Variability in dosing and costs of biologics among patients with rheumatoid arthritis (RA) is of interest to healthcare descision-makers. We examined dosing and costs among RA patients newly treated with infliximab or etanercept under conditions of typical clinical practice.Integrated pharmacy and medical claims data were obtained from 61 U.S. health plans. RA patients newly treated with infliximab or etanercept between July 1999–June 2002 were selected. A maintenance number of infliximab vials was determined after the "loading period" (2–3 infusions); those with ≥ 2 occurrences of an increase in vials or an interval between infusions of <49 days were considered to have had escalated. For etanercept patients, escalation was based on ≥ 2 instances of increased average daily dose. Multiple logistic regression analyses were conducted to assess variables associated with dose escalation. RA-related costs at one year post-initiation also were examined; comparisons were made using generalized linear models.A total of 1,548 patients were identified (n = 598 and 950 for infliximab and etanercept respectively). Infliximab recipients were somewhat older (50.5 vs. 46.6 years for etanercept). Nearly 60% of infliximab patients increased their dose at one year, compared to 18% for etanercept. Infliximab patients who escalated dose incurred a 25% increase in mean one-year costs . Costs among etanercept patients did not substantially differ based on dose escalation .Infliximab is associated with higher rates of dose escalation relative to etanercept, which contributes to substantially higher one-year medical costs. Rheumatoid arthritis is a costly and debilitating autoimmune disorder that is characterized by joint pain, stiffness, and impaired functionality. Symptoms arise from the inflammation and degradation of the synovial membrane, causing progressive disability in joint function . As the Because there is no known cure for RA, the goal of therapy is to treat the disease's symptomatology while attempting to slow or halt its overall progression. Pharmacotherapy is the cornerstone of treatment where symptoms may be treated with various combinations of nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and narcotic analgesics. In addition, disease-modifying antirheumatic drugs (DMARDs) are sometimes administered in an effort to alter the disease's progression. The effectiveness of DMARDs, however, is offset by the high levels of toxicity experienced by some patients taking these medications, and is problematic for long-term therapy .®, Amgen, Inc./Wyeth, Inc., Thousand Oaks, CA and St. David's, PA) and infliximab  which provide anti-rheumatic activity by inhibiting tumor necrosis factor (TNF), another important mediator of an inflammatory response. The use of such agents in combination with the DMARD methotrexate has been shown to be clinically superior to methotrexate alone in controlled clinical trials [In addition to the use of various DMARDs either alone or in combination with other therapies, several new DMARDs have recently been introduced for the treatment of RA. These include two biologic agents, etanercept  may provide additional benefits for patients with rheumatoid arthritis ,14. HoweGiven the wide disparity in costs between therapy alternatives and the potential impact of dose escalation on these costs, it is essential to examine the total costs of RA-related care associated with each form of therapy from a perspective of typical U.S. clinical practice. In this study, the direct costs of RA-related care were estimated on an annual basis after initial treatment with anti-TNF biologics. Resource utilization and cost estimates were also stratified by dosing status (increase in dose during follow-up vs. no increase), using retrospective claims data from commercial insurers in the US.Medical and pharmaceutical service claims were obtained from the PharMetrics Patient-Centric Database, and spanned the period from January 1999 to June 2002. At the time of this study, the database contained fully adjudicated service claims from 61 health plans across the US. Inpatient and outpatient diagnoses (ICD-9-CM format) and procedures (CPT-4 and HCPCS formats), as well as standard and mail order prescription records, are included in the data set. Reimbursed payments and charged amounts are available for all services rendered, as well as dates of service for all claims. Additional data elements include demographic variables , product type , payor type , provider specialty, and start and stop dates for plan enrollment. All patients who met the sample selection criteria specified below were included in the analyses.Patients with a diagnosis of rheumatoid arthritis (ICD-9-CM 714.XX) who newly started on infliximab or etanercept between July 1999 and June 2001 were initially selected for inclusion in the study sample. A hierarchical procedure was then implemented to stratify patients into treatment cohorts according to their first utilization of a particular medication at a specific point in time. For example, a patient initially receiving infliximab and later receiving etanercept would be classified as an "infliximab" patient for the duration of the study period. An index date for each therapy was established based on the first occurrence of a claim. Patients with no claims activity for the index therapy for six months prior to the index date were deemed "newly started".Those patients not continuously enrolled during the six-month pretreatment and 12-month follow-up periods were excluded from all analyses. Additionally, patients must have had at least five infusions or prescriptions for their index medication. Patients 65 and older who were not enrolled in a Medicare "risk" plan  were excluded from each of the analyses, as such patients may not have had fully visible utilization and cost values due to coordination of medical benefits. All medical and pharmaceutical claims spanning the period January 1, 1999 to June 30, 2002 were then extracted for eligible patients in the data set.The primary measures of interest in this evaluation were the frequency and economic impact of an escalation in biologic dose. Dose escalation was assessed for patients new to infliximab and etanercept during the study period described above. Infliximab and etanercept doses reported at the third infusion/prescription respectively were considered to be the maintenance dose levels. Subsequent utilization was then examined to determine dose escalation. Dose escalation for patients initiating infliximab was based on at the presence of least two occurrences of an increase in the number of vials reported on the infusion claim. The standard period (as indicated on the prescribing information for infliximab) between infusions following the third infusion is eight weeks. Therefore, patients with two infusions within seven weeks on two or more occasions were also considered to have an increase in dose. Dose escalation for patients initiating etanercept therapy was determined according to a change in the average daily dose (expressed in terms of mg per day); average daily dose was calculated based on data from pharmacy claims, using the following formula:metric strength(25 mg)*quantity dispensed /days suppliedPatients having two or more prescriptions with a higher average daily dose than that reported on their maintenance dose were considered to have an increase in dose. Dose escalation results were reported on an overall basis and stratified by age , geographic region , calendar year of biologic therapy initiation , and quartile of pre-index RA-related costs.In addition to dose escalation, the demographic and clinical characteristics of the sample also were assessed and stratified among patients who did and did not escalate their dose. Characteristics of interest included age, gender, health plan type, geographic region, physician specialty as of the index date, presence of selected pre-index medications, procedures, and comorbid diagnoses, co-diagnosis of Crohn's disease, and pre-index total  healthcare costs.During follow-up, the numbers of prescriptions or infusions of biologic therapy were tracked, as were the costs of all appropriate medical interventions, inpatient, outpatient, and pharmacy services. Costs were tallied for both RA-related and unrelated services and medications. Costs were deemed to be RA-related based on the presence of a relevant diagnosis, medication claim, or procedure . In addition, a multiple logistic regression model was applied to identify characteristics that were most predictive of patients experiencing an escalation in dose (including the index biologic therapy). The selection of predictors for inclusion in the models began with a univariate analysis of each variable to determine the frequency of the observations associated with each treatment cohort. An initial model run was performed using the following variables: age group, gender, region, biologic therapy group, plan type, prescribing specialty, RA-related costs during the six month pre-index period, non-RA related costs during the pre-index period, maintenance dose, dummy variables  for the receipt of other RA related medications during the pre-index period, and selected comorbidities. A stepwise method was employed to produce the final model specification using an "entry" level of α = 0.15 and a "stay" level of α = 0.05.The amount of costs  for all services previously described were calculated on an annual per patient basis for each cohort. Total RA-related, unrelated, and overall costs during the one-year follow-up period were compared controlling for differences in age, gender, pre-index RA related costs and other appropriate variables between the cohorts. A generalized linear model using a gamma distribution was used to control for these differences.Clinical and demographic characteristics of the study sample N = 1,548) are presented in Table ,548 are The use of other RA-related medications prior to biologic use differed numerically by treatment group. NSAIDs were used more frequently by etanercept users relative to infliximab (42% vs. 26% respectively), as were Cox-II inhibitors (35% vs. 27% respectively) and leflunomide (26% vs. 19% respectively). The rate of pretreatment methotrexate use was similar among the etanercept and infliximab groups (55% and 56%).Utilization of joint aspiration procedures was numerically higher during the pre-index period for patients in the infliximab group relative to the etanercept sample (35% and 28% respectively). Additionally, pre-index RA related costs also were somewhat higher among infliximab patients .Patients who initiated infliximab therapy experienced significantly higher rates of dose escalation during the first year of follow-up relative to patients who were initiated on etanercept  Table . When stModeled dose escalation results for patients who initiated infliximab or etanercept therapy are presented in Table In an effort to better understand the factors associated with infliximab dose