, found that the benefit of aspirin use only outweighed risk in subjects older than 40 years who had both diabetes and hypertension There is a wide reported variation in response to antiplatelet treatment in different ethnic groups (0.4 to 35%) The mechanisms underlying the first occurrence of occlusive vascular diseases and recurrence may be different, suggesting that the benefit of aspirin in the secondary prevention of vascular diseases may not be extrapolated to its use in primary prevention. Aspirin is a non-selective cyclo-oxygenase inhibitor; its half-life is approximately 6 hours in plasma. Although the half-life of aspirin is short, to generate the health effect from reducing the synthesis of prostaglandins in the vascular wall resulting in constricting the vessel wall and enhancing platelet adhesion to the vessel wall in healthy people takes much longer than the original exposure half-life of aspirin. The present study in vs. control, OR = 0.70, 95% CI = 0.67–0.98). These two findings suggest that subjects may need to avoid the risk of major bleeding complications during the first three years of aspirin use to enjoy the benefit of cancer prevention. That study, however, only addressed low-dose aspirin associated extracranial bleeding The advocacy of low-dose aspirin use for primary prevention has regained some momentum because it has recently been reported to protect against the development of some forms of cancer When attempting to prevent cardiovascular disease, physicians may need to be reminded that treatment with antiplatelets, including low-dose aspirin, is not the sole choice for primary prevention of cardiovascular diseases. Many other alternative preventive strategies of lifestyle changes such as regular exercise, weight loss, saturated-fat diet control, smoking cessation and sugar restriction can be used first in primary prevention to avoid unintentional harm by antiplatelet agents This study has several limitations. One limitation is that our study findings could be influenced by time-variant variables In conclusion, this study shows that low-dose aspirin use increased acute major bleeding events, particularly gastrointestinal bleeding. While this increased risk was found in both diabetes and non-diabetes patients as well as both hypertension and non-hypertension patients, it was more prominent among patients without diabetes and hypertension. Further large prospective studies are warranted to investigate the risks and benefits of low-dose aspirin use in primary prevention to help physicians make appropriate recommendations for patients without previous history of cardiovascular or cerebrovascular diseases.Table S1aICD-9-CM Diagnosis Codes for Constructing Deyo-Charlson Comobidity Index.(DOCX)Click here for additional data file.Table S2Usage of Confounding Medicine Between the Case (1-56-days) and Control Periods (57-112-days) for Major Hemorrhagic Risks by Hemorrhagic Types.(DOCX)Click here for additional data file.


Little is known about how improved depression care affects HIV-related outcomes in Africa. In a sample of depressed HIV patients in a low income, sub-Saharan country, we explored how implementing measurement-based antidepressant care (MBC) affected HIV outcomes over 4 months of antidepressant treatment.As part of a project adapting MBC for use in Cameroon, we enrolled 41 depressed HIV patients on antiretroviral therapy in a pilot study in which a depression care manager (DCM) provided an outpatient HIV clinician with evidence-based decision support for antidepressant treatment. Acute depression management was provided for the first 12 weeks, with DCM contact every 2 weeks and HIV clinician appointments every 4 weeks. We measured HIV clinical and psychiatric outcomes at 4 months.Participants were moderately depressed at baseline . All HIV clinical outcomes improved by four month follow-up: mean (range) CD4 count improved from 436  to 452 , mean (range) log-viral load decreased from 4.02  to 3.15 , the proportion with virologic suppression improved from 0% to 18%, mean (range) HIV symptoms decreased from 6.4  to 3.1 , the proportion reporting good or excellent health improved from 18% to 70%, and the proportion reporting any missed ARV doses in the past month decreased from 73% to 55%. Concurrently, psychiatric measures improved. The mean (range) PHQ score decreased from 14.4  to 1.6  and 90% achieved depression remission, while mean maladaptive coping style scores decreased and mean adaptive coping scores and self-efficacy scores improved.In this pilot study of an evidence-based depression treatment intervention for HIV-infected patients in Cameroon, a number of HIV behavioral and non-behavioral health outcomes improved over 4 months of effective depression treatment. These data are consistent with the hypothesis that better depression care can lead to improved HIV outcomes. Major Depressive Disorder (MDD) co-occurs frequently with HIV in sub-Saharan Africa, with most studies that use structured interviews reporting a prevalence ranging from 11–38%,,3 DepresHowever, little health care infrastructure and limited mental health expertise is available to support improved depression care in most of sub-Saharan Africa. The WHO estimates that in low-income countries, three-quarters of those needing mental health treatment lack access to treatment, a gap esAs a resource-efficient, scalable model for medication-based depression care for HIV-infected patients in a resource-limited setting, we adapted and pilot-tested a depression management strategy called measurement-based care (MBC) for application with depressed HIV-infected patients in Cameroon, a low income, sub-Saharan country with minimal mental health services. The model proved feasible, safe, and acceptable; was delivered with high fidelity; and led to substantial reductions in depressive severity over 3 months in nearly all patients. Here we Measurement-Based Care (MBC) is an evidence-based, resource-efficient depression management strategy designed to help psychiatric and non-psychiatric medical practitioners to deliver best-practices, guideline-concordant medication management for depression. ConsisteIn the MBC model, depressive symptoms and side effect burden are measured at regular intervals with validated instruments and compared to an evidence-based algorithm to produce specific treatment recommendations. The goal is to achieve remission—minimum depressive symptoms with return to pre-depression levels of functioning. MBC offers evidence-based guidance on depression management in situations where access to specialty mental health care is limited. With MBC, guided by a psychiatrist’s clinical supervision, social workers, nurses, or other trained members of the healthcare team can measure symptoms and treatment response, assess tolerability, and provide decision support to non-psychiatric prescribers  based on the treatment algorithm.We have previously shown in The Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial, the largest trial of depression treatment, that MBC in primary care settings can produce depression care delivery and outcomes equivalent to that observed in specialty psychiatric clinics. We have As part of an NIMH-funded grant , we adapted MBC for use in Cameroon, modifyinFollowing this adaption, we conducted a pilot study to assess feasibility, acceptability, safety, and preliminary efficacy of the adapted MBC intervention using a single DCM. From April 15-November 30, 2011, HIV-infected patients presenting for care at the Bamenda Day Hospital AIDS Treatment Center were invited to participate in the study if they met the following eligibility criteria: (1) a score ≥ 10 on the PHQ-9,20; (2) For all participants enrolling in the pilot study, the DCM provided a recommendation about antidepressant prescription to the treating HIV physician at the time of enrollment. Consistent with the standard MBC timeline,,21 the DParticipants completed in-person interviews at enrollment and at 4 months (1 month after the end of acute phase depression treatment) to measure changes in HIV-related behavioral and other health outcomes. Behavioral outcomes included self-reported antiretroviral medication adherence over the past 30 days using a Visual Analog Scale and a siWe recognized that given the small sample size, we would have limited power to assess mediators and moderators of effect. However, we hypothesized that MBC depression treatment would improve HIV behavioral and health outcomes by reducing depressive severity, which would in turn improve coping styles and self-efficacy (mediators). We measured these mediators with the Patient Health Questionnaire-9 (depressive severity),,20 the BConcerning moderators, we hypothesized that we would see larger improvements in HIV-related outcomes among those more severely depressed at baseline compared to those with moderate depression; among early vs. late responders to depression treatment ; and among those with low vs. high baseline ARV adherence.The present analysis of changes in HIV outcomes was restricted to participants already on ART at the time of study enrollment. Sample characteristics and outcome measures are described using proportions for categorical variables and means and range for continuous variables. Differences in means (for continuous variables) and proportions  were obtained, along with 95% confidence intervals (CIs), comparing the