escalation, an additional model was conducted among patients initiating infliximab only. Results are presented in Table Overall, costs for patients initiating infliximab therapy were numerically higher than for patients in the etanercept group . Much of the cost difference was due to the difference in drug costs . In addition, infliximab patients had higher costs for physician management visits ($691 vs. $381), ancillary services , and hospitalizations . Use of alternative biologic therapy  was minimal, as illustrated by extremely low average annual costs for these alternative strategies.Average annual RA-related, unrelated, and total costs are also stratified by whether patients underwent dose escalation in Table In an effort to better understand the differences in dosing patterns and costs among RA patients on biologic therapy, a retrospective analysis of pharmacy and medical claims for patients new to biologic therapy was undertaken. Dosing frequency and quantity was examined, as were RA-related costs at one year after therapy initiation.Dosing guidelines suggest that etanercept patients receive two 25 mg vials a week; the use of higher doses has not been studied. The recommended dosing for infliximab is 3 mg/kg of body weight for the first dose, and then at two and six weeks and every eight weeks thereafter. Patients experiencing an inadequate response may increase dose to 10 mg/kg; or they may receive treatment as frequently as every four weeks . The fleLastly and most importantly, differences in RA-related cost among patients new to infliximab and etanercept therapy  were manifested mainly in the treatment costs . Management and ancillary services accounted for most of the remaining difference. The difference in treatment costs may be attributed to the higher rate of dose escalation among the infliximab group. These patients had treatment expenses that were ~60% higher than patients who did not dose escalate , while infliximab patients who did not dose escalate had costs similar to patients in the etanercept group. There was little difference in drug therapy costs among etanercept patients who experienced an increase in dose and those with no change . These findings highlight the differences in treatment patterns and associated costs among patients new to etanercept and infliximab.Our study was subject to some important limitations. First, as this was a retrospective analysis of claims data, results were based on amounts billed to health plans. As a result, the unit of measurement for infliximab is billed whole vials. For example, if 1.2 vials were administered to a patient, 2 vials would be billed to the health plan. Therefore, these results may not reflect the true amount of infliximab utilized and may in fact under- or overstate the rate of dose escalation – for example, a patient who increases from 1.2 to 1.7 vials will be shown to have utilized 2 vials in both instances; in contrast, a patient moving from 1.9 to 2.1 vials will appear as having moved from 2 to 3 vials.In addition, information regarding body mass and/or patient weight was not available. As stated above, infliximab dosing levels may range from 3 mg/kg to 10 mg/kg. Dosing changes resulting from weight changes alone were therefore undetectable. Also, one method of estimation of dose change for infliximab was based on two infusions within seven weeks on two or more occasions. It is possible that some patients may have had these non-standard queuing times simply as a result of scheduling availability, and not as a result of dose escalation. Clear estimates of dose increase due to increased frequency may only be obtained through a more controlled observational study.Furthermore, no information is available in this administrative database regarding the reason for dose escalation – lack of efficacy, increase in symptoms, other reasons. Our major focus for this study was therefore to simply document that standard dosing assumptions regarding infliximab may lead to erroneous conclusions regarding its cost, given the high level of escalation seen in this and other studies. In addition, the database lacks clinical detail on levels of disease severity as well as other potentially important variables  for consideration of the full clinical and economic impact of dose escalation.As with all retrospective study, we cannot rule out the possibility that differences in disease progression and/or severity between patients who do and do not escalate dose may have influenced our findings. Nevertheless, our results remained statistically significant even after controlling for observable differences between groups, indicating that any selection bias would likely only affect the magnitude, not the direction, of our findings.Finally, while the data used represent final, adjudicated claims in a health plan setting, it is possible that the data elements used are subject to coding or misclassification error. Nevertheless, if such an error rate exists, it is likely not a systematic phenomenon – that is, there is no reason to expect that coding errors would disproportionately affect the infliximab or etanercept samples in our study.Despite the limitations noted above, we believe our study has important implications. While, the results of recent observational studies suggest that both infliximab and etanercept are highly effective in clinical practice , our finAll authors were employed by PharMetrics, Inc. at the time of this analysis, which was conducted based on an unrestricted research grant from Abbott Laboratories, Inc. No other competing interests are declared by any author, including stocks or other holdings, other financial interests, or non-financial interests.TG was involved in the conception and design of the study, oversaw and provided quality assurance on all study output, and drafted the methods and results sections of the manuscript. DS was responsible for the design and conduct of all descriptive and statistical analyses. DO was responsible for the conception and design of the study, drafting of the background, discussion, and conclusions sections of the manuscript, and all formal correspondence regarding the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:File consists of ICD-9-CM codes, CPT-4 codes, GPI drug codes to describe the various diagnoses, type of drugs and procedures.Click here for file


Aorto-atrial fistulas (AAF) are rare but important pathophysiologic conditions of the aorta and have varied presentations such as acute pulmonary edema, chronic heart failure and incidental detection of the fistula. A variety of mechanisms such as aortic dissection, endocarditis with pseudoaneurysm formation, post surgical scenarios or trauma may precipitate the fistula formation. With increasing survival of patients, particularly following complex aortic reconstructive surgeries and redo valve surgeries, recognition of this complication, its clinical features and echocardiographic diagnosis is important. Since physical exam in this condition may be misleading, echocardiography serves as the cornerstone for diagnosis. The case below illustrates aorto-left atrial fistula formation following redo aortic valve surgery with slowly progressive symptoms of heart failure. A brief review of the existing literature of this entity is presented including emphasis on echocardiographic diagnosis and treatment. A 66 year old male with history of rheumatic heart disease and aortic valve replacement (AVR)  presented with progressive worsening fatigue, exertional dyspnea and paroxysmal nocturnal dyspnea. His other medical problems included poorly controlled hypertension and hyperlipidemia. Following his second AVR 4 years prior to this presentation, a routine follow-up 2-dimensional echocardiogram (TTE) had shown preserved left ventricular and prosthetic valve function and a aorto-atrial fistula with color flow between the aorta-and the left atrium. Notably, only a soft and short ejection murmur across the mechanical prosthesis was appreciated and no continuous murmurs were heard. This was felt to be a possible postoperative complication currently not of clinical significance given his asymptomatic status and he was treated medically and did well for the last 3–4 years.Although the echocardiographic findings mimicked changes which could also be related to endocarditis (abscess around prosthesis with pseudoaneurysm formation), the absence of any obvious vegetations or prosthetic malfunction combined with lack of clinical and laboratory evidence of endocarditis favoured a more slowly progressive postoperative complication rather than an infectious process. Based on his heart failure symptoms and progressive increase in AAF size and flow, surgical correction was recommended. He underwent uncomplicated surgical repair of the AAF which was found during surgery to be inferior to the left coronary ostium. No evidence of abscess or infection was found and the prosthesis appeared intact and healthy. The echo lucent area represented a postoperative weakening of the aortic wall adjacent to the left atrium, predisposing to the fistula formation and was also repaired. Intra-operative TEE showed no residual fistula by color flow at the site of repair.Physical examination during this visit revealed an afebrile patient with a blood pressure of 138/84 mm Hg, regular pulse of 84/minute. An ejection systolic murmur (2/6 in intensity) was heard all over the precordium likely from the flow across his prosthesis. No continuous murmurs were heard. No evidence for clinical heart failure, anemia, jaundice or infection was noted. Laboratory tests revealed no leukocytosis and blood cultures were negative. Given prior echo documentation of fistula and new symptomatology suggestive of heart failure, a transesophageal echocardiography (TEE) was requested for more detailed assessment of prosthesis and AAF. TEE revealed normal left ventricular function, normal aortic prosthesis function with trivial aortic regurgitation. An echolucent area above the mechanical prosthesis, close to the left atrium near the orifice of the left coronary artery was noted. There appeared to be expansion of a portion of this lucency into the left atrium during systole suggesting communication with the aorta . Apart from antibiotic treatment for endocarditis, definitive treatment revolves around surgical correction. Since many of these patients may have had some form of surgical intervention in the past , reoperaAorto atrial fistulas are rare but important complications of many disease processes of the aorta and aortic valve. Classical clinical signs of continuous murmurs may not be present and echocardiography forms the cornerstone of diagnosis. AAF should be suspected in patients with poorly controlled heart failure and prior aortic surgery. Prompt surgical repair is usually helpful in relieving symptoms and decreasing mortality.The author declares that he has no competing interests in preparation of this mansucript and has fully contributed to preparation of this manuscript.


Although back pain is the most common reason patients use complementary and alternative medical (CAM) therapies, little is known about the willingness of primary care back pain patients to try these therapies. As part of an effort to refine recruitment strategies for clinical trials, we sought to determine if back pain patients are willing to try acupuncture, chiropractic, massage, meditation, and t'ai chi and to learn about their knowledge of, experience with, and perceptions about each of these therapies.We identified English-speaking patients with diagnoses consistent with chronic low back pain using automated visit data from one health care organization in Boston and another in Seattle. We were able to confirm the eligibility status  of 70% of the patients with such diagnoses and all eligible respondents were interviewed.Except for chiropractic, knowledge about these therapies was low. Chiropractic and massage had been used by the largest fractions of respondents , mostly for back pain . Among prior users of specific CAM therapies for back pain, massage was rated most helpful. Users of chiropractic reported treatment-related "significant discomfort, pain or harm" more often (23%) than users of other therapies (5–16%). Respondents expected massage would be most helpful  and meditation least helpful (median of 3) in relieving their current pain. Most respondents indicated they would be "very likely" to try acupuncture, massage, or chiropractic for their back pain if they did not have to pay out of pocket and their physician thought it was a reasonable treatment option.Most patients with chronic back pain in our sample were interested in trying therapeutic options that lie outside the conventional medical spectrum. This highlights the need for additional studies evaluating their effectiveness and suggests that researchers conducting clinical trials of these therapies may not have difficulties recruiting patients. Back pain is one of the most common and costly health problems in developed countries, where more than half of adults suffer from this condition each year  and 70% Prior to designing and conducting two pilot clinical trials evaluating the effectiveness of five different CAM therapies for chronic low back pain (LBP) in older (65+ years) and younger (20 through 64 years) adults, we sought to refine recruitment strategies. As part of this effort, we surveyed chronic low back pain patients about their interest in trying each of these five CAM therapies if included in their health plan benefits and as part of two separate clinical trials. In this descriptive and exploratory study, we also collected information about their knowledge of, experience with, and perceptions of each of these therapies.From April to October 2001, we conducted telephone interviews with 249 patients who were currently suffering from non-specific low back pain that had persisted at least three months. The patients were members of a non-profit managed health care system  and a large multi-specialty group practice . Our goal was to interview 150 patients from Group Health and 100 patients from Harvard Vanguard who were otherwise healthy and spoke English, with 50% of the interviews from adults 65 years or older. The Institutional Review Boards of Group Health Cooperative, Seattle WA and Harvard Pilgrim Health Care, Boston, MA approved the study.Using automated visit data, we identified and mailed letters to 787 patients with working phone numbers who visited a Group Health (n = 422) or a Harvard Vanguard (n = 365) primary care provider and had a diagnosis consistent with non-specific low back pain between 12 and 52 weeks previously. Because we were planning to use the results of the survey to help us refine recruitment strategies for two pilot randomized trials, our exclusion criteria for this study were those we planned to use in the subsequent trials. We therefore used the automated visit data to exclude the following individuals from our sample:• those whose back pain may have been due to a specific disease or condition ;• those who had other pain conditions that could complicate the interpretation of trial results ;• those who were inappropriate candidates for one or more of the clinical trial treatments  or who were unlikely to be able to give informed consent or to participate in the baseline and follow-up assessments of the trial .Before administering the in-person survey, we asked eight screening questions and then excluded individuals who did not have back pain at the time of the interview or who had not had back pain for at least 12 weeks, who had previously had low back surgery, who reported having fractured a vertebrae, who were pregnant, who were involved in back-pain related litigation, who had serious health problems, or who could not speak English.We tried to contact all mailees, but could not reach 28 (7%) persons from Group Health and 73 (20%) from Harvard Vanguard despite at least seven phone calls. Among the 394 Group Health and 292 Harvard Vanguard patients who were contacted, 57 from Group Health and 81 from Harvard Vanguard refused the interview and we could not determine their eligibility status, 195 from GHC and 104 from Harvard Vanguard were ineligible upon screening, and 142 from Group Health and 107 from Harvard Vanguard were eligible and interviewed. Thus, we were able to screen 70% of all mailees for eligibility . All screened and eligible mailees were interviewed.In both areas, individuals aged 65 and older were more likely to refuse screening  and those under 65 were less likely to be contacted by phone .Most patients were ineligible because they were not experiencing back pain at the time of the interview  or their pain had not persisted for three months .We conducted a phone interview that lasted an average of 17.7 minutes . It included questions about demographic characteristics ; back pain characteristics ; self-reported knowledge  of five CAM treatments or self-care methods ; previous use of these therapies for any reason and for back pain specifically (and helpfulness of the therapy for back pain relief); perceived harm from previous use of these therapies; expectations of helpfulness of these therapies for current back pain; willingness to try these therapies if offered by the health plan for no additional cost and for a $10 per visit co-pay; willingness to participate in two hypothetical clinical trials, one evaluating acupuncture, chiropractic, and massage and another involving massage, meditation, and t'ai chi.  Finally, respondents were asked about which treatment they most preferred among those offered in each trial. Gender and geographic location were obtained for respondents from the enrollment files of each healthplan. General definitions of each therapy were provided only for respondents who informed the interviewers they did not know what a particular therapy was. Acupuncture was described as a system of healing that involved inserting hair thin needles into acupuncture points just beneath the skin or using other methods, such as heat, to stimulate these points, whereas chiropractic was defined as a system of therapy that uses manipulation to adjusts the spine and other body parts to "promote normal nerve functions". Massage was described as the systematic rubbing and manipulation of muscle and other tissues to relieve bodily infirmities, while meditation was defined as a "self-directed practice for relaxing the body and calming the mind". Finally, tai chi was described as a Chinese martial art that uses slow and smooth body movements and is often practiced for its purported health benefits. The survey was pre-tested on a convenience sample of 15 people (both older and younger) in Seattle and 5 people in Boston.We analyzed the data using the SAS statistical software version 6.12 . Descriptive data were characterized using percentages or medians. For each of the five CAM therapies, we performed separate exploratory logistic regressions to identify specific characteristics associated with 1) high degree of knowledge  (five separate models), 2) prior use (for any reason and for back pain) (10 separate models), 3) high expectations of helpfulness for current back pain  (five separate models), 4) greatest likelihood of trying therapy for no additional cost  and for a $10 per visit co-pay  (eight separate models), and 6) greatest likelihood of participating in each of the two hypothetical clinical trials (two separate models). Thus, a total of 30 separate logistic models were created.For each of the 30 dependent variables, we identified potential predictor variables in advance and evaluated them in preliminary models. In Table Initially, we evaluated potential predictor variables in preliminary models containing five or fewer independent variables. Any independent variable associated with the dependent variable at a p value of 0.15 or less in a preliminary model was a candidate for the appropriate final model. We used a backwards elimination procedure to evaluate candidate predictor variables and to determine the final models . All varMost study participants were white, women, and had attended college Table . Most haExcept for chiropractic, most participants reported little or no knowledge of these therapies Table . In logiMore than half of the participants had tried chiropractic compared with 38% who had tried massage and substantially fewer who had tried the other therapies Table . ChiroprRespondents believed that massage would be most helpful for their current back pain (median rating of 7) and that meditation would be least helpful (median rating of 3) Table . High exMore than half of the respondents said they would be "very likely" to try acupuncture, chiropractic, or massage if provided by their health plan for no additional cost and their physician felt it was reasonable. Fewer respondents said they would be "very likely" to try meditation training (27%) or t'ai chi 41%) under those circumstances Table . In logi1% under More than half of respondents were "definitely willing" to participate in each of two hypothetical clinical trials about which they were asked and less than 5% were definitely unwilling to participate .Respondents showed a clear preference for receiving hands-on treatments delivered by a practitioner compared to attending classes that teach self-care techniques. Whether this reflects a preference for provider-oriented, more passive, therapies or the belief that classes teaching these specific self-care therapies would be less effective is not clear. Unfortunately, our interview did not include questions about yoga, which has recently received more popular press than meditation or t'ai chi as a self-care therapy for back pain ,10.Survey respondents were not enthusiastic about "meditation training" as a treatment for back pain. Relatively few of those who indicated prior use of meditation for physical or mental health problems had used the forms of meditation most commonly taught in a medical setting . Consequently, studies recruiting patients to participate in interventions including meditation training may need to carefully describe the treatment in terms of a concrete goal .There is still relatively little knowledge about and experience with acupuncture and t'ai chi even in Boston and Seattle where use of CAM therapies is generally high. In fact, about one – quarter of respondents were unable to provide an expectation of the helpfulness of acupuncture or t'ai chi. Nevertheless, substantial fractions of participants were willing to try acupuncture and t'ai chi as a treatment if their primary care provider thought it reasonable, and in the case of acupuncture, even if they had to pay a $10 co-pay each visit. Our finding that people in our sample reported being almost as willing to try acupuncture as massage, despite less knowledge of, expectations about and experience with it, is intriguing and requires further inquiry.Although participants in this study reported more knowledge of and experience with chiropractic, they were more enthusiastic about massage. A recent survey of 46,000Most survey respondents indicated they were "very willing" to participate in our two hypothetical clinical trials evaluating different CAM treatments for chronic back pain. Massage was the preferred treatment in both trials, but more than one in five survey respondents stated a preference for acupuncture and t'ai chi. In view of the long-standing popularity of chiropractic, surprisingly few respondents reported chiropractic as their top choice. Nonetheless the finding that massage was substantially more popular than chiropractic mirrors the results among acute low back pain patients in a clinical trial who were randomized to a choice of acupuncture, chiropractic, massage, or usual care or to usual care alone . In thatDespite low levels of knowledge about t'ai chi and acupuncture, the finding that over 40% of respondents indicated they were very likely to try these therapies suggests that recruiting enough subjects for clinical trials involving these therapies may be feasible if moderate to large patient populations are available. Recruiting patients for meditation trials, however, is likely to be difficult. Consequently, when we recruited patients for a pilot trial that included a stress reduction intervention based on the principles of mindfulness meditation, we chose to describe it as "Mindfulness Based Stress Reduction" rather than mindfulness meditation.We believe that clinical trials evaluating obviously different treatments for chronic low back pain, such as massage and meditation, may have problems retaining subjects who do not receive the treatment  that attracted them to the study. This problem may be exacerbated if patients have an exceptionally strong preference (or dislike) for one treatment. Inclusion of multiple CAM modalities in a single study risks tempting potential participants to sign up for the study in the hope of receiving a desired treatment, and then dropping out if they receive a different treatment.In addition, if one treatment is vastly more popular than another, it could be difficult to disentangle the effects of patient expectations and treatment efficacy per se, leading to difficulties in interpreting positive study outcomes. This problem is compounded by concerns about the subjective nature of back pain outcomes, the difficulty in masking participants to study treatment, and the strong skepticism of some researchers that CAM treatments can be effective, even when results are