four-month measurements to baseline measurements. Our primary HIV outcomes of interest were change in CD4 lymphocyte count and change in HIV RNA viral load (VL), while our primary psychiatric outcomes of interest were change in PHQ-9 score and proportion with remission of depression.To assess possible mediation and moderation, we compared the magnitude of change in outcomes between specific subgroups.Given the small sample size in this pilot study, we focus primarily on magnitude of changes in outcomes rather than tests of statistical significance, and all participants were included in the analysis in an intent-to-treat framework. All analyses were conducted using Stata version 13 .All study activities were approved by the Cameroon National Ethics Committee (No. 111/CNE/SE/09), the University of North Carolina at Chapel Hill’s Biomedical Institutional Review Board (IRB) (# 09–0852), and the Duke University Health System IRB (# Pro00016937). The study also received administrative approval from the Ministry of Public Health in Cameroon.We enrolled 55 depressed HIV participants, of whom 41 were already on ART at the time of enrollment . All furMost of those on ART were female (76%) and had attained a primary education only (63%) . The majThe mean (range) baseline PHQ-9 was 14.4 , consistent with moderate depressive severity (PHQ-9 10–14). Depression was likely to be recurrent; 34% had two prior episodes, and 54% had three or more prior episodes. Only 2% (1 participant) had ever been treated for depression .3, and mean (range) log10 VL of 4.02  copies/mL, (Participants reported a mean (range) of 6.4  HIV-related symptoms in the past week, and 18% described their overall physical health as good, very good, or excellent, at baseline. Clinical HIV measures showed a mean (range) baseline CD4 count of 436  cells/mmpies/mL, . Of noteSelf-reported ARV adherence at baseline was relatively high, with over half reporting ARV adherence of 95% or greater in the past month . HoweverParticipants had a median of one sexual partner in the past month, and 7% reported having any unprotected sex in the past month. Alcohol use overall was low .During the course of the project, 1 of the 41 participants, with a history of HIV-associated nephropathy, died of acute renal failure and pyelonephritis unrelated to study participation shortly after completing her week 4 visit. For the remaining 40 participants, there were no losses to follow-up at 4 months.All HIV clinical outcomes, including our two primary outcomes, had improved by 4-month follow-up, although by varying degrees . CD4 couMeasurement of behavioral outcomes suggested some improvement in adherence . The proAll psychiatric measures were consistent with improved outcomes . Our twoIn exploratory analyses, we assessed whether particular variables might be moderators or mediators of outcomes. These analyses were necessarily preliminary given the small sample size.When considering moderation or mediation of changes in ARV adherence, there was greater improvement in the proportion reporting >95% adherence among those with severe depression at baseline (50 percentage point improvement) than among those with moderate to moderately severe (3 percentage point improvement), and greater improvement among those with low baseline adherence (36 percentage point improvement) than among those with high baseline adherence (no change). There was also evidence of moderation in the anticipated direction by time to depression treatment response.When considering moderation or mediation of achievement of virologic suppression, there was little evidence of difference by baseline depressive severity or coping style. There was some suggestion that faster remission of depression was associated with greater likelihood of achieving suppression.In this pilot study of an evidence-based depression treatment intervention for HIV-infected patients in Cameroon, nearly all participants experienced a strong improvement in their depressive illness. Meanwhile, over the course of 4 months of depression treatment, a number of HIV behavioral and health outcomes also improved. Among study participants, mean viral load decreased by one log and the proportion with virologic suppression improved from 0% to 19% over 4 months. Self-reported HIV symptoms, overall health, and ARV adherence all improved by varying amounts. Hence, HIV improvement paralleled depression improvement, and was consistent with a model in which effective depression care yields improved HIV outcomes.This is the first study to our knowledge to provide evidence, even if only suggestive, that depression treatment may lead to improved HIV clinical outcomes among depressed HIV-infected individuals in sub-Saharan Africa. An increasing number of studies identify the high prevalence of depression among those living with HIV in Africa as well as the consistent associations between depression and low antiretroviral adherence. These findings mirror the much deeper literature on the prevalence and negative consequences of depression among people living with HIV in high-income countries. Some, althoughAlthough the preliminary nature of this study precludes any firm conclusions about the relationship between depression treatment and HIV outcomes, exploratory analyses provide some clues that treating the most severely depressed may offer marked benefits. For example, the greatest gains in ARV adherence were achieved by the most severely depressed group, who started out with the worst adherence but by 4 months had the best adherence (relative to those with moderately severe or moderate depression at baseline). Time to depression remission might also mediate virologic suppression, with a dose-response relationship observable between the time to depression remission and the likelihood of achieving virologic suppression by 4 months.There are limitations to our findings. First, this pilot study did not include a comparison group, so we cannot account for depression that would have recovered without active depression treatment. However, nearly all participants reported chronic or recurrent depression that would be unlikely to remit so uniformly on its own in the absence of treatment, and a treatment effect is further supported by simultaneous improvement in several other psychiatric measures including suicidal ideation, self-efficacy, and coping styles. Second, the sample size is small, precluding any firm conclusions about the roles of moderators and mediators of the relationship between depression improvement, ARV adherence, and HIV outcome. Third, while associated, the improvement in both depression and HIV outcomes may not be causally related. These findings are clearly preliminary. For example, HIV clinical markers may have improved due to antiretroviral treatment alone; however, nearly all participants were on stable antiretroviral therapy at the time of enrollment rather than newly initiating therapy.In summary, these data support a conceptual model in which successful depression treatment in 4–12 weeks might lead to a decreased maladaptive coping style and improved self-efficacy, resulting in improvement in ARV adherence and increased likelihood of virologic suppression. The intervention may be especially beneficial in those with more severe depression and those who achieve early remission of their depression. This pilot study provides some of the first evidence, even if only suggestive, in sub-Saharan Africa that effective depression treatment may play an important role in optimizing HIV treatment benefits among HIV-infected patients with depression. In this sample of depressed HIV-infected patients in Cameroon receiving evidence-based depression care management, both depressive and HIV outcomes improved over 4 months of evidence-based depression treatment. These data are consistent with the hypothesis that better depression care can lead to improved HIV outcomes. Subsequent large scale prospective trials can test whether these relationships hold true.


Human induced pluripotent stem cells (hiPSCs) are considered as one of the most promising seed cell sources in regenerative medicine. Now hiPSC-based clinical trials are underway. To ensure clinical safety, cells used in clinical trials or therapies should be generated under GMP conditions, and with Xeno-free culture media to avoid possible side effects like immune rejection that induced by the Xeno reagents. However, up to now there are no reports for hiPSC lines developed completely under GMP conditions using Xeno-free reagents.Clinical-grade human foreskin fibroblast (HFF) cells used as feeder cells and parental cells of the clinical-grade hiPSCs were isolated from human foreskin tissues and cultured in Xeno-free media. Clinical-grade hiPSCs were derived by integration-free Sendai virus-based reprogramming kit in Xeno-free pluriton™ reprogramming medium or X medium. Neural cells and cardiomyocytes differentiation were conducted following a series of spatial and temporal specific signals induction according to the corresponding lineage development signals. Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the “Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III”.We have successfully derived several integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture media in line with the current guidance of international and national evaluation criteria. As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC). The hiPSC lines are pluripotent and have passed the safety evaluation. Moreover, one of the randomly selected hiPSC lines was capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media.The clinical-grade hiPSC lines therefore could be valuable sources for future hiPSC-based clinical trials or therapies and for drug screening.The online version of this article (doi:10.1186/s13287-015-0206-y) contains supplementary material, which is available to authorized users. Human pluripotent stem cells (hPSCs) can differentiate into any type of cells in the body, such as functional neural progenitor cells or cardiomyocytes, and therefore have enormous value in regenerative medicine. The increasing incidence of degenerative diseases, limitations of traditional therapeutic methods, and the shortage of isolated human functional cells have urged scientists to turn to stem cell-based cell replacement therapies. Although the translation from basic discoveries to clinical settings comes with great challenges, intensive stem cell-based clinical trials are emerging from around the world. For human embryonic stem cells (hESCs), a clinical trial of spinal-cord injury treatment using immature glial cells derived from hESCs by the Geron Corporation  has recommenced after it was brought to a halt in 2011 . When taOct4, Sox2, Klf4, and c-Myc [c-Myc may initiate tumorigenesis [Initially, the generation of hiPSCs involved integrated retrovirus expressing nd c-Myc , 6. Howeigenesis . To circigenesis , episomaigenesis , mRNAs [igenesis , minicirigenesis , microRNigenesis , and proigenesis . AlthougMost of the hESC lines collected by the National Institutes of Health (NIH) have been reported ineligible for future therapeutic products use because their derivation processes did not follow the “Tissue Donor Guidance” . PrecautWe define hiPSCs intended to be used for potential clinical applications as clinical-grade hiPSCs. Theoretically, clinical-grade hiPSCs should meet the following requirements. First, parental cell donors should meet the requirements of the “Tissue Donor Guidance”. Second, the cell handling processes should be conducted under GMP-controlled environments and with Xeno-free reagents. Third, the derived clinical-grade hiPSCs should be integration-free and biologically safe.Previously, a group reported the derivation of hiPSCs under fully defined conditions. However, the biological safety of the obtained hiPSC lines is questionable. Furthermore, the group did not describe whether their protocol was within the “Tissue Donor Guidance” . SeveralHere, we report successful generation of several clinical-grade hiPSC lines. A series of pathogenic microorganism tests were conducted on foreskin tissue donors according to the “Tissue Donor Guidance” of China. The human foreskin fibroblast (HFF) cells used for reprogramming and as feeder cells were isolated and cultured using Xeno-free reagents in a GMP-grade laboratory and were confirmed negative for mycoplasma and certain pathogenic microorganisms. Furthermore, the biological safety of the HFF cells is validated by the National Institutes for Food and Drug Control (NIFDC). All processes of the hiPSC generation were conducted under GMP-controlled conditions using Xeno-free media and reagents. The resulting hiPSCs are validated as integration-free, and are able to randomly differentiate into three germ layers in vitro and in vivo as well as directly differentiate into functional neural cells and cardiomyocytes in Xeno-free media. The biological safety of these hiPSCs is further validated. These hiPSC lines, generated following all of the requirements of clinical-grade hiPSCs strictly, might serve as valuable cell sources for future clinical applications.The foreskin tissues were donated by males undergoing foreskin operation in Beijing Children’s Hospital with the approval of the Animal and Medical Ethics Committee of Institute of Zoology, Chinese Academy of Sciences. Written consent was obtained from the donors’ parents. The content of the consent includes the potential use of cells derived from the foreskin  and the rights and obligations of the donors. After donation, a thorough assessment of the donors’ medical history and infectious diseases were performed. Based on the results, donors were selected. All information is secured to protect the privacy and confidentiality of donors. No financial benefits were involved in the donation process. All cell isolation and culture procedures followed closely the guidelines legislated and posted by the Ministry of Health of the People’s Republic of China.All animal experiments were approved in advance by the Animal and Medical Ethics Committee of Institute of Zoology, Chinese Academy of Sciences.3 and transferred to T75 flasks precoated with MesenCult-XF attachment substrates  without cell culture medium. After 24 hours of inverted culturing in a humidified incubator , 4 ml FibroGRO™ Xeno-Free Human Fibroblast Expansion Medium  was added to each flask. After 7 days of culture, fibroblasts could outgrow the tissues and be routinely passaged. The obtained fibroblasts were used as parental cells for hiPSC induction and as feeder cells after being irradiated with gamma rays.Foreskin tissues were collected into 50 ml centrifuge tubes containing Dulbecco's Phosphate Buffered Saline (DPBS)-CTS  and transported to the laboratory on ice. After sufficient washing with DPBS-CTS, fat and connective tissues were removed from the foreskin. The foreskin was then cut into small pieces with a volume of about 1 mmHFF cells were cultured in FibroGRO™ Xeno-Free Human Fibroblast Expansion Medium (Millipore) on dishes precoated with Mesencult-XF attachment substrates according to the manufacturer’s instructions. Clinical-grade hiPSCs were initially cultured on gamma ray-inactivated human feeder cells in pluriton™ reprogramming medium  or X medium (configured in our laboratory). The formulations of X medium were as follows: clinical-grade hESC basic medium and clinical-grade N2B27 medium mixed at 1:1 ratio supplemented with 10 ng/ml human leukemia inhibitory factor  and human basic fibroblast growth factor . The clinical-grade hESC basic medium contained knockout Dulbecco’s modified Eagle’s medium (DMEM)-CTS (Life Technologies), 20 % knockout serum replacement (KOSR)-CTS (Life Technologies), 100 μM none essential amino acid , 2 mM GlutaMAX-CTS (Life Technologies), 55 μM β-mercaptoethanol (Life Technologies), and 1000 U/ml penicillin/streptomycin (Life Technologies). The clinical-grade N2B27 medium was a mixture of knockout DMEM-CTS supplemented with N2-CTS (Life Technologies) and Neurobasal Medium-CTS (Life Technologies) supplemented with B27-CTS (Life Technologies) at 1:1 ratio plus 100 μM NEAA, 2 mM GlutaMAX-CTS, 55 μM β-mercaptoethanol, and 1000 U/ml penicillin/streptomycin. At passages 10–12, the cells were transferred to E8 medium (Life Technologies) without feeder cells.HFF cells were cultured in six-well sterile culture plates precoated with MesenCult-XF Attachment Substrate in FibroGRO™ Xeno-Free Human Fibroblast Expansion Medium without antibodies. The CytoTune®-iPS Sendai Reprogramming Kit (Life Technologies) was used for deriving integration-free iPSCs according to the manufacturer’s instructions. The medium used in the reprogramming process was pluriton™ reprogramming medium or X medium. Colonies with typical hPSC morphology were picked 20 days post transduction for further expansion.Alkaline phosphatase staining was performed using the Alkaline Phosphatase Assay Kit  according to the manufacturer’s instructions.Immunofluorescence staining was conducted as described previously . The priWhen hiPSCs reached 60–70 % confluency, karytoyping and G-binding analysis were carried out by Chinese Academy of Medical Science & Peking Union Medical College.RNA isolation and reverse transcription were performed as described previously . Real-tiThe embryoid body (EB) formation assay was conducted as described previously  with the6 cells in 20–30 μl DPBS were injected into each testis of severe combined immunodeficiency (SCID) mouse under a sterile stereo microscope. The mice were sacrificed 8 weeks after injection for teratoma examination following the guidelines of the Institutional Animal Care and Use Committee (IACUC). The teratomas were fixed, sliced, and stained with hematoxylin and eosin for further analysis.Confluent cells in a six-well plate were harvested using EDTA and suspended in DPBS-CTS. Then 105 cells/cm2 on day 0. Initially, the cells were cultured in a medium of knockout DMEM-CTS supplemented with 15 % KOSR-CTS, 100 μM NEAA, 2 mM GlutaMAX-CTS, 55 μM β-mercaptoethanol, and 1000 U/ml penicillin/streptomycin. On day 5, the medium containing KOSR-CTS was gradually transitioned to N2 medium-CTS as described previously [Neural cell differentiation was performed as described previously  apart freviously . On day 5 cells/cm2. Then the cells were grown in E8 medium for 2–3 days to reach 100 % confluency. To improve the efficiency of single cell survival, 10 μM Y27632 were added on the first day. Culture medium used in the differentiation process was RPMI 1640 (Life Technologies) with a combination of chemically defined components (modified in our laboratory and data not published) which we named Car-CDM. From day 0 (the day for differentiation medium adding) to day 8, Car-CDM without insulin was used as differentiation