impressive. Masking patients to treatment is quite difficult in studies of many types of conventional as well as CAM treatments if the treatments involve a physical modality, such as massage, or active participation of the patient in the treatment, as in t'ai chi. In such circumstances, using masked outcomes assessors is important to minimize bias. We also suggest that patient (and provider) expectations for treatment and prior experience with each treatment, be measured and, if appropriate, controlled for in the analyses. Finally, if a particular therapy is shown effective in clinical trials in different populations, mechanistic studies will be important for determining how these therapies achieve their effects. Such studies are especially important to convince skeptics that CAM therapies actually have specific effects. In the meantime, the high and rising public interest in CAM therapies, especially for musculoskeletal conditions , highligMost patients in our sample were interested in trying options for treating chronic back pain that lie outside the conventional medical spectrum, even within the context of a clinical trial. This was true even among patients who had relatively little knowledge of or experience with the therapy. Given our limited knowledge about the effectiveness of most CAM therapies, there is a clear need for additional studies evaluating their effectiveness. Fortunately, our results suggest that researchers will not find it difficult to recruit patients interested in participating in clinical trials of many of the CAM therapies.None declared.KJS participated in the development of the questionnaire and took primary responsibility for the analysis of the data and for drafting the manuscript. DCC was the PI on one of the grants funding the study, participated in the development of the questionnaire and the analysis of the data and played a major role in drafting the manuscript. MTC participated in the development of the questionnaire and the analysis of the data. JE and JBS coordinated the project and oversaw the data collection. RBD participated in the analysis of the data and provided statistical oversight. DME was the Principal Investigator on one of the grants funding the study, participated in the development of the questionnaire and the analysis of the data. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


Most analyses of risks to health focus on the total burden of their aggregate effects. The distribution of risk-factor-attributable disease burden, for example by age or exposure level, can inform the selection and targeting of specific interventions and programs, and increase cost-effectiveness.For 26 selected risk factors, expert working groups conducted comprehensive reviews of data on risk-factor exposure and hazard for 14 epidemiological subregions of the world, by age and sex. Age-sex-subregion-population attributable fractions were estimated and applied to the mortality and burden of disease estimates from the World Health Organization Global Burden of Disease database. Where possible, exposure levels were assessed as continuous measures, or as multiple categories. The proportion of risk-factor-attributable burden in different population subgroups, defined by age, sex, and exposure level, was estimated. For major cardiovascular risk factors  43%–61% of attributable disease burden occurred between the ages of 15 and 59 y, and 87% of alcohol-attributable burden occurred in this age group. Most of the disease burden for continuous risks occurred in those with only moderately raised levels, not among those with levels above commonly used cut-points, such as those with hypertension or obesity. Of all disease burden attributable to being underweight during childhood, 55% occurred among children 1–3 standard deviations below the reference population median, and the remainder occurred among severely malnourished children, who were three or more standard deviations below median.Many major global risks are widely spread in a population, rather than restricted to a minority. Population-based strategies that seek to shift the whole distribution of risk factors often have the potential to produce substantial reductions in disease burden. Analysis of 26 major global health risk factors by age, sex, exposure level, and epidemiological subregion reveals that many of them are widely spread in a population rather than restricted to a minority Reliable and comparable analysis of risks to health is an important component of evidence-based policies and programs for disease prevention ,2. An imUnderstanding the distribution of risk-factor burden is particularly important for targeting interventions. For example, the large number of road traffic accident injuries and deaths among young adult males may be largely associated with binge alcohol consumption by this group. Interventions that focus on this population subgroup and their specific drinking behaviors may be more effective or cost-effective than, for example, raising alcohol taxes, which would have a more diffuse impact on alcohol consumption. On the other hand, the majority of effects from risk factors such as blood pressure have been found to be among those at moderately elevated levels, motivating interventions beyond those intended for clinical hypertension ,4,5.The distributions of the health effects of risk-factor exposure by age and sex or by exposure level have been studied in specific cohorts and for specific risk factors ,7,8. MosThe methods and data sources for individual risk factors and for estimating population attributable fractions (PAFs) and disease burden attributable to them have been fully described elsewhere ,2 and arRR(x) is relative risk at exposure level x, P(x) is the population distribution of exposure, P′(x) is the alternative or counterfactual distribution of exposure, and m is the maximum exposure level.The alternative  scenario used in this work is the exposure distribution that would result in the lowest population risk, referred to as the theoretical minimum-risk exposure distribution  1,2]. T,2. T1,2]World Health Report, was expressed in disability-adjusted life-years (DALYs) with methods and assumptions described elsewhere [Estimates were made for eight age groups , both sexes, and 14 Global Burden of Disease subregions . PAFs welsewhere . Aggregalsewhere , though The distribution of deaths and DALYs attributable to the risk factors by age and sex is shown in The estimated disease burden from childhood and maternal undernutrition; unsafe water, sanitation, and hygiene; and global climate change  was almost exclusively among children under 5 y of age. For these risks, more than 85% of total attributable burden occurred in this age group, with the exception of iron deficiency, for which 30% of burden was borne by women of childbearing age. Only a small fraction of disease burden from the risk factors considered was among the 5–14 y olds. This was because some of the leading diseases of this age group  have complex causes that could not easily be included in the current risk-based framework . For othThe disease burden from other diet-related risks, tobacco, and occupational risks (except injuries and back pain) was almost equally distributed among adults above and below the age of 60 y. For example, 43% of disease burden due to blood pressure and 61% of burden due to tobacco occurred in the 15–59-y age group. More than 90% of disease burden attributable to lack of contraception, illicit drugs, occupational ergonomic stressors, risk factors for injury, and childhood sexual abuse occurred in adults below the age of 60 y. Similarly, 67%–87% of the disease burden attributable to alcohol, unsafe sex, and unsafe health-care injections arose from events occurring between 15 and 59 y of age. Most of the risks whose hazards are concentrated in the younger adults are causes of injuries, neuropsychiatric diseases, maternal conditions, and HIV/AIDS. Assessment by the level of economic and demographic development illustrated that, with the exception of alcohol, which has global presence, the majority of disease burden from risks for mortality and disease among young adults is concentrated in developing countries . This was similar to or greater than the amount of disease burden occuring among individuals with risk-factor levels above the commonly used, but arbitrary, thresholds for hypertension, hypercholesterolemia, and obesity indicated in age-specific BMI levels in those countries [Despite the above finding on the important role of moderately elevated levels in total disease burden, the actual patterns of how disease burden is distributed among exposure levels varied across different regions and risk factors . For exaountries . This isThe findings reported here should be considered within the context of limited available data and are subject to uncertainty, which varies across risk factors and is generally most marked in developing countries. Full discussion of uncertainty in the basic data sources and parameters is provided elsewhere and includes the uncertainties in estimates of disease incidence, duration, severity, and disability weighting ,2. UncerOf particular relevance to the current analysis is the fact that, due to data limitations, some risks were measured using categorical variables  even though the health effects occur along a continuum. Other risk factors were represented using indirect or aggregate indicators  that do not allow quantification of risks along continua of exposure. Two important sources of uncertainty with implications for interventions are correlations among multiple risk factors and the skewness of risk-factor distribution. Because risks are often correlated , the contributions of high-exposure groups are likely to be underestimated. In addition to being a source of underestimation at higher exposure levels, risk-factor correlation implies that the same individuals and groups are at the high end of multiple risk factors. Positive (rightward) skewness of exposure distribution, not modeled here, would also lead to an underestimation of events occurring in those who were hypertensive, hypercholesterolemic, or obese. The importance of skewness is emphasized by the observation that the recent increase in the proportion of people who are overweight and obese  has involved not only a shift in the distribution, but also increasing skewness, which has extended the high-exposure tail. Coupled with risk-factor correlation, this should motivate more systematic data collection and reporting to determine mean exposure as well as the complete shape of distribution.The temporal nature of risk-factor exposure also has implications for the current cross-sectional estimates. There is an expectation that the size and rank order of risk-factor burden will alter in coming decades as a result of changes in exposure levels and delays between exposure and hazard. For example, it is predicted that by 2020 the leading risks to health will be 1) unsafe sex, principally because of HIV/AIDS and driven by increasing exposure, and (2) tobacco, because of market expansion of tobacco products in developing countries and delayed temporal effects on major health outcomes such as lung cancer [ unsafe sThis analysis in multiple age and exposure categories, or along a continuum of exposures, showed that, globally, a considerable proportion of the disease burden attributable to major risk factors occurred among those with only moderately raised levels, not the extremes such as those that define hypertension, obesity, or severe malnutrition. Further for many chronic-disease risk factors, such as tobacco and high blood pressure, as well as risk factors for injuries and sexual and reproductive health, significant proportions of disease burden occurred from events in middle ages, especially in developing countries. The concentration of disease burden from such a large number of risk factors in those below 60 years of age illustrates the large, and at times neglected, disease burden from risks that affect young adults in developing nations, with important consequences for economic development .The distribution of risks and their determinants in a population have major implications for strategies of prevention. As stated by Rose, risk typically increases across the spectrum of a risk factor . Rose's There were distinct patterns for the distribution of disease burden across risk factors, and across regions at various stages of development. At the extreme of these diverse patterns, for risk factors with acute exposure and acute outcomes, the underlying relationship is considerably more complex. For example while in many societies most alcohol-attributable injury  involves people who on average drink moderate amounts , these pIn summary, the analysis presented in this paper confirms and extends to a global level previous work indicating that the impact of many major risks is important across their exposure levels, not just among people with particularly high levels . This anHealth policy makers must understand existing health risks and which diseases cause the most health problems. The Global Burden of Disease database, maintained by the World Health Organization, collects information from around the world on risk factors such as malnutrition, childbirth, tobacco use, and cholesterol levels, as well as on diseases such as depression, blindness, and diarrhea. This information can be used to target health interventions.These researchers determined for 26 major risk factors the distribution of disease burden by age, sex, and exposure level. They found that many risk factors (such as high blood pressure and high cholesterol levels) occur across populations, and are not confined to one sex or age group. And for most risk factors, exposure to moderate risks  is responsible for causing most disease.Measures that reduce exposure to risk factors across whole populations, if only by a modest extent, can achieve large reductions in disease burden. This information is important for people who set global health policies and priorities.There are many challenges in estimating the impact of different major risks, each of which has distinct effects on a number of diseases. In addition, exposure to some risk factors today will cause disease only many years from now, so the picture will change over time. The major finding of the global distribution of many major risks is secure, but the exact extent of this remains uncertain due to the paucity of data in developing countries.


Ruptures of the anterior cruciate ligament (ACL) are common injuries to the knee joint. Arthroscopic ACL replacement by autologous tendon grafts has established itself as a standard of care.Data from both experimental and observational studies suggest that surgical reconstruction does not fully restore knee stability. Persisting anterior laxity may lead to recurrent episodes of giving-way and cartilage damage. This might at least in part depend on the method of graft fixation in the bony tunnels. Whereas resorbable screws are easy to handle, pins may better preserve graft tension. The objective of this study is to determine whether pinning of ACL grafts reduces residual anterior laxity six months after surgery as compared to screw fixation.SPOT is a randomised, controlled, patient and investigator blinded trial conducted at a single academic institution. Eligible patients are scheduled to arthroscopic ACL repair with triple-stranded hamstring grafts, conducted by a single, experienced surgeon. Intraoperatively, subjects willing to engage in this study will be randomised to transplant tethering with either resorbable screws or resorbable pins. No other changes apply to locally established treatment protocols. Patients and clinical investigators will remain blinded to the assigned fixation method until the six-month follow-up examination.The primary outcome is the side-to-side  difference in anterior translation as measured by the KT-1000 arthrometer at a defined load (89 N) six months after surgery. A sample size of 54 patients will yield a power of 80% to detect a difference of 1.0 mm ± standard deviation 1.2 mm at a two-sided alpha of 5% with a t-test for independent samples.Secondary outcomes  will be handled in an exploratory fashion.SPOT aims at showing a reduction in anterior knee laxity after fixing ACL grafts by pins compared to screws. Anterior cruciate ligament (ACL) rupture belongs to the most common musculoskeletal injuries in the western world. In the United States, 100,000 new cases occur each year, with 10% of all injuries leading to occupational disability .An ACL deficient knee is at risk of developing secondary damage to the cartilage and is liable to undergo progressive intra-articular worsening. Roughly half of all acute ACL disruptions attend meniscus damage. Arthroscopic reconstruction surgery by autologous grafting emerged as the therapy of choice. Predictions call for 175,000 ACL replacements performed yearly in the United States. In Germany, around 50,000 patients undergo ACL repair each year.Selecting the ideal graft remains an issue of debate. Randomized controlled trials suggest a lower degree of persistent laxity with bone-patellar-tendon-bone (BPTB) comparing with two-, three- or four-bundle hamstring  transplants (HT).However, the biomechanical advantage does not frame higher patient satisfaction, or differences in scoring after long-term follow-up . In contAvailable data from randomized, quasi-randomized and uncontrolled trials signal a weighted mean difference of 2.28 mm  in anterior laxity between the injured and healthy knee with HT reconstruction  screw that contains both resorbable poly-L-lactid and osteoconductive tricalcium phosphate , with the knee in a smoothly flexed position. A common way to anchor the tibial end of the graft is by titanium or biodegradable interference screws, for example, the BioCryl® (Smith & Nephew) or RigidFix® (DePuy Mitek) provided better strength than did the interference screws [® over other tethering methods is a splicing of strands, tightening the contact between the tendon surface and the bony tunnel over the entire graft circumference  in its German translation, 2000 revision . BesidesConfirmatory testing will apply for the primary endpoint only. All secondary endpoints will be addressed in an exploratory fashion.SPOT is a patient and investigator blinded, randomised controlled trial conducted at a single academic institution.Randomisation is carried out in the operating theatre shortly before transplant fixation, with random codes drawn from sealed envelopes. We use block-randomisation with five subjects a block following a computer-generated random list .Men and women (providing that they are not pregnant) being at least 18 years old are recruited to this trial. Subjects may engage in this study if theyfirst one-sided total or subtotal rupture of the anterior cruciate ligament, proven either by arthroscopy or MRI-scanning- faced a - had met an acute knee distorsion event likely to have caused the index injury at least six weeks before scheduled repair- have been physically examined in the ambulatory of the study hospital before assigning an admission date, and were screened and considered suitable to enter this trial by one of the investigatorsAlso, patients must be able to give voluntary written informed consent, and to comply with the post operative follow up regimeWe exclude patients- with related lower limb fractures- with active infection affecting the limb subject to needed treatment- who have previously took part in this investigation or who are taking part in another clinical investigation- with contraindications for MRI-scanning This protocol and all accompanying documents were approved by the local Institutional Review Board (IRB).According to IRB recommendations and requirements, information leaflets explicitly note that "a benefit from participation in this trial cannot be guaranteed."We will stress the principle of randomisation as "treatment assignment by chance, without the possibility of the investigator, other health care professionals involved in this study, or the patient influencing the choice of treatment." We also tell patients that, as long as they keep agreement in participation, they will not know their assigned treatment until the six-month follow-up visit.We will notify the IRB of any significant changes to the protocol. Also, we will notify the IRB within ten working days of the discovery of any severe adverse events which occur during this investigation.Confidentiality of subject data will always be maintained. Subject anonymity will be guaranteed and all documentation about a subject (including radiographs) will be kept in secure location.th World Medical Assembly in 1964 and its later revisions, as well as to principles of GCP, developed within the Expert Working Group (Efficacy) of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH).This investigation strictly adheres to the relevant articles of the Declaration of Helsinki as adopted by the 18® screw and RigidFix® cross pins. Also, postoperative care and rehabilitation programs do not differ from those employed outside a clinical trial.All devices and instrumentations used in this clinical investigation bear the CE mark. They belong to the regular implants used for ACL repair at the study hospital since 2002. Except different graft anchoring, similar treatments apply to patients in both study groups. All participants undergo internationally accepted surgically procedures by a single surgeon (D.C.) with extensive experience in ACL repair using both the BioCrylAll repairs are carried out under general anaesthesia, with the patient in a supine position. Perioperative antibiotic prophylaxis comprises 2 g of cefotiam. Patients receive 40 mg of enoxaparin daily for prophylaxis of thromboembolic events until full weight bearing.The knee joint is accessed through two to three standard portals. Meniscal injuries are addressed with partial resection or repair.Hamstring tendons are harvested via a small incision over the insertion of the pes anserinus at the anterior medial tibia by a closed tendon stripper, and prepared as triple-stranded grafts.Tibial and femoral tunnels are drilled to approximate graft thickness  with the use of a guiding wire. Grafts are fixed with the knee in 30° flexion to achieve firm tension.® orthosis for six weeks. Patients are allowed partial weight bearing with walking crutches. Subjects are prescribed intense physical therapy for motion exercise, and to strengthen thigh muscles. Normally, full range of motion and weight bearing is achieved until week 12 after surgery.Postoperatively, the knee is stabilized for three days by a zero-degree splint. Afterwards, flexion is limited to 90° by a Secutecexperimental group have their grafts secured by tibial and femoral RigidFix® pinning. Patient in the control group receive tibial and femoral BioCryl® screws.Patients in the Each subject considered eligible for entry into this investigation has the following information and procedures recorded at the pre-investigational examination:- Demographic details including date of birth and gender- Medical history, coexisting diseases, and accompanying medication- Physical examination, including circumferential measurement of both legs at defined landmarks, Lachman and pivot shift tests, KT-1000 arthrometer objectifying of instability, one-legged hop test, Lysholm, Tegner, and IKDC scores, knee and kneeling pain measured by visual analogue scales- Radiographic examination, including a conventional roentgenogram of the injured knee in anteroposterior and lateral projection, and a preoperative MRI scan according to local standardsDuring surgery, we record procedure details in the electronic CRF. We assess the duration of surgery (from cut to skin closure), and operating theatre time . A clinical knee examination is performed and documented with the subjects under general anaesthesia and relaxation. Arthroscopy findings  are recorded by video and/ or hard copy images.We document eventual blood loss, and any other adverse event occurring during surgery.The responsible surgeon judges the handling of implants and his overall satisfaction with the intraoperative result using five-point Likert scales. We detail any problems or complications on an Adverse Events form.Patients are appointed outpatient visits as part of the clinical investigation at 3, 6, and 12 months postoperatively. For study purposes, except quality of life measurements, patients do not undergo any diagnostic or other procedure not belonging to the common repertoire of assessments carried out after ACL repair. Specifically, we avoid invasive procedures, blood sampling, or