medium. In this period, the medium was changed on days 0, 1, 3, and 5. From day 0 to 1, 4 μM were added, whereas 2 μM Wnt-C59 (Sigma) was added from day 3 to 8 (Sigma). From day 8 onward, the differentiation medium was changed to Car-CDM plus insulin and the medium was changed every other day up to day 14. Beating cardiomycytes generally appeared on day 12 of differentiation.Confluent hiPSCs were digested into single cells by accutase (Life Technologies) and seeded on vitronectin-coated dishes at a density of 1.25 × 10Endotoxin testing was performed using the ToxinSensor™ Gel Clot Endotoxin Assay Kit  according to the manufacturer’s instructions.Mycoplasma testing was performed using the PlasmoTest™ Reagent Kit  according to the manufacturer’s instructions.Chinese Pharmacopoeia (third edition).The defined pathogenic microorganisms tested were human papilloma virus (HPV), human parvovirus B19, human immunodeficiency virus (HIV), John Cunningham virus (JCV), Epstein–Barr virus (EBV), human hepatitis C virus (HCV), human hepatitis A virus (HAV), human cytomegalovirus (HCMV), human T-lymphotropic virus I (HTLV-I), human hepatitis B virus (HBV), bovine virus, and porcine virus. These viruses were detected using corresponding kits produced by Zhongshandaan Company, Guangzhou, Guangdong, China. Some of the viruses were reviewed by Beijing Ai Puyi Medical Testing Center, Beijing, China. The hiPSCs were also inoculated into the chorioallantoic membrane and yolk sac of chicks according to the methods described in the Safety, traceability, and reproducibility are three basic criteria for cells to be used in clinical trials or therapies. To meet these criteria, GMP environments are required for the whole process of cell manipulation. We have established a GMP-controlled cell culture laboratory and it has passed the Beijing Institute for Drug Control  test. The room was cleaned and tested regularly to ensure its cleanliness Fig. .Fig 1OveThe legal aspect is another fundamental requirement for cell-based therapy products. In China, the Guidelines for Human Somatic Cell Therapies and Quality Control of Cell-based Products stipulate that cell donation for clinical applications must comply with national requirements the same as for blood donation. In America, the Food and Drug Administration (FDA) “Tissue Donor Guidance (2007)” states that the potential tissue donor must test negative for a series of infectious microorganisms prior to donation. Screening for tissue donors who have signed the informed consent is therefore an important step to ensure the suitability of the cells generated from the donated tissue for clinical applications. After explaining the significance and potential applications of cells that would be generated from the donated tissue to children undergoing foreskin resection surgery and their parents, some patients and parents agreed to donate their tissues and signed the informed consent. The cases and medical reports of the patients were then strictly evaluated to assess their eligibility for donation. Finally, a 6-year-old boy’s foreskin tissue was chosen Fig. .Serum-containing medium are often used for fibroblast derivation and culture. To establish fibroblasts free of animal-source reagents in our laboratory, several types of Xeno-free or chemical defined human fibroblasts culture media were trialed (Data not shown). The result showed that FibroGRO™ Xeno-Free Human Fibroblast Expansion Medium provided the optimum support for HFF cell growth. As a potential clinical-grade hiPSC and Xeno-free feeder cell source, biosecurity of the HFF has to be validated. In this case, a series of biosecurity-related projects were carried out. The results of these projects are presented in Additional file The clinical-grade HFF cells of passage 8 were used for integration-free Sendai virus-based reprogramming. The reprogramming procedure was similar to the instructions of the CytoTune®-iPS Sendai Reprogramming Kit except that Xeno-free media were used in our protocol. Pluriton™ reprogramming medium is a type of Xeno-free medium which can efficiently support the generation of hiPSCs in mRNA-based reprogramming. The X medium, which was defined in our laboratory, could greatly improve the induction efficiency of pig and human iPSCs , 26 and Oct4 (Pou5f1), Sox2, Nanog, Rex1, and E-Cadherin at similar levels to hESCs , mesoderm (Enolase and Ostenectin), and endoderm (Nicastrin and Alpha-fetoprotein)  Fig. . The terers Fig. .Fig 3ClihPSCs including hESCs and hiPSCs cannot be used directly in cell replacement therapies as they may induce tumor formation. However, terminally differentiated functional cells derived from hiPSCs can be used for clinical applications. Therefore it is important to evaluate the differentiation ability of our clinical-grade hiPSCs into specific cell types under Xeno-free conditions. Neural cell and cardiomyocyte differentiations were chosen for this purpose.For neural cell differentiation, hiPSCs were directed to forebrain dopamine neurons following a series of spatial and temporal specific signal inductions according to the neural lineage development process . Twenty-For cardiomyocyte differentiation, the Car-CDM medium modified in our laboratory was used. The differentiation process followed a temporal WNT signal activation and inhibition as described previously . BeatingPharmacopoeia of the People’s Republic of China, 2010 edition, Volume III (Additional file To evaluate whether our clinical-grade hiPSCs are biologically safe, we performed a series of tests according to the Guidelines for Human Somatic Cell Therapies and Quality Control of Cell-based Products. The results showed that the cells were negative for mycoplasma and free of serious pathogenic microorganisms such as HIV and HPV (Additional file Clinical-grade hiPSCs and GMP environments are essential in order to ensure the safety, effectiveness, traceability, and reproducibility of hiPSC-based clinical trials or therapies , 28. To The hESCs used in the clinical trials approved by the US FDA as mentioned earlier were not derived under GMP conditions. Instead they were converted from research-grade lines which were derived and maintained in the presence of animal products . It is uOncogenicity is a major concern for the application of hiPSCs in clinical settings. Nevertheless, an increasing number of studies in mouse models suggest that iPSCs are safe for transplantation . For exaReprogramming efficiency is another important factor for future hiPSC-based therapies because parental cells from old patients which are difficult to be reprogrammed may be required. We compared the reprogramming efficiency of two Xeno-free mediums and found that the number of colonies yielded from X medium, defined in our laboratory, are three times that from pluriton™ reprogramming medium, suggesting that our X medium could be used as a high-efficiency clinical-grade reprogramming medium.Envisaging the potential translation from hiPSCs to cell replacement therapies, Xeno-free and GMP conditions are essential for the differentiation of hiPSCs into functional cells. We demonstrated the feasibility of differentiating one clinical-grade hiPSC line to functional neurons and cardiomyocytes in Xeno-free mediums according to the monolayer differentiation methods. As for monolayer differentiation, each hPSC line has an optimal differentiation method, and hence further work will be focused on optimizing the monolayer differentiation method to obtain functional neurons, cardiomyocytes, and other cell types from the clinical-grade hiPSC lines.The incidence of degenerative diseases such as Parkinson’s disease and Alzheimer’s disease which cannot be treated effectively by traditional medical methods is increasing worldwide. Theoretically, replacement of degenerated cells with normal functional cells in patients paves the way for new treatment strategies. hiPSCs can provide an unlimited number of cells for autologous or closely HLA-matched transplantation upon differentiation which significantly reduces the risk of host immune response in patients. Only by standardizing and normalizing all processes during stem cell-based therapies can we ensure therapy safety and obtain objective and reliable results. These processes involve acquisition, cultivation, characterization, and purification of stem cells, testing tumorigenicity and immunogenicity of stem cells, in vivo reinfusion, efficacy evaluation of stem cells, and so on. In the near future, stem cell-based therapies will create a landmark achievement in the field of regenerative medicine and provide the theoretical basis and technical supports for conquering particular “human ills” by following strict regulations.hiPSCs hold great promise in regenerative medicine. To ensure clinical safety and obtain reliable therapeutic results, standardized and normalized processes must be followed during hiPSC-based therapies. Herein, integration-free clinical-grade hiPSC lines were derived under GMP-controlled conditions and with Xeno-free reagent culture in line with the current guidance of international and national evaluation criteria, and proved to be biologically safe and capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media. These results provide that the clinical-grade hiPSC lines could be valuable sources for future hiPSC-based clinical trials or therapies, especially in neural and heart-related diseases.