imaging tests exposing subjects to radiation or contrast agents. The investigators consider the possible burden caused by extra clinical tests negligible.We assess the following items at the scheduled visits:- Physical examination, including circumferential measurement of both legs at defined landmarks, Lachman and pivot shift tests, KT-1000 arthrometer objectifying of instability, one-legged hop test, Lysholm, Tegner, and IKDC scores, knee and kneeling pain measured by visual analogue scales- Any complications or complaints raised by the patient, GP/ ambulatory surgeon, or Clinical Investigators- Resumption of work- MRI scan according to local standards- Physical examination, including circumferential measurement of both legs at defined landmarks, Lachman and pivot shift tests, KT-1000 arthrometer objectifying of instability, one-legged hop test, Lysholm, Tegner, and IKDC scores, knee and kneeling pain measured by visual analogue scales- Any complications or complaints raised by the patient, GP/ ambulatory surgeon, or Clinical Investigators- Resumption of work and leisure activities- MRI scan according to local standardsafter completing the six-month CRF.Patients and Investigators responsible for follow-up examinations may learn about the assigned treatment - Physical examination, including circumferential measurement of both legs at defined landmarks, Lachman and pivot shift tests, KT-1000 arthrometer objectifying of instability, one-legged hop test, Lysholm, Tegner, and IKDC scores, knee and kneeling pain measured by visual analogue scales- Any complications or complaints raised by the patient, GP/ ambulatory surgeon, or Clinical Investigators- Resumption of work and leisure activities- MRI scan according to local standardsRadiological evaluation comprises- Tunnel widening- Impingement- Transplant sufficiency, morphology of transitional areas and tendon-bone-interfaces- Degree of degradation of screws and pins- Degree of inflammation (synovitis) and effusion- Presence or progression of arthrofibrosis- Changes in cartilage and meniscal morphologyBefore physical examination, both knees are prepared by applying opaque dressings to hide scars and to blind the examining doctors for the managed side.Examination is performed independently by two of three board-certified surgeons  who had not operated on any of the patients in the study. The responsible surgeon (D.C.) conducts a third clinical examination after completion of the case report forms. We document his examination findings separately and consider them as the diagnostic reference standard. We assess both interobserver agreement by kappa statistics and the accuracy of measurements taken by independent observers comparing with those of the responsible surgeon.Objective translation measurements comprise a defined load (89 N). We measure the anterior translation of the injured and healthy side (in mm), as well as the difference between both knees.We define an adverse event as 'any undesirable clinical instance in a subject whether it is considered treatment related or not'. In addition, an adverse device effect, undesirable side effect, is defined as 'a device related adverse event'.all adverse events, including details of the nature, onset, duration, severity, relationship to the device, relationship to the operative procedure and outcome, will be made on the relevant section of the subject's CRF. The subject will be questioned about any adverse event at each later follow-up assessment visit.A record of An adverse event or an adverse device effect may be mild, moderate or severe and are usually unexpected.A severe adverse event or adverse device effect is defined as any experience that- is fatal or life-threatening- is permanently disabling- needs or prolongs in-patient hospitalization because of a potential disability, danger to life or forces an interventionimmediately by telephone or facsimile toAll severe adverse events or adverse effects which occur during this investigation must be and will be reported Bundesinstitut für Arzneimittel und Medizinprodukte, Kurt-Georg-Kiesinger-Allee 3, 53175 Bonn, Phone: ++49 30 228 207 30, Fax: ++49 30 228 207 5207® Database, run on a mobile computer separately from the hospital documentation and the intranet. Data collection and storage comply with orders fixed by the data safety board of the Unfallkrankenhaus Berlin, and follow German laws for data safeguard and protection . We ensure data storage for five years.For data collection, we set up a Microsoft XP Professional AccessFor study documentation, we assign patients an identification number. Electronic sources do not contain names or addresses of participants. Linking lists are stored in a study folder with copies of adverse events forms.Since this study runs at a single centre, we do not appoint an external monitor for data handling and management. We regularly (at least twice a month) check datasets for consistency, completeness and plausibility.We will conduct all analyses following the intention to treat principle .We will express measurements as means, medians or proportions with their proper distribution indices . In case of skewed distributions, normalizing will be achieved by logarithmic transformation, where necessary.As pointed out earlier, we will address only the primary hypothesis in a confirmatory fashion, whereas all other results will be evaluated in a plain exploratory intent.We will employ the student's t-test for independent samples to test for the difference in anterior laxity between both fixation methods at six months of follow-up.For secondary endpoint analysis, we will calculate cross tables, 95% confidence intervals for normally or binomially distributed data, and differences in means, proportions, and ratios. In case of obvious benefits or harms with either device in a certain subgroup of patients, we will use stratified analyses .Where statistically and/ or clinically sound, we will consider linear and logistic regression analyses, or more sophisticated regression models for correlated data . We will, however, respect the small sample size of this study, and limit statistical analyses to the necessary minimum.In case of missing data, we will use both a last observation carried forward approach, and imputation methods by regression or semi-Bayesian modelling. Separate analyses will be performed for raw and modelled data.After ACL repair, most patients rarely recognize slightly weakened anterior knee stability in everyday life. However, subjects with a high recreational and sporting activity and physically strenuous professions often suffer from recurrent events of giving-way, especially on hastened movements. This poses a high risk for secondary knee injury. Of note, muscular training cannot compensate for residual laxity, outbalancing the anticipated benefit from surgical repair.® tray is ten times higher than that of BioCryl® screws. Obviously, the more expensive implant must prove a distinct clinical advantage over the common one to justify its further use. Unfortunately, there is lack of robust evidence on the effectiveness of either fixation method beyond laboratory and animal experiments.Thus, attempts to optimize the surgical technique may be valuable. Currently, surgeons performing ACL reconstruction use screws, pins, buttons, and cramps for graft fixation because of individual preference, or institutional orders. The latter are chiefly driven by cost considerations. For example, the purchase price of a RigidFix® over screws. The investigators consider the equipoise principle fulfilled, since it is unclear whether screw or pins lead to better long-term stability, or show any measurable differences at all. We hope that the results from this pragmatic study can clarify this issue.Although conceptually impressive, there is no comparative study that proved a clinically measurable advantage of RigidFix® International, and received project-related funding that does not apply to this work. No support in any form was provided, or will be provided by third parties to set up the trial protocol, or to conduct this study. This study aims at investigating biomechanical principles, not certain implants.MW, DS and AE have worked as independent scientific consultants for DePuyThe members of the SPOT Group have no financial or non-financial competing interests in this work.DS drafted the manuscript. KB and GM edited the manuscript.DC is in charge of all surgical procedures. DS is responsible for statistical analyses.All authors participated in the design of this study, and approved the final manuscript.The pre-publication history for this paper can be accessed here:


While BaP and ANTH selectively injure glomerular mesangial cells, and CHRY targets cortico-tubular epithelial cells, binary or ternary mixtures of these hydrocarbons markedly reduce the overall cytotoxic potential of individual hydrocarbons.Benzo(a)pyrene (BaP), anthracene (ANTH) and chrysene (CHRY) are polynuclear aromatic hydrocarbons (PAHs) implicated in renal toxicity and carcinogenesis. These PAHs elicit cell type-specific effects that help predict toxicity outcomes To study the biochemical basis of these antagonistic interactions, renal glomerular mesangial cells were challenged with BaP alone (0.03 – 30 μM) or in the presence of ANTH (3 μM) or CHRY (3 μM) for 24 hr. Total RNA and protein will be harvested for Northern analysis and measurements of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activity, respectively, to evaluate cytochrome P450 mRNA and protein inducibility. Cellular hydrocarbon uptake and metabolic profiles of PAHs were analyzed by high performance liquid chromatography (HPLC).Combined hydrocarbon treatments did not influence the cellular uptake of individual hydrocarbons. ANTH or CHRY strongly repressed BaP-inducible cytochrome P450 mRNA and protein expression, and markedly inhibited oxidative BaP metabolism.These findings indicate that antagonistic interactions among nephrocarcinogenic PAHs involve altered expression of cytochrome P450s that modulate bioactivation profiles and nephrotoxic/ nephrocarcinogenic potential. These findings indicate that the toxicity and carcinogenicity profiles of PAHs may be defined on the basis of their ability to regulate Cyp1a1 and Cyp1b1 at either the mRNA or protein level.In mesangial cells,NTH Figs . In contNTH Figs , a profiCyp1a1 and Cyp1b1 mRNAs by PAHs and polychlorinated biphenyls (PCBs) has been investigated in wildtype and AhR-deficient C57BL/6J mice [Cyp1a1 is AhR-dependent, Cyp1b1 is constitutively expressed in various organs in male and female Ahr (+/+) and Ahr (-/-) mice. Cyp1b1 is of interest because it encodes for AHH and EROD, the predominant PAH-metabolizing activities in rodent embryos [Cyp1b1 in PAH carcinogenesis have been proposed by Gonzalez and co-workers [Cyp1b1 knock-out mice expressing significant levels of Cyp1a1 are highly resistant to lymphoma formation by 7,12-DMBA. In vitro human studies with recombinant enzymes have shown that Cyp1b1 is more active (about 10-fold) than Cyp1a1 in the formation of BaP-7,8-diol [The tissue-specific induction of /6J mice . While e embryos . Importa-workers -20 who o7,8-diol .Cyp1a1 mRNA, but induction at the mRNA level was not associated with corresponding increases of enzymatic activity. The greater potency of BaP as an inducer of enzymatic activities compared to ANTH and CHRY is consistent with previous observations showing that BaP is the most toxic PAH to renal mesangial cells [Cyp1a1 and Cyp1b1 inducibility by BaP, but EROD and AHH activities were reduced in binary mixture. CHRY, on the other hand, modestly inhibited Cyp1a1 and Cyp1b1 mRNA inducibility by BaP and also reduced AHH activity. Because both ANTH and CHRY antagonize the mesangial cell response to BaP (3), these findings implicate AHH as the primary enzymatic target for antagonistic interactions among nephrotoxic PAHs.Measurements of EROD and AHH were used to monitor kidney microsomal catalytic activities Figs  and 4. Tal cells . The proThe above interpretation is supported by the marked inhibition of 3-OH BaP formation seen in cells co-treated with BaP and ANTH or CHRY Table . A role A major implication of our findings is that despite similarities in gene and protein inducibility among structurally-related PAHs, their behavior may be influenced by the reactivity of oxidative intermediates generated during the course of cellular metabolism. As such, PAH substrates in complex mixtures may compete for, and inhibit, the same metabolizing enzymes that act upon them to give rise to antagonistic interactions that protect against further chemical toxicity. Interactions of this nature are among the most commonly encountered in environmental mixtures , but the


Patients with asthma demonstrate circadian variations in the airway inflammation and lung function. Pinealectomy reduces the total inflammatory cell number in the asthmatic rat lung. We hypothesize that melatonin, a circadian rhythm regulator, may modulate the circadian inflammatory variations in asthma by stimulating the chemotaxins expression in the lung epithelial cell.Regulated on Activation Normal T-cells Expressed and Secreted) and eotaxin expression were measured using ELISA and real-time RT-PCR, eosinophil chemotactic activity (ECA) released by A549 was measured by eosinophil chemotaxis assay.Lung epithelial cells (A549) were stimulated with melatonin in the presence or absence of TNF-α(100 ng/ml). RANTES  and eotaxin . Melatonin(10-10 to 10-6M) alone didn't change the expression of RNATES (204.97 ± 32.56 pg/ml) and eotaxin (55.28 ± 6.71 pg/ml). However, In the presence of TNF-α (100 ng/ml), melatonin promoted RANTES  and eotaxin  expression in a dose dependent manner in A549 cells . The increased release of RANTES and eotaxin in A549 cells by above treatment were further confirmed by both real-time RT-PCR and the ECA assay.TNF-α increased the expression of RANTES (307.84 ± 33.56 Taken together, our results suggested that melatonin might synergize with pro-inflammatory cytokines to modulate the asthma airway inflammation through promoting the expression of chemotaxins in lung epithelial cell. Regulated on Activation Normal T-cells Expressed and Secreted) are C-C chemotaxins that can recruit eosinophils to the airway in asthma[Eosinophils are known to be the important effector cells in asthmatic airway inflammations. Previouin asthma. A variein asthma.Pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1 are released in the early stage of allergic inflammation. In endothelial and epithelial cells, TNF-α induces an influx of eosinophils into tissues through the increased expression of adhesion molecules,7. AlthoMelatonin(N-acetyl-5-methoxytryptamine) is a key regulator of circadian rhythm homeostasis in humans,10. It aThe circadian variations of lung function in nocturnal asthma are associated with the increased airway inflammation during night. As a key regulator in human circadian rhythm homeostasis as well as an immunomodulator in allergic diseases, melatonin may regulate the circadian airway inflammation in asthma through modulating the expression of chemotaxins in the airway epithelial cells.In order to test this hypothesis, we conducted the present study to answer two questions. First, whether melatonin is able to up-regulate RANTES and eotaxin expression in the lung epithelia cell line-A549. Second, what is the combinatory effect of melatonin and TNF-α on RANTES and eotaxin expression and whether this effect increases the eosinophils chemotactic activity (ECA) released in A549. The answers to these questions might provide new insights into the pathophysiology of asthma.This study was approved by the medical ethics committee of the West China Hospital of Sichuan University. Informed consents were obtained from all subjects in the study.2 at 37°C in DMEM medium  supplemented with 10% heat-inactived fetal bovine serum (GIBCO BRL) and penicillin-streptomycin , at 1 × 106 cells/ml. A549 cells were then plated onto 6-well, flat-bottom tissue culture plates  at a density of 1 × 106 cells/ well in DMEM medium. The medium was changed every 2 d until the cells became confluent and then the cells were used for the experiments.A549 cells, human type II-like epithelial lung cells, were obtained from ATCC . The cells were cultured in tissue flasks incubated in 100% humidity and 5% CO-10, 10-9, 10-8, 10-7, 10-6M, the physiology concentration are 10-9 to 10-7 M during day and night[-10, 10-9, 10-8, 10-7, 10-6M) and TNF-α (100 ng/ml). The epithelial cell layers were then washed three times with Hanks' balanced salt solution (GIBCO BRL) and incubated for 48 h. Cell-free culture supernatants were collected. RANTES and eotaxin were assayed using enzyme-linked immunosorbent assay (ELISA) kits according to the instructions of the manufacturers. Assay kits for RANTES and eotaxin were purchased from R&D Systems , and the minimum detectable concentration of RANTES and eotaxin was 5 pg/ml. Experiments were performed at least three times with the similar results.As IL-1β and TNF-α have similar effect on the expression of many chemotaxins,15, we cand night)  is shown as the total number of migrated eosinophils counted in 10 high-power fields under a light microscope  at × 400 magnification.Eosinophil chemotaxis assay was performed as described previously. BrieflyData were expressed as means ± SD. Differences between groups were assessed by one-way ANOVA followed by the LDS significant difference test. A value of p < 0.05 was considered statistically significant.-10 to 10-6M. However, TNF-α induced RANTES release in A549 increased significantly by incubation with melatonin (from10-10 to 10-6M). Similarly, eotaxin released from A549 cells also increased significantly when the cells incubated with TNF-α; Melatonin alone had no effect on eotaxin released from A549 at dose range from10-10 to 10-6M. However, eotaxin released from A549 increased significantly when the cells incubated with melatonin and TNF-α . Melatonin alone didn't have this effect on A549 in dose from1000 ng/ml.-10, 10-9, 10-8, 10-7, 10-6M) and TNF-α (100 ng/ml). Melatonin alone did not change the RANTES and eotaxin mRNA expression in A549. TNF-α can promote the RANTES and eotaxin expression in A549 cells. When stimulated with TNF-α, melatonin synergistically increased the RANTES and eotaxin expression in a dose dependent manner  alone didn't have this effect. When stimulated with TNF-α (100 ng/ml) and melatonin, ECA released increased in A549 cells in a dose dependent manner , ECA released by A549 cells increased significantly. Melatonin  and the minimal lung function occurring near 4:00 AM (0400 hours). An episode of nocturnal asthma is characterized by an exaggeration in this normal variation in lung function from daytime to nighttime, with diurnal changes in the pulmonary function generally of > 15%. A recent study showed that the circadian variability in pulmonary function in asthma was related to changes in the airway eosinophils recruitment and activation. AlthougRANTES and eotaxin expression are regulated by two important transcriptional factors: active protein-1 (AP-1) and nuclear factor kappa B(NFκB). Benis et al found thThe role of melatonin as an immunomodulator is poorly understood and, in some cases, contradictory results have been reported. For example, Shafer's study showed that melatonin has no effect on the activity of stimulated macrophages. HoweverMelatonin alone did not change eotaxin and RANTES protein level and gene expression in A549 cells, and had no effect on ECA released by A549 cells. However, when A549 cells were stimulated with melatonin, together with TNF-α, the mRNA expression and protein release of eotaxin and RANTES increased significantly. This result suggested that combined with pro-inflammatory cytokines, melatonin may play a role in the airway inflammation through up-regulation of the eotaxin and RANTES expression in the lung epithelial cells.FML conceived of the experiment, carried out all experiments and prepared the manuscript. XJL conceived of the experiment and performed RNA extraction and real-time RT-PCR. SQL conceived of the experiment and assisted in collection and analysis of ELISA samples. CTL performed cell culture and provided expert advice and interpretation of the study's results. WZL participated in the study's design, coordination and final revisions of the manuscript. All authors read and approved the final manuscript.