Pro-anorexia communities exist online and encourage harmful weight loss and weight control practices, often through emotional content that enforces social ties within these communities. User-generated responses to videos that directly oppose pro-anorexia communities have not yet been researched in depth.The aim was to study emotional reactions to pro-anorexia and anti-pro-anorexia online content on YouTube using sentiment analysis.Using the 50 most popular YouTube pro-anorexia and anti-pro-anorexia user channels as a starting point, we gathered data on users, their videos, and their commentators. A total of 395 anorexia videos and 12,161 comments were analyzed using positive and negative sentiments and ratings submitted by the viewers of the videos. The emotional information was automatically extracted with an automatic sentiment detection tool whose reliability was tested with human coders. Ordinary least squares regression models were used to estimate the strength of sentiments. The models controlled for the number of video views and comments, number of months the video had been on YouTube, duration of the video, uploader’s activity as a video commentator, and uploader’s physical location by country.The 395 videos had more than 6 million views and comments by almost 8000 users. Anti-pro-anorexia video comments expressed more positive sentiments on a scale of 1 to 5  than did those of pro-anorexia videos . Anti-pro-anorexia videos also received more likes  than pro-anorexia videos . Negative sentiments and video dislikes were equally distributed in responses to both pro-anorexia and anti-pro-anorexia videos.Despite pro-anorexia content being widespread on YouTube, videos promoting help for anorexia and opposing the pro-anorexia community were more popular, gaining more positive feedback and comments than pro-anorexia videos. Thus, the anti-pro-anorexia content provided a user-generated counterforce against pro-anorexia content on YouTube. Professionals working with young people should be aware of the social media dynamics and versatility of user-generated eating disorder content online. Over the last decade, the emergence of pro-anorexia  online communities has become a growing public health concern. With the help of information technologies, such communities are easily accessible and interactive, while encouraging harmful weight loss and weight control practices -6. AccorThe rationale for the use of sentiment analysis here lies in the rapid expansion of social media content. YouTube has more than 1 billion users with 300 hours of video being uploaded every minute . One of Those users commenting on videos concerning a shared interest can be considered part of a self-selected online community whose starting point is the online content itself. Online communities are defined as groups of individuals that interact through an online medium, regardless of the existence of explicit friendship links ,16. ThisPro-anorexia communities are found on various social media sites, including Facebook , YouTubeIn this study, we examined emotional reactions that pro-anorexia and anti-pro-anorexia videos received on YouTube among registered users. YouTube was selected for this study because it is the most popular social media site characterized by publicly available videos and comments. Both easy access and popularity make YouTube a significant source for information concerning anorexia. The study is built around 3 research questions: (1) what are the general characteristics of the pro-anorexia and anti-pro-anorexia videos and video uploaders, (2) what is the strength of both positive and negative emotional feedback received by these videos, and (3) how do the videos’ background information  associate with the comments posted?The user profile selection was conducted October 15-29, 2014. During that time period, we retrieved YouTube videos using 2 search terms, namely “pro-ana” and “anti pro-ana.” Based on Google Trends queries, these were the most popular words used to describe the positive and negative stance toward anorexia. Using additional synonyms would not have changed the search results because of the YouTube search engine’s use of query expansion, which involves applying synonyms of search terms to increase recall. We received similar results using “pro-ana,” “proana,” and “pro-anorexia” and their “anti” counterparts with more than 90% coverage. These search terms were able to cover part of “thinspiration,” “thinspo,” “anti-thinspiration,” and “anti-thinspo” hits as well.We selected the most popular profiles that included uploaded video material from the past 24 months concerning either the pro-anorexia or anti-pro-anorexia stance on anorexia, limiting both profile lists to 25 on the basis of their popularity. The popularity of profiles selected was determined by both video views and channel subscriptions. Notably, the YouTube search engine is also likely to list these channels first due to these criteria. All of the selected profiles were in English as were the included video comments posted during the previous 24 months. In addition, we checked that the profiles were either pro-anorexia or anti-pro-anorexia. The most popular material was created by individuals who often included video blogs concerning their opinion on anorexia. Our data did not include profiles that might be considered official profiles of governmental or nongovernmental organizations.The second phase of data collection involved the retrieval of information about videos and comments through an automatic crawler using the YouTube Data Application Programming Interface . On NoveUser information included the date when the user joined YouTube, their physical location (country), and the number of subscribers to their channel. The gender of profile users was checked separately by viewing profile pages and videos . Video information included upload time, duration, number of video views, numbers of video likes and dislikes, and the total number of video comments and commenter usernames.Video comments were assessed with the SentiStrength automatic sentiment analysis tool, which uses an algorithm to estimate the sentiment content of texts based on lexical information consisting of a list of sentiment words (approximately 3000) and grammatical categories  -28. SentWe ran interrater reliability checks throughout the data collection period. Interrater reliability of the YouTube profile users and video contents were assessed by 2 independent raters. Average interrater agreement was 90.68% (Cohen’s κ=.80).The reliability of the SentiStrength tool was tested in the study. We ran the test with a random sample of 1000 comments with 3 blind reviewers who were given instruction to code the comments on both the positive and negative axis. The raters had, on average, 47.03% (470/1000) full agreement with SentiStrength on the positive scale and 63.70% (637/1000) on the negative scale. In all, 87.20% (872/1000) of their answers showed high similarity  on the positive scale and 88.30% (883/1000) on the negative scale. Mean correlation between raters and SentiStrength was ρ=.63 on the positive scale and ρ=.69 on the negative scale.U test, the difference between SentiStrength and raters 1 and 2 was not statistically significant on the positive scale . However, this difference was significant between SentiStrength and rater 3 . On the negative scale, there was no statistically significant difference between SentiStrength and rater 3 . However, raters 1 and 2 were statistically different on the negative scale . Overall, our results indicate that SentiStrength’s precision is within human-level accuracy in estimating sentiments from text.U test for the comparison of pro-anorexia and anti-pro-anorexia communities. Our main analyses were based on ordinary least squares (OLS) regression models. We analyzed both positive and negative feedback to the videos by investigating the content of comments and video likes and dislikes. Both negative and positive SentiStrength scales as well as video likes and dislikes were used as dependent variables.Our key interest was to analyze responses to both pro-anorexia videos and anti-pro-anorexia videos. First, we provided descriptive statistics on both video types. These included a Mann-Whitney 2-sample Our regression models controlled for the number of video views, number of comments, number of months the video had been available on YouTube, and video duration. In light of the skewed distribution of these variables, we used logarithmic transformation  in the analysis. We also controlled for the country of the video uploader  and activity of the uploader as a commentator . The models predicting positive and negative sentiments were adjusted to account for the clustering of observations on the level of videos. This procedure had an impact on standard errors. Therefore, we report the adjusted predictions on the regression models (with the other variables in the model set at their means) for both positive and negative sentiments as well as for video likes and dislikes.Of 50 uploaders, 46 were female (92%) and the rest represented the category of unknown or “other” gender . The uploaders came from 13 different countries, almost half from the United States ; 74% (37/50) were from English-speaking countries. Their user profiles were, on average, 4 years old  and they had a mean of 2208 subscribers . Uploaders were not active in writing comments to other videos. In addition, pro-anorexia profile users and anti-pro-anorexia profile users did not interact in our dataset. There were only 2 comments written by a pro-anorexia uploader to anti-pro-anorexia videos, whereas there were only 4 comments vice versa.Z=–3.21, P=.001) and they also received more positive comments based on SentiStrength analysis .Our data included 133 pro-anorexia videos and 262 anti-pro-anorexia videos. As Our OLS regression models were used to estimate the difference between responses to pro-anorexia and anti-pro-anorexia content after controlling for selected independent variables. The adjusted predictions are presented in B=–0.12, P<.001 and B=–0.13, P<.001, respectively). Longer videos gained more positive comments. Furthermore, the videos were less likely to receive positive sentiments if their uploader had commented on them. Country did not have any impact on comments. The number of anti-pro-anorexia video comments was associated with positive sentiments. On the negative axis, the number of comments was likely to increase the negativity of the comments for both pro-anorexia and anti-pro-anorexia videos .Our OLS regression models  showed tOur aim was to provide new insights into user reactions regarding pro-anorexia and anti-pro-anorexia content on YouTube using a sentiment analysis approach. We found that anti-pro-anorexia video comments expressed more positive sentiments than those of pro-anorexia videos. They also had more views and a higher number of video likes than pro-anorexia videos. Our analysis based on negative sentiments and video dislikes showed that there were no statistically significant differences between pro-anorexia and anti-pro-anorexia videos. Consistent with earlier findings showing that negativity is the fuel of online conversation ,30, we aOur results showed that there was little interaction between pro-anorexia and anti-pro-anorexia video commenters, with only 1% of users having commented on both pro-anorexia and anti-pro-anorexia videos. Similarly, a comparison of pro-anorexia and pro-recovery groups on Flickr showed that most of the interaction took place within each group . HoweverOur results also deviate from a recent Facebook study in which a pro-anorexia group was found to be more active and better organized than an anti-pro-anorexia group . The difOur study has its limitations. First, we examined only communities and videos within YouTube; therefore, the results cannot be generalized to other online communities that may function differently depending on varying social media user interfaces. Second, our work is limited to English-language content because it was used as a selection criterion. Thus, results might not apply to videos and comments written in other languages. Third, we limited our analysis to 50 uploaders who were selected based on their popularity. However, despite limiting ourselves to 50 users, the analysis involved a total of 1163 videos, representing a large dataset through limited sources. Fourth, our study focused on 2 contrasting search words that were most commonly used to express either a “pro” or “anti” statement toward anorexia. Despite this limited search criteria, the YouTube search engine optimization guaranteed coverage of synonyms. Thus, our search results are not compromised by using only 2 search words.Social media has been recognized as a valuable medium for health behavior identification and communication with adolescents and young adults . One speEarlier studies have shown that pro-anorexia communities are active online and encourage unhealthy behavior -6. Our s


Preclinical evidence suggests that aspirin may inhibit lung cancer progression. In a large cohort of lung cancer patients, we investigated whether low-dose aspirin use was associated with a reduction in the risk of lung cancer-specific mortality.We identified lung cancer patients from English cancer registries diagnosed between 1998 to 2009 from the National Cancer Data Repository. Medication usage was obtained from linkages to the UK Clinical Practice Research Datalink and lung cancer-specific deaths were identified from Office of National Statistics mortality data. Hazard ratios (HR) and 95 % confidence intervals (CI) for the association between low-dose aspirin use (before and after diagnosis) and risk of lung cancer-specific mortality were calculated using Cox regression models.A total of 14,735 lung cancer patients were identified during the study period. In analysis of 3,635 lung cancer patients, there was no suggestion of an association between low-dose aspirin use after diagnosis and cancer-specific mortality . Similarly, no association was evident for low-dose aspirin use before diagnosis and cancer-specific mortality . Associations were comparable by duration of use and for all-cause mortality.Overall, we found little evidence of a protective association between low-dose aspirin use and cancer-specific mortality in a large population-based lung cancer cohort. The ug cancer , 18 and g cancer . Only ong cancer  reportedg cancer . No diffg cancer . These 3g cancer , 21, didIn a large population-based cohort of cancer-registry confirmed lung cancer patients utilising detailed prescribing records, we aimed to investigate whether low-dose aspirin use, either before and after diagnosis, was associated with a reduced cancer-specific mortality.This study utilised record linkages between the National Cancer Data Repository (NCDR), the United Kingdom (UK) Clinical Practice Research Datalink (CPRD) and the Office of National Statistics (ONS) death registration data. The NCDR contains data on cancer patients diagnosed in England including the date and site of primary cancer diagnoses, as well as information on cancer treatments received. The CPRD is the world’s largest computerised dataset of anonymised longitudinal primary care records covering approximately 7 % of the United Kingdom population. It comprises general practice records of documented high quality , 24 contBetween 1998 and 2009, all patients newly diagnosed with primary lung cancer  were identified from the NCDR. Patients with a previous NCDR cancer diagnosis were excluded, with the exception of in situ neoplasms and non-melanoma skin cancers. Using ONS death registration data, deaths were obtained up until January 2012 and lung cancer specific deaths were identified using an underlying cause of death ICD code C34.General practitioner (GP)-recorded aspirin prescriptions, according to the British National Formulary , were clrd Edition). Data on lifestyle factors including smoking, alcohol and BMI was derived from the closest GP records within 10 years prior to diagnosis. Clinical GP-recorded diagnoses were used to determine comorbidities prior to diagnosis, and comprised those which were included in a recent adaptation of the Charlson Comorbidity index [Clinical data on tumour histology, and receipt of cancer treatments including surgery, chemotherapy and radiotherapy within 6 months after diagnosis was obtained from the NCDR. Tumour histology was based on cancer registry recorded International Classification of Diseases for Oncology codes , lung cancer patients who died in the first year after diagnosis were excluded  as it is likely that these patients had stage IV disease and it seemed unlikely that short term post-diagnostic drug use would influence such deaths. Patients were therefore followed up from one year after diagnosis until death, end of registration with the general practice, last date of data collection from general practice or end of ONS follow-up. Time dependent Cox regression models were used to produce unadjusted and adjusted hazard ratios (HRs) and 95 % confidence intervals (CIs) for the association between low-dose aspirin use and lung cancer-specific mortality. Low-dose aspirin use was treated as time-varying, with users not considered to be exposed until after a lag of six months following their initial prescription. Other medications including statins and beta-blockers were treated in a similar manner. The use of a lag is recommended  and was In the analyses of low-dose aspirin use before lung cancer diagnosis, follow-up began from diagnosis until death or censoring (as described earlier). Patients who died in the first year after diagnosis were not excluded. Cox regression models were used to calculate unadjusted and adjusted HRs and 95 % CIs based upon prescriptions in the year prior to diagnosis, among patients with at least 1 year of CPRD records prior to diagnosis. To prevent over-adjustment in the analysis of pre-diagnosis low-dose aspirin use, adjustments were only made for potential confounders recorded prior to cancer diagnosis , 32 . The analysis of aspirin use before diagnosis included 13,433 patients, after excluding 1,302 patients with less than 1 year of CPRD records prior to diagnosis. In the analysis of aspirin use after diagnosis average follow-up was 3 years (maximum 14 years) and in the analysis of aspirin use before diagnosis, average follow-up was 1 year (maximum 14 years).Table P for interaction = 0.034). Results from sensitivity analyses were comparable to that of the main analysis, Table There was no evidence of an association between low-dose aspirin use after diagnosis and lung cancer-specific mortality , as shown in Table Overall, no association between aspirin use prior to diagnosis and lung cancer-specific mortality was observed  and no dose–response relationship was apparent in analyses by increasing prescriptions or tablets, Table P = 0.05). The authors hypothesised that the observed benefit in all-cause mortality may be due to an improvement in cardiovascular-related mortality, as the reduction in risk was most evident after 3 years [In this population-based study, we did not find evidence of a protective association between low-dose aspirin use and cancer-specific or all-cause mortality in a large cohort of lung cancer patients. Only one previous study has assessed the impact of aspirin on lung cancer-specific outcomes. An American study of 643 non-small cell lung cancer patients conducted by Wang et al.  observed 3 years . In cont 3 years . Our stu 3 years . FurtherIn sensitivity analyses, we observed a non-significant 28 % reduction in risk of lung cancer-specific mortality with post-diagnostic aspirin use in patients diagnosed with small cell lung cancer. Caution however is required in the interpretation of this finding as this was not an a priori defined subgroup analysis and was based on substantially smaller numbers compared to the main analysis .Our study had a number of strengths and limitations. This is the first study to evaluate the impact of low-dose aspirin use and lung cancer-specific mortality. The cohort was identified from the NCDR, a large population-based resource which allowed for robust verification of cancer diagnoses. Similarly, deaths could be confirmed using ONS. Some misclassification of deaths could have occurred but evidence from methodological comparative studies suggest that risk estimates are unlikely to be greatly affected . The useIn this population-based study, low-dose aspirin use was not associated with an improvement in cancer survival in a large cohort of cancer-registry confirmed lung cancer patients.Informed patient consent was not required for this study.


Previous studies of problem Internet gamblers have failed to distinguish whether their problem gambling relates to Internet or land-based gambling modes. Therefore, characteristics and help-seeking behaviors of people whose gambling problems relate specifically to Internet gambling are unknown, but could inform the optimal alignment of treatment and support services with the needs and preferences of problem gamblers.This study aimed to compare (1) characteristics of problem Internet gamblers and problem land-based gamblers and (2) uptake of different types and modes of help between problem Internet gamblers and problem land-based gamblers. Hypothesis 1 was that problem Internet gamblers are less likely to seek help. Hypothesis 2 was that problem Internet gamblers are more likely to use online modes of help.A sample of 620 respondents meeting criteria for problem gambling was drawn from an online survey of 4594 Australian gamblers. Respondents were recruited through advertisements on gambling and gambling help websites, Facebook, and Google. Measures consisted of gambling participation; proportion of gambling on the Internet; most problematic mode of gambling; help seeking from 11 different sources of formal help, informal help, and self-help for gambling problems; psychological distress (Kessler 6); problem gambling severity ; and demographics.21=28.3, P<.001, φ=0.21), younger , have lower psychological distress , and experience problems with sports and race wagering . Uptake of help was significantly lower among problem Internet compared to problem land-based gamblers , including from face-to-face services, gambling helplines, online groups, self-exclusion from land-based venues, family or friends, and self-help strategies. Both problem Internet and problem land-based gamblers had similarly low use of online help. However, problem land-based gamblers  were significantly more likely to have sought land-based formal help compared to problem Internet gamblers .Problem Internet gamblers were significantly more likely than problem land-based gamblers to be male (χThe findings suggest that more targeted and innovative efforts may be needed to increase use of gambling help by problem Internet gamblers. Alternatively, their lower PGSI and K6 scores suggest Internet problem gamblers may have less need for gambling-related help. This is the first known study to classify problem Internet gamblers as those whose problem gambling specifically relates to Internet gambling. Further research is needed to better understand why help-seeking rates are lower among Internet problem gamblers. Problem gambling is characterized by difficulties in limiting time and/or money spent on gambling which leads to adverse consequences for the gambler, others, or the community . ProblemAlthough use of Internet gambling is increasing internationally and several studies have examined Internet gamblers, little is known about their preferences and likelihood of seeking help for gambling problems. This knowledge is lacking because most studies have classified problem Internet gamblers as Internet gamblers who meet criteria for problem gambling, regardless of whether their problem gambling is related to Internet or land-based gambling modes -11. TherThe present study aimed to (1) compare the characteristics of problem Internet gamblers to problem land-based gamblers, differentiated according to their most problematic mode of gambling and (2) compare the uptake of different types and modes of help between problem Internet gamblers and problem land-based gamblers.To our knowledge, this study is the first with a nonclinical sample to classify problem Internet gamblers as individuals whose problem gambling is specifically associated with Internet gambling. Understanding the comparative similarities and differences in characteristics and help-seeking behaviors of those nominating Internet (vs land-based) gambling modes can guide the optimal alignment of treatment and support services with the needs and preferences of problem Internet gamblers. This should improve rates of gambling help seeking from their currently low base rate. This is relevant given only 5%-10% of problem gamblers are in professional treatment at any 1 time and only small proportions access other supports such as self-exclusion (in which people voluntarily bar themselves from gambling venues or websites for a specified time period), peer support groups, informal assistance from family and friends, and self-help -20.Research has not yet established whether the help-seeking behavior of problem Internet gamblers differs from that of problem land-based gamblers because the most problematic mode of gambling has not been considered previously. However, several reasons are discussed subsequently that support the 2 hypotheses tested in this study:Hypothesis 1: Problem Internet gamblers are less likely to seek help for problem gambling compared to problem land-based gamblers.Hypothesis 2: Problem Internet gamblers are more likely to seek online help for problem gambling compared to problem land-based gamblers.Our first hypothesis was proposed based partly on the different sociodemographic and psychological profile of problem Internet gamblers compared to problem land-based gamblers. A large representative Australian telephone survey conducted in late 2011 (with different participants than this study) found that Internet moderate-risk/problem gamblers were more likely to be younger, male, married, and to have lower levels of psychological distress compared to moderate-risk/problem land-based gamblers . AnotherDifferences in types of problematic gambling between Internet gamblers and land-based gamblers also lend support for our first hypothesis that problem Internet gamblers are less likely to seek help for problem gambling. An Australian telephone survey found that Internet problem gamblers were more likely to experience problems with sports and race wagering, and land-based problem gamblers were more likely to experience problems with EGMs . AnotherOther features of Internet gambling may also impede help seeking for problem gambling. Use of electronic money, accounts, and credit may delay problem recognition and acknowledgment in contrast to land-based cash gambling where losses are immediately apparent ,29. LackFurther, the negative consequences of gambling appear to differ between problem Internet and problem land-based gamblers. One study found that moderate-risk/problem land-based gamblers are more likely to experience more serious gambling consequences than their Internet gambler counterparts . These cIn support of our second hypothesis that problem Internet gamblers are more likely to seek online help for problem gambling compared to problem land-based gamblers, preferred mode of help  may align with preferred mode of gambling. Studies suggest that Internet gamblers more frequently access Internet-based help and this may be a preference. Wood and Griffiths’  2007 evaApproval for this study was obtained from 2 university human research ethics committees . An online survey was used to reach a large national sample, and for its anonymity and privacy which enhance response rates, response accuracy, and survey completeness, especially when focused on sensitive topics such as gambling ,35. AlthAdvertisements were placed on 46 websites likely to be visited by Australians interested in gambling and obtaining help for gambling problems. These websites included those of 18 regulated gambling operators, 12 gambling-related sites (containing information and research), 7 government departments which regulate gambling, 6 gambling help organizations, and 3 sporting associations. Paid advertisements were also displayed on Facebook and Google. A sample of 4594 respondents was obtained between May and December 2012. Inclusion criteria were aged 18 years or older, gambling at least once in the previous 12 months, and English literacy. Most respondents  were alerted to the survey via advertisements on online wagering/lottery sites, Facebook , and Google .Gambling behavior was measured, including participation during the previous 12 months in 10 gambling forms: instant scratch tickets; lottery, lotto, or pools tickets; sports betting; betting on horse or dog races; bingo; keno; poker; casino table games not including poker; games of skill not including poker; and EGMs. For each type of gambling, respondents were also asked the percentage of their “purchases or play” during the previous 12 months that “was done over the Internet.” Global measures to assess the proportion of all gambling money and time spent online were also included. Two questions were asked about use of alcohol and use of recreational drugs while gambling, with response options from “never” to “almost always.”The Problem Gambling Severity Index (PGSI)  is widelProblem gamblers (based on PGSI scores) were asked which form of gambling had contributed most to any problems that they had experienced from gambling. Respondents could choose 1 of 10 forms listed previously, “other,” or “I have not experienced problems from my gambling.”Problem gamblers (based on PGSI scores) were asked “What type of gambling medium has contributed MOST to any problems you may have experienced from your gambling?” The following response options were provided: Internet via computers, Internet via mobile phone, Internet via portable/wireless device, Internet TV, land-based gambling, and betting via telephone. The first 4 options were recoded into “Internet gambling” to classify most problematic gambling mode.Problem gamblers were asked if they had ever sought help from 11 different sources encompassing the most common forms of formal help, informal help, and self-help . Formal Psychological distress was assessed by the 6-item Kessler Psychological Distress Scale (K6) , which aSex, age, place of residence, household type, highest educational qualification, work status, income, debt, and cultural background were self-reported.U), chi-square tests of independence (with post hoc pairwise comparisons using z tests for multiple degrees of freedom tests), or independent samples t tests. A binary logistic regression was also conducted and this is detailed in the results section. Finally, repeated measures nonparametric analyses were conducted for expenditure using the Wilcoxon signed rank test. Nonparametric tests were used for ordinal frequency data or for expenditure data where extreme values resulted in excessive variance. Alpha was set at .05 for all analyses unless stated otherwise.Problem Internet gamblers were defined as those meeting criteria for problem gambling as measured by the PGSI  and who U=37,845.5; z=4.47, P<.001).Of the 4594 respondents to the survey, 70.51% (3239/4594) had gambled using the Internet at least once during the previous 12 months (designated as Internet gamblers), whereas 29.49% (1355/4594) had gambled only on land-based modes (designated as land-based gamblers). Among the 4594 respondents, 643 scored as problem gamblers on the PGSI, representing 14.20% (460/3239) of Internet gamblers and 13.51% (183/1355) of land-based gamblers in the sample. Problem land-based gamblers exhibited significantly higher PGSI scores  compared to problem Internet gamblers  nominated Internet modes as their most problematic either via computers, mobile phones, portable/wireless devices or interactive television (designated as problem Internet gamblers). The remaining 335 respondents (54.0%) nominated land-based modes as their most problematic (designated as problem land-based gamblers). Thus, problem Internet gamblers were past-year problem gamblers who nominated an Internet gambling medium as contributing most to their gambling problems. Problem land-based gamblers were past-year problem gamblers who nominated a land-based gambling medium as contributing most to their gambling problems.The sample of problem gamblers (n=620) was predominantly male , with a mean age of 37.6 years (SD 13.1). The most common marital status was never married  followed by married  or de facto/living with partner . At least 70.0% (434/620) of the sample reported living with others and 62.6% (388/620) reported living in a major metropolitan city. Most  worked full-time and 52.7% (327/620) reported completing some form of tertiary study. Most  were born in Australia and 81.0% (502/620) spoke English at home as their primary language. Median household income was AU$60,000-AU$69,999 per annum, with median household debt reported to be AU$20,000.Problem Internet gamblers were compared to problem land-based gamblers for demographic characteristics, mental health, gambling behavior, and most problematic gambling form.21=28.3 P<.001; φ=0.21) and significantly younger  compared to problem land-based gamblers .Problem Internet gamblers were significantly more likely to be male  compared to problem land-based gamblers , location of residence (approximately 62% of both groups lived in major metropolitan cities), level of education, work status, household income or debt, country where the respondent was born, or language spoken at home.21=5.4, P=.02; φ=0.09).Problem land-based gamblers were significantly more likely to be classified as having high psychological distress according to the K6 than were problem Internet gamblers  and spent more money  on those forms than did problem land-based gamblers. In contrast, problem land-based gamblers gambled on EGMs significantly more frequently  and spent significantly more money per month on them  than did problem Internet gamblers. Repeated measures Wilcoxon signed rank tests were used to compare these 3 forms of betting for expenditure (reported here as z tests). Problem Internet gamblers spent more money on horse or dog race betting than sports betting  or on EGMs , but there was no significant difference between sports betting and EGMs . Problem land-based gamblers reported spending significantly more money on EGMs than on horse or dog racing  or sports betting , and more on horse or dog race betting than on sports betting . No significant differences were found between the 2 groups for alcohol or drug consumption while gambling.A significantly higher proportion of problem Internet gamblers participated in sports betting and horse and dog race betting, whereas a significantly higher proportion of problem land-based gamblers took part in EGMs, with no significant differences for lottery-type or other forms of gambling . Further24=228.5 P<.001; φ=0.61)  .21=6.9, P<.001; φ=0.11) and supported our first hypothesis that problem Internet gamblers are less likely to seek help for problem gambling.Among the 620 problem gamblers, 342 (55.2%) reported having sought at least 1 type of help for their gambling. Among the problem Internet gamblers, 141 of 285 (49.5%) had sought at least 1 type of help compared to 201 of 335 (60.0%) problem land-based gamblers. This difference was statistically significant  had sought formal land-based help, 103 of 620 (16.6%) had sought formal online help, and 101 of 620 (16.3%) had sought formal telephone help. There was some overlap between land-based, online, and telephone help seeking . All comt618=3.25, P=.001; d=0.26), but there was no difference in age between those who had and had not sought online help or telephone help.Those who sought land-based help were significantly older  compared to those who had not sought land-based help . However, no significant differences between the groups were observed for use of online help  or for use of telephone help . Therefore, our second hypothesis that problem Internet gamblers are more likely to seek online help for problem gambling compared to problem land-based gamblers was not supported.Problem land-based gamblers  were significantly more likely to have sought land-based formal help compared to problem Internet gamblers  or problem land-based gambler (coded as 2). The predictors were the 11 help-seeking variables , gender , and age in years (treated as a continuous predictor). The analysis was run as a binary logistic regression with all predictors entered in 1 step. Before conducting the logistic regression, a linear regression was run to check for tolerance issues. The lowest tolerance value obtained was 0.72, indicating little overlap between predictors. Alpha was set to .05 for all predictors.213=99.5, P<.001) and successfully predicted 63.9% (182/285) of problem Internet and 64.8% (217/335) of problem land-based gamblers. Results for the predictors are shown in The overall logistic regression model was significant (χTo our knowledge, this study is the first to identify the characteristics of problem Internet gamblers classified according to most problematic mode of gambling. Consistent with earlier reports, the current study found that problem Internet gamblers were significantly more likely to be male and younger compared to their land-based counterparts ,42,43. HOur results lend support to previous findings that Internet gamblers are more involved and diverse gamblers compared to non-Internet gamblers ,11,42,44This study has revealed new information about help seeking among problem Internet gamblers. In support of our first hypothesis, it found that problem Internet gamblers were significantly less likely to access help than their land-based equivalents. This was reflected in their significantly lower uptake of professional face-to-face help and through a gambling helpline, self-exclusion from a land-based venue, from family or friends, and through self-help strategies. Problem Internet gamblers also had lower usage of online support groups or discussion boards compared to problem land-based gamblers. The results provide some indication that problem Internet gamblers were more likely to have self-excluded from a gambling website, but this was the only type of help used more than problem land-based gamblers. This greater use is unsurprising given that online gambling sites created the greatest problems for this cohort. It is possible that website self-exclusion is an adequate intervention for some Internet gamblers to maintain control over their gambling. Nevertheless, less than 1 in 10 problem Internet gamblers had used this intervention.The most popular types of help among the problem Internet gamblers were face-to-face gambling counseling, followed by a gambling helpline and use of self-help and support from family and friends. Uptake of diverse forms of help may indicate that provision of a wide range of help options best caters for the varying preferences of problem Internet gamblers. However, slightly more than half had never sought any type of help for their gambling. These findings may reflect the greater promotion of help services advertised in land-based venues compared to online gambling websites. As suggested in prior studies, further publicity of formal help services, self-help tools and resources, and encouragement to use family and social support may be needed by online gambling sites to improve help-seeking rates among Internet gamblers ,34,45-48Additional explanations are possible for the lower uptake of help among problem Internet gamblers. Lower uptake may reflect greater reticence to use gambling help among males and younger people ,48, whicHowever, problem Internet gamblers in this study were found to have lower levels of both gambling problems and psychological distress, and thus fewer reasons to seek help. Therefore, they may have less severe negative consequences from their gambling and may also be less likely to gamble for escape and dissociation, which are well-known motivations for EGM gambling among problem land-based gamblers ,17. In cThis study also compared use of formal online, telephone, and face-to-face help by most problematic gambling mode. Researchers have suggested that problem Internet gamblers are more likely to use online than land-based help -34 and tFurther research is needed to explain why help-seeking rates appear to be lower among problem Internet gamblers compared to problem land-based gamblers. It may be that they have less need to seek help or that some find self-exclusion from gambling websites adequate to maintain control over gambling. Alternatively, barriers to help seeking may be different for problem Internet gamblers than those found among problem land-based gamblers, such as stigma, shame, problem denial, a belief that one can handle the problem alone, and false hope in the ability to win back losses or regain control ,61-65. RLow numbers of Internet gamblers in the population necessitated a targeted approach to recruitment. Thus, although the study sample was large, it was not necessarily representative of the general population of problem Internet gamblers or of problem land-based gamblers among whom use of the Internet may be low. This convenience sampling may explain the high rates of help seeking found in this study compared to previous estimates that approximately 10% of problem gamblers in Australia seek professional help for their gambling problem . RecruitClassifying problem Internet and problem land-based gamblers based on most problematic gambling mode appears an advantageous approach to analyzing their characteristics and behaviors because it removes the confounding issue that some Internet gamblers experience most problems with a land-based mode of gambling. By using this approach, this study was able to identify distinguishing characteristics of problem Internet gamblers as being more likely to be male, younger, with lower levels of psychological distress than their land-based counterparts, and to most likely have problems with sports and race wagering. Further, this approach identified lower uptake of help by problem Internet gamblers compared to problem land-based gamblers and a similarly low use of online help among the 2 groups.Findings suggest that targeted, more innovative, and widespread efforts may be needed to increase use of gambling help by problem Internet gamblers, including through their promotion on Internet gambling websites. Internet gambling operators could also ensure that harm minimization measures, such as deposit limits, credit limits, and self-exclusion, are available and prominently advertised. Further, promotion of gambling help could be tailored to the predominantly young male profile of problem Internet gamblers and focus on risks associated with online sports and race wagering in addition to the current focus of most public health initiatives on EGM gambling. Online self-help resources should also be further developed to cater to Internet gamblers and be promoted to both Internet and land-based gamblers. However, additional research is needed to further understand why help-seeking rates appear to be lower among problem Internet compared to problem land-based gamblers. Whether this group of problem gamblers has the same need for gambling help as their land-based counterparts is not currently known.