Bacillus Calmette Guerin (BCG) lymphadenitis is a well known entity. Disseminated BCG infection usually presents as generalized lymphadenopathy, skin rash and hepatosplenomegaly and at times, can pose a diagnostic challenge to clinicians. There are only a few published studies on the cytological findings of BCG lymphadenitis. In this letter we report the fine needle aspiration cytology (FNAC) of BCG lymphadenitis clinically masquerading as Langerhans cell histiocytosis (LCH). FNA smears showed sheets of foamy macrophages and many polymorphs in a dirty necrotic background with many macrophages as well as polymorphs showing negatively stained rod like structures within their cytoplasm. Zeihl Neelson stain revealed that these cells were heavily loaded with acid fast bacilli (AFB). In the index case, AFB were also seen within the cytoplasm of polymorphs, which has not been documented earlier in the literature. Bacillus Calmette Guerin (BCG) vaccine has been included in the immunization programmes of the developing countries including India. Immunization with BCG is done on the first day of life. The most common response to BCG vaccine is sub clinical. However, some infants may develop regional lymphadenitis or a subcutaneous abscess at the vaccination site . DissemiEnlargement of regional lymph nodes is a well-known complication of BCG vaccination and occurs within 6 months of vaccination . It may


Sc. In a cell-based assay, quinacrine inhibits the conversion of normal host prion protein (PrPC) to PrPSc at a half-maximal concentration of 300 nM. While these data suggest that quinacrine may be beneficial in the treatment of prion disease, its penetration into brain tissue has not been extensively studied. If quinacrine penetrates brain tissue in concentrations exceeding that demonstrated for in vitro inhibition of PrPSc, it may be useful in the treatment of prion disease.Prion diseases are caused by the accumulation of an aberrantly folded isoform of the prion protein, designated PrPOral quinacrine at doses of 37.5 mg/kg/D and 75 mg/kg/D was administered to mice for 4 consecutive weeks. Plasma and tissue  samples were taken over 8 weeks: 4 weeks with treatment, and 4 weeks after treatment ended.Sc in cell culture. Particularly extensive distribution was observed in spleen (maximum of 100 μg/g) and liver (maximum of 400 μg/g) tissue.Quinacrine was demonstrated to penetrate rapidly into brain tissue, achieving concentrations up to 1500 ng/g, which is several-fold greater than that demonstrated to inhibit formation of PrPC) to PrPSc will be critical toward its potential benefit.The documented extensive brain tissue penetration is encouraging suggesting quinacrine might be useful in the treatment of prion disease. However, further clarification of the distribution of both intracellular and extracellular unbound quinacrine is needed. The relative importance of free quinacrine in these compartments upon the conversion of normal host prion protein (PrP While the precise site of antiprion action is unknown (intracellular versus extracellular) adequate quinacrine penetration into brain tissue might result in an effective agent for the treatment of prion diseases.Prion diseases, while rare, invariably result in fatal neurodegeneration. At present, no therapy has been proven to be useful in the treatment of prion disease. However, acridine and phenothiazine derivatives have been evaluated ,2 using 0 μg/ml) . HoweverThe objective of this study was to characterize the achievable plasma concentrations and associated tissue  penetration associated with quinacrine.ad lib as a chocolate flavored liquid diet. From weeks 5–8 animals were given regular feed without quinacrine. At weekly intervals, 3 animals of each strain were euthanized and tissues were collected for analysis.The protocol was approved by the institutional animal care and use committee. The Animal Facility of the Institute for Neurodegenerative Diseases provided animal samples from 2 different strains of mice, FVB and CD1. Twenty four animals of each strain  were fed 37.5 mg/kg/D and 24 animals of each strain were fed 75 mg/kg/D of oral quinacrine over a 4-week period (weeks 1–4), given Quinacrine dihydrochloride (purity: 98.6%) was purchased from Fluka, while sulfadimethoxine sodium salt (purity: 98%) which served as the internal standard (IS) was obtained from Sigma. Quinacrine stock solution was prepared as 1 mg/ml in methanol. Sulfadimethoxine stock solution was prepared as 1 mg/ml in 50% methanol. The working solutions were prepared by diluting the respective standard and control stock solutions with 50% methanol and 0.1% formic acid to 2 μg/ml and 100 ng/ml, respectively. All solutions were stored in a 4°C refrigerator in silinized brown glass containers.3OH/H2O/TFA (45:55:0.05) with 1 mM ammonium formate. The flow rate was 0.8 ml/min with 1/4 split into the mass spectrometer. The injection volume was 5–10 μl with a run time of 3.5 min.The HPLC system employed a Shimadzu LC-10 AD pump and a Waters intelligent Sample Processor 717 Plus autosampler/injector. A BDS Hypersil C18 column, 50 × 4.60 mm, 5 μm particle size was directly coupled to a Micromass Quattro LC Ultima triple quadrupole tandem mass spectrometer using electrospray ionization in positive ion mode. The sample cone voltage and collision energy were 25 V and 20 eV respectively for both quinacrine and the internal standard and the source block and desolvation temperature were 100°C and 400°C, respectively. The mass scanning mode employed multiple reaction monitoring (MRM) with the singly charged quinacrine ion selected at m/z 400.5 giving a fragment ion at m/z/142.0, and the internal standard at m/z 311.0→156.0. The mobile phase consisted of CHAll samples were stored at -70°C until analyzed. Each tissue sample was subjected to a specific method, as described below, for drug extraction and for the determination of concentration. All samples were analyzed by LC/MS/MS.Accuracy and precision was demonstrated throughout the working range with interday and intraday coefficient of variation and relative error <10%.Each plasma sample was thawed at room temperature for 10–15 min; then 20 μl of plasma was aliquotted to a new test tube. To each tube 200 μl of 70% acetonitrile solution, containing 0.1% formic acid and 50 ng/mL of internal standard, was added. The test tubes were vortexed at high speed for 1 min and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred into the autosampler for LC/MS/MS analysis. A set of standard curves with a duplicate set of quality control (QC) samples was generated for sample analysis.Prior to the in vivo study, the stability of quinacrine in mouse brain tissue was determined. Using 3 different doses of quinacrine , mouse brain tissue was soaked in 100% methanol for 7 days. Samples were taken on days 0,1,2,3, and 7 to analyze quinacrine concentrations using LC/MS/MS. Complete equilibration was observed by Day 1 and no degradation in quinacrine was noted through day 7.Brain samples were thawed (in plastic tubes) at room temperature for 15–20 min and weighed. To each sample 0.5 ml of 0.9% NaCl was added followed by incubation at room temperature for 1 h. For the standard curve and quality control samples, a brain tissue sample from an untreated mouse was spiked with different amounts of quinacrine and incubated at room temperature for 1 h. After the addition of 100 μl of 1 μg/ml internal standard solution in 50% methanol and 0.1% formic acid, 5 ml of 100% methanol was added into each sample and soaked at 4°C for two weeks. On the last day, 200 μl was aliquotted from each brain sample and placed into the autosampler for analysis via LC/MS/MS.Liver samples were thawed at room temperature for 15–30 min and weighed. Considering the increased size of the liver samples and that the increased connective tissue in liver samples could potentially prevent the complete distribution of quinacrine into methanol, homogenization was used for these samples. To each sample 100% methanol was added (10 ml/g of tissue) and the tissue was homogenized in ice water for 1 min at speed 3 . The internal standard  was added to 0.2 ml of each homogenized liver sample in a glass test tube. Samples were vortexed for 1 min, centrifuged at 3000 rpm for 10 min and 20 μl of each supernatant was transferred into a new test tube. Each sample was further diluted with 4 ml of 50% methanol, vortexed, and 200 μl was transferred to the autosampler for LC/MS/MS analysis. A set of standard curve with a duplicate set of QC samples was generated for sample analysis.Spleen sample preparation was similar to the preparation of brain samples using a methanol soak. Prior to the in vivo study, the stability of quinacrine in mouse spleen tissue was determined. Using 3 different doses of quinacrine , mouse spleen tissue was soaked in 100% methanol for 7 days. Samples were taken on days 0, 1, 2, 3, and 7 to analyze quinacrine concentrations using LC/MS/MS. Complete equilibration took place by Day 1 and no degradation in quinacrine was observed through day 7.Samples from the in vivo analysis were soaked for 14 days at 4°C. On day 14, 50 μl was aliquotted from each spleen sample and diluted with 1 ml of 50% methanol. Each sample was vortexed for 1 min and 200 μl was transferred to the autosampler for LC/MS/MS analysis. A set of standard curves with a duplicate set of QC samples was generated for sample analysis.The mass spectrum of quinacrine Q1) and its tandem spectrum Q3), showing the fragment ion selected for MRM, are shown in Figure  and its , showingIn plasma, quinacrine concentrations with the 37.5 mg/kg/D dose ranged from 75 to 175 ng/ml for both FVB and CD1 strains mice and remained at this level during the 4-week dosing interval. Once discontinued, quinacrine was completely eliminated from plasma, with no drug detectable within one week. At 75 mg/kg/D quinacrine, plasma concentrations reached >2 × those observed with the lower dose, averaging 300 to 400 ng/ml.Quinacrine levels in brain tissue for both mice strains were determined to be substantially greater than those achieved in plasma. Similar to observations with plasma, steady-state levels in the brain with the 37.5 mg/kg/D dose were achieved by the end of the first week, averaging 400 to 600 ng/g brain tissue. Figure th week. Similarly, spleen tissue levels in both mice strains were very elevated, averaging 5 to 10 μg/g with the 37.5 mg/kg/D dose and 40 to 100 μg/g with the 75 mg/kg/D dose. Figure The analysis of liver samples showed particularly high quinacrine levels, at many times those observed in plasma and brain. At the end of the first week of treatment with 37.5 mg/kg/D, steady-state levels of 70 to 90 μg/g were achieved in liver tissue in both mice strains, which remained throughout the course of treatment. Figure Of note, in contrast with plasma and brain tissue, in which dose dependency was somewhat linear, increasing quinacrine doses in spleen and liver were associated with substantially greater tissue levels. In addition to the isolation of quinacrine, numerous metabolites were identified in all tested tissue samples (data not shown).The current study is the first comprehensive evaluation of quinacrine distribution in plasma and brain tissue. Shannon et al. evaluated the pharmacokinetics of quinacrine in the treatment of malaria . Using ain vitro study, the EC50 for quinacrine was 300 nM, which approximates 120 ng/ml. We report here that quinacrine levels in brain tissue averaged 400 to 600 ng/g with the 37.5 mg/kg/D dose and 1500 ng/g with the 75 mg/kg/D dose, concentrations that exceed the in vitro EC50 by 3- to 10-fold.The concentrations we report in brain in terms of concentration/gm of tissue are several-fold greater than that shown for effective antiprion activity in ScN2a cells [in vitro, including clearance of PrPSc (5) and/or inhibition of PrPSc formation [in vitro benefit from quinacrine, found the drug to be ineffective in the animal model under the conditions employed [Others have reported the antiprion function of quinacrine ormation . Barret employed . In contemployed ,8.50, it important to note critical limitations in their interpretation. Quinacrine has been determined to be highly protein-bound [While the current study has confirmed brain tissue concentrations in excess of the reported ECin-bound , measurein-bound . The brain-bound  evaluatein vitro antiprion activity similar to or greater than that of the parent compound. These tests warrant further evaluation.In addition to steady-state levels of quinacrine, we observed a number of quinacrine metabolites in all tissue samples studied, which raises the possibility that these metabolites may have in vitro activity, quinacrine may be a useful agent in the treatment of prion disease. However, more detailed tissue distribution analyses linked with the critical sites of prion action are warranted.Based upon its oral bioavailability, favorable tissue distribution characteristics, and The author(s) declare that they have no competing interests.LY, YH, ETL developed the assay, performed sample analyses, contributed to the writing of the paper. PL, GL, MB, CR developed the protocol and performed the animal model experiments. SBP conceived of the study and participated in its coordination. BJG drafted the manuscript, participated in its coordination and mentored LY. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: