Recent findings suggest a role of oxidative stress in the pathogenesis of Behcet's disease (BD), but the utility of oxidative stress-associated assays in offering diagnostic information or in the monitoring of disease activity is largely unassessed.We aimed to measure oxidative and inflammatory markers, along with the markers of reactive nitrogen species, S-nitrosothiols and 3-nitrotyrosine, in BD patients (n = 100) and healthy volunteers (n = 50). These markers were evaluated in regard to their role in the pathogenesis of BD as well as their relation to clinical presentation, disease activity and duration.Median values for erythrocyte sedimentation rate (ESR), C-reactive protein, leukocyte count, and IL-18 levels, as well as myeloperoxidase (MPO) activity, were statistically higher in the patient group compared to controls. Some inflammation markers  were statistically higher (p < 0.05) in the active period. In contrast, oxidative stress-associated measures , revealed no statistically significant differences between the median values in BD patients versus healthy control subjects , nor was there any difference in median levels of these oxidative stress markers in active disease versus disease remission. S-nitrosothiols and 3-nitrotyrosine were undetectable in BD plasma.The application of oxidative stress-associated measures to BD blood samples offered no supplemental diagnostic or disease activity information to that provided by standard laboratory measures of inflammation. S-nitrosothiols and 3-nitrotyrosine appeared not to be markers for active BD; thus the search for biochemical markers that will indicate the active period should be continued with larger studies. Behcet's disease (BD) is a multisystem, chronic inflammatory, relapsing disorder that is characterized by oral and genital ulcerations and ocular, arthritic, vascular and neurological involvement. Its diagnosis is generally clinical with a higher endemic rate in Turkey, Iraq, Iran, Korea and Japan. There is no biochemical parameter showing the active phase of the disease, other than nonspecific tests of CRP, erythrocyte sedimentation rate and increased leukocyte count . Althoug2-), nitrate (NO3-), S-nitrosothiols (RSNOs) and 3-nitrotyrosine (3-NT). Recently increases in nitrite and nitrate concentrations have been shown in BD patients and were associated with disease activity [Vascular inflammation is one of the important features of BD ,4. NO isactivity ,8. HowevIn the present study we assessed, in BD patients, standard measures of inflammation , proinflammatory cytokine levels , Trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP), total antioxidant enzyme activity (AOA), catalase (CAT), superoxide dismutase (SOD) and myeloperoxidase (MPO) activities. RSNO and 3-NT levels were also investigated. All these markers were evaluated in regard to their relation to the disease activity and clinical presentation in BD patients, as well as their role in the pathogenesis of BD. It was anticipated that the results could help to identify a parameter that can be helpful in clinical practice to determine disease activity.One hundred patients with BD , and 50 sex-matched  and age-matched (38.0 ± 6.6 years) healthy volunteers as control subjects were included in the study. All BD patients fulfilled the diagnostic criteria of the International Study Group for BD [Blood samples were collected in heparin-containing tubes, EDTA-containing tubes and anticoagulant-free tubes after an overnight fast. Plasma and serum were separated immediately. Erythrocyte lysates were prepared  and polyTNF-α and IL-18 levels were measured using enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturers' instructions. AOA  was determined spectrophotometrically . A solutTEAC was determined spectrophotometrically . ABTS 2,3 (20 mM) was added to the serum sample and incubated at room temperature for 30 min. Readings were done at 620 nm.FRAP was determined spectrophotometrically . A mixin• released was measured in the presence of the spin trap complex (MGD)2- Fe2+, in which MGD is N-methyl-D-glucamine dithiocarbamate. In order to concentrate S-nitrosothiols and further improve the detection limit for S-nitrosothiols, the EPR method was modified using different techniques such as ultrafiltration (Whatman ultrafiltration devices), incubation of the samples at different temperatures  and different time intervals  and enzymatic proteolysis was carried out using proteinase K and pronase. Each procedure was performed several times.RSNO concentrations were measured using a previously described EPR spectrometry method  in 15 plNitrotyrosine (3-NT) concentrations were investigated with an enzyme-linked immunosorbent assay using a commercial kit . This 3-NT ELISA test kit is based on the sandwich method. All samples were diluted 1:20 prior to conducting the assay.o-dianisidine (in phosphate buffer). After addition of hydrogen peroxide solution (20 mM), absorbances of samples were recorded at 492 nm with a microplate reader during 3 minutes with 15 s intervals. Protein concentration was measured according to Lowry's method [Heparinized blood samples were diluted with an equal volume of phosphate buffered saline (PBS) and then 4 mL of Ficoll Hypaque (Sigma-Aldrich-1077) was added to each sample and centrifuged for 20 min at 1200 g. After centrifugation, the leukocyte pellet was taken and mixed with 0.9% NaCl. This mixture was centrifuged for 5 min at 1500 g. The cell pellet that contained the PMN was then resuspended in phosphate buffer and stored at -80°C until analysis. Leukocyte MPO activities were measured according to the modified method of Grisham et al. [s method .After separation of plasma, the packed erythrocytes were washed twice with 9 g⁄L NaCl and haemolysed with ice-cold water  Erythrocyte SOD (eSOD), eythrocyte catalase (eCAT) and erythrocyte TBARS (e-TBARS) activities were determined immediately in haemolysates. The haemoglobin values were measured by Drabkin's method .Erythrocyte SOD (eSOD) activity was measured spectrophotometrically using a commercial kit . This method is based on the use of xanthine and xanthine oxidase (XOD) to generate superoxide radicals which react with a chromogen solution to form a red formazan dye. SOD activity is then measured by the degree of inhibition of this reaction by means of the decrease in absorbance at 490 nm. This result was normalised to the Hb concentration, and SOD activity was expressed as units/g Hb.2O2 was recorded spectrophotometrically at 240 nm. One unit of CAT was defined as the amount of enzyme that decomposes 1 μm0l H2O2 /min.Erythrocyte CAT (eCAT) activities were determined as described by Sozmen et al. , in whicPolyunsaturated fatty acids are oxidized due to increased oxidant stress resulting in high plasma TBARS levels. Erythrocyte TBARS (eTBARS) levels were determined according to the method previously reported . After dAll statistical analyses were performed using GraphPad Prism v5.04. Data were not normally distributed, and were expressed as median values with interquartile ranges. Data were analyzed by the Kruskal-Wallis test, and Mann-Whitney U test to compare the median values of two groups. Correlations between variables were tested using Spearman's rank correlation. P values less than or equal to 0.05 were evaluated as statistically significant.The results of all parameters, when comparing the group of healthy subjects (n = 50) with all BD patients analysed n = 100) are given in Table 00 are giResults of all parameters analysed in regard to active n = 26) and remission (n = 74) phases of the disease are given in Table 6 and remPositive correlations were found between the concentrations of established markers of inflammation  in patients with BD. R values for the correlations of all markers are given in Figure Neither inflammatory parameters nor oxidative stress-associated parameters showed any statistically significant differences when patients were evaluated in regard to ocular, vascular, articular or mucocutaneous involvement (data not shown). There were also no significant correlations between any of the measured parameters and disease duration.In the present study, we explored the potential utility of measures of oxidative stress in monitoring disease activity, and in the diagnosis of complications, in BD patients. We applied a broad panel of widely adopted oxidative stress-associated measures to plasma, serum and erythrocyte samples. These measures were carried out in conjunction with both standard laboratory assays of the inflammatory response, such as CRP and ESR, and clinical assessments. We aimed to assess the additional clinical information that measures of oxidative stress might provide in terms of disease activity or the diagnosis of complications.As expected, the group of BD patients showed evidence of a substantial inflammatory response, as indicated by high median levels of CRP, ESR, blood leukocyte count, MPO and IL-18, compared with healthy, age- and sex-matched control individuals. Moreover, when the BD group was subdivided into active disease and remission subgroups, some of these inflammatory measures were clearly elevated in the active disease subgroup compared with the remission subgroup. In contrast - and as expected - measures of inflammation provided no obvious indication of clinical complications or disease duration. Thus, our findings with standard inflammatory measures are consistent with their known nonspecific increase in BD. None of these markers can be defined to be a specific measure for disease activity or an indicator of any complication. TNF-α is an important proinflammatory cytokine and it has been implicated in the pathogenesis of many inflammatory and autoimmune diseases such as sepsis, inflammatory intestinal disease  and RA . SimilarRecently, Musabak and colleagues  and Lee Increased neutrophil activation is observed in BD patients and this can directly be determined with MPO activities. In accordance with previous studies ,26, leukThe strong negative correlation between leukocyte MPO activity and FRAP  in this study shows the importance of the oxidant/antioxidant balance in BD patients. In inflammatory diseases such as BD, MPO production from activated neutrophils is increased, which could be consistent with oxidative stress. Also consistent with oxidative stress, plasma antioxidant activity in BD is reportedly decreased .Although increased oxidative stress has been suggested to be another hallmark of BD ,28,29, tIn the present study, when patients were grouped for ocular, vascular, articular and mucocutaneous involvements, the measured parameters did not show statistically significant differences between groups. In contrast, Taysi et al.  have shoIt has been proposed that NO plays a role in the pathogenesis of BD. Laroux et al.  showed tRecently 3-NT, which is produced by nitration of tyrosine by reactive nitrogen species has been suggested to be a useful marker for inflammation and NO-mediated tissue injury ,39. BothIn conclusion, in agreement with previous studies ,23,25,27The authors declare that they have no competing interests.YDA, FSG, EYS, participated in the study design and experimental work and drafting the manuscript, ET, IK and PGW, participated in the experimental work and drafting the manuscript, KA and GK provided the patients and participated in drafting of the manuscript. All authors read and approved the final manuscript.


This study in patients with rheumatoid arthritis (RA) treated with infliximab describes prospectively the course of (anti)infliximab levels within an infusioncycle to assess at what moment patients develop low/no infliximab trough levels and/or detectable anti-infliximab levels.Infliximab treated RA patients were included in this descriptive open-label cohort study. During one infusioncycle (anti-)infliximab levels were assessed just before and one hour after infusion, and subsequently at 50%, 75% and at the end of the infusioncycle (pre-infusion).27 patients were included. The median infliximab levels decreased from 77.0 mg/l (p25-p75: 65-89) one hour after the infusion to pre-infusion levels of 0.0 mg/l (p25-p75: 0.0-3.1). In 7 (26%) patients pre-infusion anti-infliximab antibodies were detected; these antibodies were already present halfway through the infusioncycle in 5 of the 7 individuals. Patients with detectable pre-infusion anti-infliximab antibodies have significantly more often low/no infliximab levels (< 1 mg/l) halfway trough the infusioncycle (in 5/7 patients) compared to patients without detectable pre-infusion anti-infliximab antibodies .Most anti-infliximab forming patients have detectable anti-infliximab antibodies halfway through an infusioncycle, which implies that these patients are exposed to nontherapeutical infliximab levels during more than halve of their infusion cycle. As none of the patients without anti-infliximab antibodies had no/low-infliximab levels halfway through the infusioncycle, the presence of pre-infusion anti-infliximab antibodies seems a sensitive and specific predictor for no/low infliximab-levels Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by inflammation of synovial tissue leading to progressive articular cartilage and bone destruction. To prevent progression of joint damage and functional disability, early introduction of effective disease modifying antirheumatic drugs (DMARDs) is considered to be essential in the treatment of patients with rheumatoid arthritis (RA). Besides traditional DMARDS like methotrexate, tumour necrosis factor (TNF) antagonists have been proven to reduce disease activity, suppress radiographic joint damage and decrease functional disability in patients with recent onset ,2 and esHowever, these results also implicate that up to 60% of patients with RA do not reach the clinical relevant 50% improvement. Therefore, non-responders (both primary as secondary non-responders) should be identified as early as possible. Firstly, a shorter period of high disease activity minimises chances of joint destruction ,8.Because valid prediction models are not available at this point, close monitoring of individual disease activity and adapting the treatment (dose) is the first available step to improve the efficacy of RA-therapy ,10. AlthPharmacokinetic data with infliximab indeed show that some patients achieve improvement and low disease activity during therapy with infliximab, although this response could most likely not be attributed to infliximab as these patients had no- or low-infliximab trough levels. These reduced levels could partially be explained by the formation of human antichimeric antibodies (HACAs) which occurs in 8% to 43% of the RA patients -14. The Clinically, it is relevant to know whether patients with serum trough anti-infliximab antibodies also have these antibodies present early in a treatment cycle or whether they appear only at the end of a treatment cycle. Patients with "early" anti-infliximab detectable antibody formation would have a long window wit nontherapeutical levels of infliximab. The alternative scenario, appearance of HACA's predominately at the end of the infusion cycle would be less important as adequate infliximab levels would be present during the majority of time between infusions. However, until now, it is unknown what the relationship is between trough anti-infliximab antibody levels and (anti-)infliximab antibody throughout the treatment cycle.This study therefore prospectively describes the course of (anti)infliximab levels within an infusioncycle in patients with rheumatoid arthritis in order to assess at what moment patients develop low/no infliximab trough levels and/or detectable anti-infliximab levels.Patients with RA, according to the ACR 1987 revised criteria, treated at the Sint Maartenskliniek  for at least 3 months with 3 mg/kg infliximab (irrespective of dose frequency) were included in this observational, descriptive open-label pharmacokinetic cohort study. No other inclusion or exclusion criteria were used. In the Sint Maartenskliniek all RA patients receive 3 mg/kg infliximab, with dose intervals adjusted to patient's disease activity. Patients were treated according to the local disease activity guided protocol, When a patient does not reach low disease activity on 3 mg/kg/4 wks the patient is switched to another DMARD or biological.Patients were enrolled between February and April 2008. Ethical approval was obtained from the Ethics Committee Nijmegen-Arnhem and all participants gave written informed consent before screening. A standardized chart review form was used to collect data on demographics, previous medication and clinical benefit of infliximab.Serum samples for the measurement of (anti-)infliximab levels were collected during one treatment cycle at five time-points: one hour prior to the first infusion, one hour after the infusion, at 50% and 75% of the infusioncycle, and just before the next infusion. The Disease Activity Score (DAS28) was assessed at the same time points excluding the time point just after the first infusion .Infliximab- and anti-infliximab antibody levels in serum were determined by an enzyme-linked immunosorbent assay and a radioimmunoassay respectively ,15. We c2-tests to evaluate differences in proportions, and Student's t-tests to evaluate differences in means. Non-parametric variables were analyzed using the two-sample paired sign test. The threshold for significance was set at p = 0.05. To test whether the relationship between the formation of anti-infliximab antibodies and inflximab serum trough levels is influenced by confounders, the baseline variables of anti-infliximab formers and non-formers were compared in a univariate analysis and in a multivariate logistic association model using the presence of no/low-infliximablevels as dependent variable, antibody formation as central determinant and all variables with a significant univariate association with both the dependent and independent variable of p < 0.1 as potential confounder. Confounders were kept in the model when the beta for the central determinant changed with > 10% after addition of the confounderDescriptive statistics were provided using mean (+/- SD) or median (p25-p75) values depending on the (non-) parametric distribution of the variables. We used Mantel-Haenszel xTwenty-seven patients were enrolled in the study; their demographic and clinical data at baseline are summarized in table Table Anti-infliximab antibodies were significantly (p < 0.01) more frequent in rheumatoid factor negative patients (4 of 6 patients (67%)) compared to rheumatoid factor positive patients (3 of 21 patients (14%)). Patients with detectable antibodies were also significantly younger (mean age: 53.5 (± 13.6) years) compared to patients without detectable antibodies (mean age: 64.5 (± 6.7) years; p < 0.01). Other baseline variables were not associated with the formation of anti-infliximab antibodies.Concomitant DMARDs were used in 6 (86%) of the patients with detectable anti-infliximab antibodies and in 17 (85%) of patients without anti-infliximab antibodies and remission or low disease activity was present in 4(57%) of the patients with detectable anti-infliximab antibodies compared to 9 (45%) patients without detectable antibodies. A logistic regression association model showed that age and the presence of rheumatoid factor did not act as confounder on the relation between anti-infliximab antibodies formation and infliximab serum trough levels.Disease activity (DAS28) and EULAR-disease activity classification percentages are shown in table Our results indicate that anti-infliximab antibodies are frequently found in patients with low and moderate disease activity and that these antibodies are already detectable in most of these patients halfway through an infusioncycle. This implies that the presence of anti-infliximab antibodies at the end of an infusioncycle seems a good predictor for low infliximab-levels throughout most of the infusioncycle.The internal validity of our study appears to be adequate. Although the number of patients in the present study is limited, this does not necessarily hamper the validity of this study as a higher number of patients would only increase precision. Confounding was considered because the presence of rheumatoid factor was related to both anti-infliximab formation and no/low infliximab levels, however a multivariate regression model could not demonstrate confounding.The design of this study is not suitable to draw conclusions about the correlation between pharmacokinetic- ((anti)-infliximab levels) and the pharmacodynamic- (disease activity) parameters. Patients were treated according to the local disease activity guided protocol, which automatically excluded the majority of non-responders in this observational cohort. This could lead to a selected study population, in which pharmacokinetic parameters could not be correlated with non-response. Due to this selection bias, this study also can not be used to assess possible risk factors for the formation of anti-infliximab antibodies.This study has two significant clinical implications. First, we found that in patients with low infliximab trough levels, the presence of serum trough anti-infliximab anti-bodies could be a specific and sensitive indicator for absence of serum infliximab level during at least half of the infusioncycle.Secondly, our finding that one fifth of the patients treated with infliximab have already non/low infliximab-levels halfway through the infusion suggests that these patients do not benefit from infliximab at all. One could argue that the effect of infliximab therapy may be  determined by peak levels or time integrated AUC rather than by minimal inhibitory concentration (MIC), implying that measuring serum trough levels is not indicative for clinical effect. This is however not likely as subcutaneous anti-TNF agents demonstrate similar efficacy without high peak serum levels . HoweverIn this study, we found an increase in disease activity at the end of the infusion interval. This could lead to an underestimation of the effect of infliximab when disease activity is only measured just before infusion. However, previous research demonstrated that fluctuations in disease activity also importantly and independently contribute to radiologic progression . This imThis study demonstrates that a substantial proportion of RA-patients treated with infliximab are already exposed to no/low-infliximab levels during more than halve of their infusion cycle. The presence of pre-infusion anti-infliximab antibodies could be used as a sensitive and specific predictor for no/low infliximab-levels halfway the infusioncycle.The authors declare that they have no competing interests.BvdB, AdB, GW, YH, PR, BB and FH conceived and designed the study. All authors were involved in carrying out the study and interpreting the results. BvdB performed the statistical analysis and drafted the manuscript. All authors read, commented on and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/12/prepub


We analysed whether a combination of bone- and fall-related risk factors (RFs) in addition to a recent non-vertebral fracture (NVF) contributed to subsequent NVF risk and mortality during 2-years in patients who were offered fall and fracture prevention according to Dutch fracture- and fall-prevention guidelines.834 consecutive patients aged ≥50 years with a recent NVF who were included. We compared subgroups of patients according to the presence of bone RFs and/or fall RFs . Univariable and multivariable Cox regression analyses were performed adjusted for age, sex and baseline fracture location (major or minor).57 (6.8%) had a subsequent NVF and 29 (3.5%) died within 2-years. Univariable Cox regression analysis showed that patients with the combination of bone and fall RFs had a 99% higher risk in subsequent fracture risk compared to all others (Hazard Ratio (HR) 1.99; 95% Confidence Interval (CI) 1.18-3.36) Multivariable analyses this was borderline not significant . No significant differences in mortality were found between the groups.Evaluation of fall RFs contributes to identifying patients with bone RFs at highest immediate risk of subsequent NVF in spite of guideline-based treatment. It should be further studied whether earlier and immediate prevention following a NVF can decrease fracture risk in patients with a combination of bone and fall RFs. For persons above fifty years of age, a history of fracture doubles the risk of a subsequent fracture. This riFracture risk reduction has only been shown with specific anti-osteoporosis medication such as bisphosphonates, denosumab, raloxifene and recombinant PTH,10-17. FIn the field of post fracture care, a Fracture Liaison Service is one of the initiatives to integrate evaluation of bone- and fall-related risk factors in patients attending the hospital with a recent clinical fracture.The aim of the Fracture Liaison Service is to evaluate bone- and fall-related risk factors, to initiate fall prevention programs, adequate calcium and vitamin D supplementation and specific anti-osteoporosis medication when needed in order to reduce subsequent falls, fractures and mortality,11,19-21In this study, patients with a recent clinical fracture were assessed at the FLS at Maastricht University Medical Center for bone- and fall related risk factors and we hypothesised that over a 2-year follow-up period the subsequent fracture risk and mortality would be highest in patients with a combination of bone- and fall-related risk factors, even though these patients received anti-osteoporosis treatment and/or fall prevention.The Fracture Liaison Service is a collaboration between the department of surgery, orthopaedics and internal medicine (rheumatology and endocrinology) and is based on the consensus guideline osteoporosis of the Dutch Institute for Health Care Improvement (CBO). The FraBetween September 2004 and September 2006 all consecutive patients older than 50 years with a recent non-vertebral fracture, who entered the level one trauma centre in the south of the Netherlands were invited to participate. Patients with pathological or vertebral fractures or living outside the postal area were excluded. All patients were prospectively followed for two years. The hospital database was searched for radiographically confirmed first and subsequent NVF, fracture location and date of occurrence. All NVFs (baseline and subsequent) were categorised according to International Classification of Disease (ICD)-9 and then pooled into 2 groups: major , and minor  fractures. All groThe study was approved by the medical ethical committee of the hospital (MEC 03–194).All patients, who were able and agreed to evaluate their fracture risk assessment, were invited to attend the Fracture Liaison Service. Medical history, current and past medication use, living situation, conditions concerning the occurrence of the fracture, dietary calcium and vitamin D intake were assessed. Additionally, bone- and fall- related risk factors were systematically assessed, and bone mineral density was measured by dual energy X-ray absorptiometry  at the lumbar spine and femoral neck. Based on criteria of the World Health Organisation osteoporosis was classified as T-score of ≤ −2.5, osteopenia as T-score between −1.0 and −2.5, and normal BMD as T-score of >−1.0.According to the national osteoporosis guideline the following bone- and fall-related risk factors were evaluated: a history of clinical fracture after the age of 50 years, family history of hip fracture, low body weight (<60 kg), glucocorticoid use and immobility (<4 hours per day). VertebrAccording to the national guideline on fall prevention the following fall-related risk factors were evaluated: a previous falls in the last 12 months , the presence of Parkinson’s disease, current use of psycho-active medication, urinary incontinence (defined as involuntary loss of urine) and articular complaints. Additionally, the Groningen Activity Restriction Scale (GARS) was used to estimate the disability in activities of daily living (ADL). PatientAccording to the Dutch guidelines on osteoporosis and fall prevention patients started with Calcium and Vitamin D or a bisphosphonate in the presence of osteoporosis.For the analyses, patients were categorised into subgroups according to the presence, combination or absence of bone- and fall-related risk factors: (1) patients with only bone-related risk factors, (2) patients with combination of bone- and fall-related risk factors, (3) patients with only fall-related risk factors, and (4) patients without bone- or fall-related risk factors. The rationale behind these groups is that there is a known treatable risk factor in group 1 and 2, but not in group 3 because in fracture prevention only bone targeted therapy has shown to reduce fracture risk and not fall targeted therapies.Differences between the groups were analysed using the chi-square or Fisher’s exact test for categorical variables. ANOVA and independent samples t-test for numerical variables. Kaplan-Meier and multivariable Cox regression analyses were performed using subsequent fracture and mortality as dependent variables (events), adjusted for age, sex and baseline fracture location (major/minor). For subsequent fracture as dependent variable, follow-up time started at time of current fracture (time = 0) and was defined as time between current fracture and subsequent fracture (= event), death or end of 2-year follow-up period (= censored). For mortality, follow-up time was calculated as time between current fracture and death (= event) or end of 2-year follow-up period (= censored). Schoenfeld residuals were used to check the proportional hazards assumption and, if violated, time-dependent Cox regression was used. Linearity was checked for continuous variables and, if violated, centered quadratic terms were included. A two-sided p-value ≤ 0.05 was considered statistically significant. All analyses were performed using SPSS for Mac .In total 834 patients with a NVF were included. Fifty-seven (6.8%) patients sustained a subsequent NVF and 29 (3.5%) died within two years.Table Table Univariable Cox analysis showed that patients with a combination of risk factors had a significantly higher subsequent fracture risk than all other patients   or multivariable analyses . However, the plot of the Kaplan-Meier curves indicated that the subsequent fractures occurred immediately after the current fracture in the combination group, while this was not the case for the patients with only bone-related risk factors . In the multivariable model, the subsequent fracture risk was no longer significantly higher .No significant differences were found between the groups on subsequent fracture risk within 2-years after univariable  and multivariable cox regression analyses .Within two years after baseline fracture 26 patients were deceased (3.5%). In univariable Cox regression analysis, no significant difference was found between patients with a combination of bone- and fall-related risk factors (group 2) and all other groups, as well as between group 2 and all groups separately. Due to the small number of deceased patients in the present study, no multivariable regression analyses were performed. However, this only reflects the very selected nature of our cohort, as the majority of patients were still alive within 2 years after their baseline fracture.The subsequent non-vertebral fracture risk in patients with a combination of bone- and fall-related risk factors was nearly double the risk of all other patients in univariable analysis (HR 1.99 (95% CI: 1.18-3.36). In multivariable analysis this tendency was also shown, but it did not reach significance . This is partly explained by the low event rate. There was a time-dependency when comparing patients with a combination of risk factors to patients with only bone-related risk factors (data not shown). This indicates that, in spite of Fracture Liaison Service assessment, patients with a combination of risk factors still had a high fracture risk at short term. Many studies have shown that the risk of subsequent fracture is highest immediately after a fracture, such as repeat vertebral, hip and non-vertebral fractures,23. HoweThere was a time-dependency with regard to subsequent fracture risk in patients who had both bone- and fall-related risk factors with the highest risk immediately after the initial fracture. This might be caused by (new) falls and, since the bone is already vulnerable due to the initial fracture, subsequent fractures mostly occur in this subgroup short-term after the initial fracture.Therefore, patients with a combination of bone- and fall-related risk factors, are definitely candidates for immediate fracture prevention combined with fall prevention.However, of the patients without additional risk factors 6.1% had a subsequent fracture within 2-years of follow-up. Presumably other risk factors that are not captured by the bone- and fall- related risk assessment in this study play a role in the occurrence of subsequent fractures. On the other hand, all patients in this study had a recent fracture, which is by itself a major independent risk factor for subsequent fractures. As a consequence, this might imply that all patients with an initial NVF, whether or not additional risk factors are present, deserve subsequent fracture prevention regardless of other risk factors. This is also proposed in the UK guideline.No difference was found between the groups for mortality rate. This could be the result of the low mortality rate (n=29) in this study in combination with the relatively short follow-up period (two years). A study with a longer follow-up period in a larger population is necessary to further study these findings. Treatment of osteoporosis with bisphosphonates might reduce mortality rate. The mechanism by which this operates remains unclear, but its effect on subsequent fracture risk or on extra skeletal sites might be two possibilities,26,27. IThe strength of this study is that all consecutive patients with a recent non-vertebral fracture who attended the Fracture Liaison Service were included in the analyses. Furthermore, a dedicated fracture nurse performed the assessments of well-defined clinical, bone- and fall-related risk factors according to the national Osteoporosis and Fall Prevention Guidelines,21.A limitation of this study is that there are no data on prescription and adherence of the proposed osteoporosis treatment and fall prevention. Persistence might be low, as has been shown in a recent publication for oral anti-osteoporosis medications in the Netherlands. Due to In patients with a recent non-vertebral fracture who had a combination of bone- and fall-related risk factors, the risk of subsequent fractures was almost doubled compared with the other patients. These patients also had a higher subsequent fracture risk immediately after the initial fracture compared with patients in whom only bone-related risk factors were present.Therefore, not only bone- but also fall-related risk factors should be assessed in order to identify patients at highest immediate risk of subsequent NVF.The authors declare that they have no competing interests.KH participated in the design of the study, performed the data acquisition and statistical analyses. TG, PG, BW participated in the design of the study, and interpretation of analyses and data. SH, JB, PW, PB participated in the design of the study and interpretation of data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/121/prepub


Bladder radiotherapy is a management option for carefully selected patients with muscle-invasive bladder cancer. However, the inability to visualize the tumor site during treatment and normal bladder movement limits targeting accuracy and increases collateral radiation. A means to accurately and reliably target the bladder during radiotherapy is needed.Eighteen consecutive patients with muscle-invasive bladder cancer (T1–T4) elected bladder-preserving treatment with maximal transurethral resection (TUR), radiation and concurrent chemotherapy. All underwent endoscopic placement of 24-K gold fiducial markers modified with micro-tines  into healthy submucosa 5-10 mm. from the resection margin, using custom-made coaxial needles. Marker migration was assessed for with intra-op bladder-filling cystogram and measurement of distance between markers. Set-up error and marker retention through completion of radiotherapy was confirmed by on-table portal imaging.before starting treatment; 16/18 completed chemo-radiotherapy. All (100%) markers were visible with all on-table , fluoroscopy, plain-film, and CT-scan imaging. In two patients, 1 of 4 markers placed at the tumor site fell-out (voided) during the second half of radiotherapy. All other markers  were present through the end of radio-therapy. No intraoperative  or post-operative complications  occurred. Use of micro-tined fiducial tumor-site markers afforded a 2 to 6-fold reduction in bladder-area targeted with high-dose radiation.Between 1/2007 and 7/2012, a total of 89 markers (3–5 per tumor site) were placed into 18 patients of mean age 73.6 years. Two patients elected cystectomy Placement of the micro-tined fiducial markers into the bladder was feasible and associated with excellent retention-rate and no complications. All markers were well-visualized during radiotherapy with all imaging modalities. Bladder fiducial markers improve targeting accuracy, and may increase treatment efficacy and reduce morbidity from collateral radiation. Radical cystectomy with urinary diversion remains the gold-standard treatment for localized muscle-invasive bladder cancer. However, bladder preservation with tri-modality therapy  remains an alternative for patients who have been thoroughly counseled regarding the attendant risks and benefits of all management options, including radical cystectomy Two key challenges to radiotherapy targeting accuracy and precision are that the location of the bladder moves constantly, and, that the tumor-site cannot be identified using CT-scan imaging or on-table imaging. Consequently, the location of the bladder tumor site during treatment cannot be predicted by the planning-CT.essential to improve clinical outcomes using radiotherapy for bladder cancer. The inability to localize the bladder walls at time of daily treatment using current “on table” imaging modalities , results in: 1. Under-treatment because the entire target (bladder), or a portion (bladder tumor site), are not treated consistently throughout the  4–6 weeks of radiotherapy, and 2. Over-treatment: delivery of radiation to collateral non-bladder tissues, or, delivery of high-dose radiation to the bladder outside of the tumor site target area.The need to improve the radio-therapeutic window is Twenty-Four karat gold prostate fiducial markers (placed into the apex and base of the prostate gland) have been shown to significantly improve targeting and decrease collateral radiation during radiotherapy for prostate cancer Beginning in January 2007, we designed a fiducial marker and placement protocol to mark the tumor site in 15 consecutive patients with localized muscle invasive (≥T1NxM0) transitional cell bladder cancer. Patients were thoroughly counseled regarding the risks and benefits of all management options, including the gold-standard– radical cystectomy and urinary diversion, in addition to bladder preserving multi-modality treatment. All markers were placed endoscopically into intact bladder submucosa surrounding the tumor resection site.The present work is a feasibility study, and describes our fiducial marker design, placement technique, and outcomes of our early experience.All patients gave written informed consent to undergo treatment and participate in this study, and approval for this study was obtained from our institution's  Committee for Human Research.Between January 2007 and July 2012, a total of 18 consecutive patients with focal, T2, NX, M0 urothelial carcinoma, elected bladder-preserving multi-modality treatment and underwent endoscopic placement of fiducial markers into the bladder wall . A total of 89 fiducial markers were placed into healthy bladder submucosa ∼0.5 cm. from the resection margin.micro-tines, and was re-sterilized before placement. . Each was modified in our laboratory to have four lengthwise rows of acement. Figure 1For the most recent 5 patients in our series, following marker placement at the tumor resection site, fiducial markers were also placed at the anatomic boundaries of the bladder , under combined direct and fluoroscopic guidance.diluted contrast/saline solution, to allow visualization of the bladder lumen margins, a cystoscope, delivery needle, and fiducial markers, under fluoroscopy. Care was taken to ensure that the C-arm fluoroscope remained fixed in place relative to the patient at all times. The cystoscope was then inserted into the bladder, and under fluoroscopic guidance, the tip was positioned at the desired anatomic margins . A single fiducial marker was deployed at each site under fluoroscopy.Technique: A 16 Fr. urethral catheter was placed per urethra. The bladder was filled with 60 ml. of For the first 12/18 patients in our series, all underwent intra-operative Fluoroscopy and post-operative pelvis pelvic X-Ray imaging to confirm placement and location of all markers. Figure 4In the last 6 patients, to confirm that the markers tracked the bladder wall throughout filling/emptying and did not migrate intraoperatively, we also performed an intra-operative contrast filling study under C-arm fluoroscopy. The C-arm was fixed in place over the patient's pelvis and the bladder was imaged empty and then during filling and emptying . A spot fluoroscopic image was taken after each 60 ml. change (+/−) in volume. This was performed first with saline, followed by a repeat series using diluted contrast Figure 5For each bladder filing volume (i.e. 0→60→120→180→240→240→180→120→60→0 ml.), we compared the location of each marker in the contrast-filled versus saline-filled bladder image  to measure the distance (mm.) between matched pairs of fiducual markers at any given equal volume of saline vs. contrast.We then used an image-based anatomic measurement smartphone App that we (MMG) designed  distance between matched marker pairs. . (seeVideo S1& ). We also annotated any anatomic abnormalities . Each fiducial marker was numbered on this map and was referenced in the operative report. The “bladder map” was then placed into each patient's electronic medical record, for easy reference by our Radiation Oncology colleagues. All patients received an oral prophylactic antibiotic for 5 days post-operatively. All patients were contacted by telephone 24, 48, and 72 hours post-operatively for follow-up assessment.We used a schematic bladder-map of the bladder to indicate (intraoperatively) the tumor resection location in 3-dimensions, and, the location of all fiducial markers  scan imaging for dosimetry planning otherapy Figure 7At treatment, patients were aligned ‘on-table’ with one of two techniques: 1. Daily anterior-posterior (AP) and lateral-view portal images were taken and the patient's gold markers were aligned to the gold markers on the reference digitally recreated radiograph, or, 2. Daily MV cone-beam CT was performed and the patient was aligned to the gold markers on the planning CT images.To assess whether the presence of the markers improved targeting, for the last 5 patients in our series, we measured set-up error on the first 3 days of treatment as follows: patients were aligned daily to skin marks using a 3-point set up. Portal images were taken and overlaid with the planning Digitally Reconstructed Radiograph (DRR). The position of the gold fiducial markers on the planning CT and the daily on-table portal image were compared. All patients were treated with IMRT using 6 MV photons.A total of 89 markers were placed into 18 patients Table 1.In the last 5 patients in this series, 2–4 fiducial markers were also placed at one or more bladder anatomic boundaries . In all (18/18) patients, all markers  were clearly visible on intra-operative fluoroscopy, and post-op pelvic X-ray Figure 4during placement . In both instances, the marker was retrieved with an endoscopic grasping instrument, and immediately re-placed. To ensure that the open portion of the beveled needle-tip is completely submucosal, we found it most helpful to withdraw the needle very slightly- simply to allow visualization of muscoa around the needle tip, before advancing the stylet.No (0%) intraoperative or post-operative complications occurred. This includes hematuria beyond what can be expected flooring transurethral tumor resection , UTI, injury to collateral pelvic structures, development of bladder calculi, or chronic pain. In our series, we used electrocautery to control mucosal bleeding only once. We limited its use to avoid tissue necrosis and delayed sloughing at the marker site – which would result in delayed marker loss. In our experience, bleeding at the marker site is generally minimal, and resolved spontaneously with the aid of a urinary catheter left in place post-op and removed prior to same-day discharge from hospital. Hematuria resolved within 24 hours for the majority of patients, and within 48 hours post-operatively for the remainder. At follow-up cystosopy, no patient demonstrated bladder calculi. For each of two patients, one marker fell-out Two of 18 patients elected to undergo cystectomy before initiating chemo-radiotherapy. The remaining 16 patients (82 markers) completed chemo-radiation therapy. In two patients early in our series, two of the 4 markers placed fell out during the last quarter of radiation therapy, resulting in a net marker-loss rate of 2/82 markers = 2.4%. All other markers  were visible and remained in place through the end of radiation therapy.See Video S1) The MedMeasure! App © was used to calculate the mean change in location of each marker on fluoroscopic images taken during bladder filling with equal volumes of saline versus contrast, which was 2.32 mm. . Then, without the aid of on-table spot-imaging, the Radiation Oncologist attempted to predict the location of each of the gold fiducial markers within the patient's pelvis by consulting the archived All patients underwent cystoscopy approximately midway through radiotherapy treatment and then every 3–6 months following treatment. In all patients, the bladder mucosa covered the fiducial markers (i.e. markers were not visible), and no patient (0%) demonstrated bladder calculi.after the maximum discrepancy was detected) that was <1 mm., it is unlikely that the markers were actually migrating, but rather, that the variance in inter-marker distance reflects changing position of the bladder wall at the marker site(s).The bladder wall is a mobile structure and is not well-visualized using on-table radiographic imaging available during radiotherapy. An inability to visualize the exact location of the tumor resection site precludes targeting it with high-dose radiation during daily radiotherapy. We have shown that the novel gold fiducial markers we describe can be easily delivered endoscopically into bladder submucosa and appear to remain in place to identify the tumor site and bladder-margins throughout radiotherapy. Our intra-operative volumetric cystogram under fluoroscopy that the distance between a matched-pair of markers varies generally little (mean 2.32 mm.) but can vary up to ∼1 cm. Because all patients had at least 1 inter-marker distance measurement  during dosimetry planning is over-sized, which increases collateral radiation with HDR sphere is equal to 4/3π r3 . Our fiducial marker protocol speaks to this need: the intraoperative bladder-map informs the radiation oncologist(s) about what was seen intraoperatively , what tumor border each marker identifies, and, the number and location of all markers. Also, if a marker subsequently falls-out, it can be readily identified with this bladder map. The images from the dilute-contrast filling study we now routinely perform are regularly used by our radiation oncology colleagues during dosimetry planning.Dose Volume Histogram (DVH), made before marker placement (i.e. blinded), so that this can be compared to the dosimetry-planning CT after marker placement. Future work should also explore the potential role of fiducial markers in combination with newer treatment technologies, such as, for example, Cyber Knife. Lastly, future work should report on clinical outcomes among patients treated with fiducial-marker targeted radiotherapy (work underway by our group), to potentially further refine placement protocols and device design.The present work serves principally to demonstrate the feasibility and safety of our fiducial marker placement technique. Future studies, with a greater number of patients and combined with inter-fraction plain imaging, are needed to more rigorously exclude marker migration. Future studies are also needed to assess clinical outcomes among patients treated with bladder fiducial markers (a report by our group on this is in progress). To formally assess benefit regarding targeting accuracy and collateral radiation, future studies should include an extra dosimetry-planning CT scan and Video S1Three markers were placed to mark the tumor site of a patient with unifocal T2 disease. Immediately after placement, under Fluoroscopy, the empty bladder was filled and then emptied  with dilute contrast in serial increments of 60 ml.  A Fluoroscopic image of bladder was recorded at each volume. So that the location of all markers could be easily compared relative to one another and to bony landmarks, the position of the Fluoroscopy unit and patient were fixed in space throughout imaging, so that the only source of movement was the patient's bladder and GI motility. Comparisons of images of the fiducial markers within the bladder while the latter was filled with an equal volume of either saline versus diluted contrast confirms that the markers move with the bladder-wall as the bladder expands and contracts during filling and emptying. Also, rapid serial images of the bladder were captured while holding intravesical volume constant. These images show how both the abdominal wall, intestines and the bladder-wall move in 3D with visceral peristalsis and inspiration.(MOV)Click here for additional data file.


Circumscript, progressing lipoatrophy at the insulin injection sites is an unexplained, however rare condition in diabetes mellitus.®) in a woman with type-1 diabetes mellitus. After 11 months of progressing lipoatrophy at two spots on the abdomen, low-dose prednisone (5-10 mg) p.o. was given at breakfast for 8 months, whereby the atrophic lesions centripetally re-filled with subcutaneous fat tissue (confirmed by MRI) despite ongoing use of insulin lispro. However, 4 weeks after cessation of prednisone, lipoatrophy relapsed, but resolved after another 2 months of low-dose prednisone. No further relapse was noted during 12 months of follow-up on insulin-pump therapy with Humalog®.We report a case of severe localised lipoatrophy developing during insulin pump-treatment (continuous subcutaneous insulin infusion) with the insulin analogue lispro , triggered by an unknown endogenous mechanism.Consistent with an assumed inflammatory nature of the condition, low-dose oral prednisone appeared to have cured the lipoatrophic reaction in our patient. Our observation suggests a temporary intolerance of the subcutaneous fat tissue to insulin lispro (Humalog So-called insulin-induced lipoatrophy is a rare, albeit feared condition mostly in patients with type-1 diabetes mellitus. It leads to a total loss of subcutaneous fat tissue at the sites of insulin injections. Neither the pathogenesis of the condition is known, nor the apparent female preponderance . The cli®, Eli Lilly Deutschland, Bad Homburg, Germany) was used for insulin pump treatment . There was no co-morbidity except for Hashimoto's thyreoiditis, requiring 100 μg levothyroxine once daily for substitution. There was no evidence for diabetic retinopathy, nephropathy or neuropathy, respectively. HbA1c  was around 8%, body mass index was 23.8 kg/m2. In November 2007 and February 2008, vaccinations against hepatitis A and B were carried out with a vaccine containing traces of thiomersal . Commencing in May 2008, a lipoatrophic defect developed at the catheter insertion site on the right side of the abdominal wall, and after changing to the opposite side, a second lesion developed there. The size of both lesions increased steadily. In March 2009 hollows of 6 × 7 cm had developed in the subcutaneous fat tissue , without obvious benefit. In consideration of an undefined immunologic pathogenesis, low-dose immunosuppressive treatment with 10 mg prednisone once daily was commenced on June 23, 2009 . As an immediate effect, no new lipoatrophic hollows developed at the catheter insertion sites in use. In August 2009, the existing hollows started to re-fill, beginning at the margins  to insulin lispro , while the lipoatrophic hollows continued to re-fill with subcutaneous fat tissue rather than fibrous scar tissue , who was diagnosed with type-1 diabetes mellitus at the age of 13 years. Since 1984 she was treated by continuous subcutaneous insulin infusion with a portable insulin pump using human regular insulin, without any adverse effects. Since 2004, insulin lispro . This process is different from the local inflammatory reaction that regularly occurs in the subcutaneous fat tissue after injection of insulin, insulin analogues or other compounds ,10,19,20In conclusion, the case of our patient supports the use of prolonged low-dose corticosteroid treatment to cure progressive insulin-induced lipoatrophy. A careful adaptation of the insulin dose is mandatory in order to avoid corticosteroid-induced hyperglycaemia.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.EC conceived the idea, compiled the data and drafted the paper. RP and JP contributed patient data and the photographs. LWP provided the MR imaging analysis and participated in writing the paper. All authors read and approved the final manuscript.


Loss of heterozygosity (LOH) is a common event in malignant cells. In this work we introduce a new approach to identify patients with loss of heterozygosity in the HLA region either at first diagnosis or after HLA mismatched allogeneic HSCT. Diagnosis of LOH requires a high purity of recipient target cells. FACS is time consuming and also frequently prevented by rather nonspecific or unknown immune phenotype. The approach for recipient cell enrichment is based on HLA targeted complement-dependent cytotoxicity (CDC). Relative fluorescent quantification (RFQ) analysis of HLA intron length polymorphisms then allows analysis of HLA heterozygosity. The approach is exemplified in recent clinical cases illustrating the detection of an acquired allele loss. As illustrated in one case with DPB1, distinct HLA loci in donor and patient were sufficient for both proof of donor cell removal and evaluation of allele loss in the patient's leukemic cells. Results were confirmed using HLA-B RFQ analysis and leukemia-associated aberrant immunophenotype (LAIP) based cell sort. Both results confirmed suspected loss of HLA heterozygosity. Our approach complements or substitutes for FACS-based cell enrichment; hence it may be further developed as novel routine diagnostic tool. This allows rapid recipient cell purification and testing for loss of HLA heterozygosity before and after allogeneic HSCT in easily accessible peripheral blood samples. The graft versus leukemia (GvL) reaction is a major component of the immunotherapy after allogeneic hematopoietic stem cell transplantation (HSCT). T cell-mediated immune responses depend on the antigen presentation by HLA molecules. After HLA haploidentical hematopoietic stem cell transplantation, the mismatched HLA molecules themselves become potent targets of the GvL reaction. Being in the center of any specific immune response, loss of antigen presentation is an appealing mechanism for immune escape. However, diminished HLA expression, as seen, for example, in infections with some herpes viruses, prompts the attack of natural killer (NK) cells. Therefore immune escape by altered HLA expression was assumed to be highly improbable in vivo , 2.The acquired loss of heterozygosity (LOH), resulting in duplication of oncogenes or loss of tumor suppressor genes, is a well-established mechanism in tumor evolution , 4. In cIn addition, loss of HLA heterozygosity was also described prior to any treatment , 6–10 inRelative fluorescent quantification (RFQ) is a technique used in a variety of fragment analysis applications that require peak area comparisons across samples. Microsatellites or single nucleotide polymorphisms, aneuploidy assay, or the detection of large chromosomal deletions , 12 can The applicability of this approach is outlined in three cases where testing for HLA-LOH should be mandatory.μL) .The isolation of genomic DNA was performed using the GenoM6 automated DNA isolation system together with the EZ1 blood kit (350 High-resolution HLA typing was performed by a homemade sequence-based typing (SBT) approach, as described previously , 15. LowThe evidence of chimerism was observed by means of sequence-specific primer amplification . SSP kits were used according to the manufacturer's instructions.To direct the RFQ analysis to the tumor cells, there was the need to remove the paternal cells from the peripheral blood cell mixture. Therefore, we depleted the paternal cells in analogy to complement-dependent cytotoxicity testing (CDC). Antibody-mediated complement-dependent cytotoxicity (CDC) refers to the classical method of complement activation that is triggered by opsonization of cells by antigen-specific antibodies  resulting in target cell-specific lysis. CDC is used in the daily routine in our HLA lab, monitoring patients awaiting solid organ transplantation or HSCT. This method enables either cross matching between recipient and donor using T and B cells or detection and specification of HLA antibodies.μL Hank's buffer. With respect to the HLA mismatches between the patient and his father  was added and the sample was incubated for 1 h at room temperature. Before usage, each lot of complement was tested for specificity; there was cytotoxicity in the presence but not absence of specific HLA antibodies. To remove the lysed paternal cells, the cell suspension was then again layered over 3 mL of FicollPaque. After centrifugation at 1500 g the remaining cells were counted in a Neubauer hemocytometer and finally 350 μL of the cell suspension was transferred to a sterile tube for DNA preparation.Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation. 4 mL of whole blood mixed with 3 mL of sodium chloride 0.9% solution was layered over 3 mL of FicollPaque . The blood/Ficoll mixture was centrifuged for 10 min at 1500 g. Following centrifugation and washing, the PBMCs were resuspended in 500 s father , two mons father . 250 μL Patients awaiting solid organ transplantation or HSCT are routinely HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 typed and their sera are tested for the presence of HLA antibodies to prevent antibody-mediated rejection. Well-known immunizing events leading to HLA antibody production are pregnancy, blood or platelet transfusions, and previous organ transplantations. Due to the standards of EFI , HLA antibody testing of patients' sera is periodically performed and updated. Thus, a collection of ancient, well-characterized antisera from immunized patients is available in our lab. The two monospecific HLA sera used for the depletion of the paternal cells were derived from those patients. According to these EFI standards, sera are routinely screened for the presence of HLA classes I and II antibodies by CDC according to a standard protocol and by Luminex, a very sensitive solid phase-based method . This technology uses microbeads coated with class I or class II HLA antigens and a fluorescence flow analyzer. These methods are not only used for detection but also for specification of HLA class I and class II antibodies.Thus, the A and B antisera used for donor cell depletion were well characterized by CDC and Luminex-based screening and specification for HLA antibodies. Both are monospecific for HLA-A2 and HLA-B13, respectively, without any detectable HLA class II antibodies.+/CD56+ cells were separated and DNA was isolated. Sorted CD34−/CD56− cells served as controls.In some patients, leukemic cells can be distinguished from healthy cells by an unusual expression pattern of surface antigens that is fairly exclusive to the patient's leukemic blasts , 16. ThiWe modified the microsatellite-based relative fluorescent quantification approach slightly and focused on the HLA gene length variations. In the majority of cases, virtually all of the short HLA-length polymorphisms (HLA-LP) in the relevant HLA loci are located in the intron sequences. This fact hinders heterozygous sequencing, but these circumstances allow us to discriminate between two or more alleles.μL of the GeneScan 500 ROX size standard, the PCR products were separated using an ABI Prism 310 Genetic Analyzer (AB). Analysis of the data was carried out using GeneMapper 5.0 (AB). All the samples were tested in triplicate.To obtain RFQ signals for the fragments, portions of differently HLA-typed alleles were amplified by PCR with the use of an AB 9700 thermocycler (Applied Biosystems (AB), Foster City, CA, USA) using NuLight547 or 6Fam 5′ fluorescent dye-labelled oligonucleotides . The amplification products were analyzed by ethidium bromide-stained agarose gel electrophoresis. The PCR products were diluted 1 : 30 with HiDi formamide (AB), and, after the addition of 1 To assess the sensitivity of our RFQ approach and to estimate the minimal required proportion of homozygous LOH bearing cells for detection, we set up a titration series . The folRFQ analysis was performed on DNA samples derived from the second patient's (Case B) peripheral blood cells, containing 34% blasts, the patient's peripheral blood cells after the CDC-based depletion of the paternal cells, the sorted CD34/CD56-positive bone marrow-derived cells and, as controls, the cells from the HLA-identical brother, and the patient's sorted CD34/CD56-negative bone marrow-derived cells.The patients' characteristics and cornerstones of treatment prior to diagnosis of HLA-LOH are summarized in A patient relapsed from acute myeloid leukemia (AML) after HLA haploidentical HSCT from his sister . To ensuhttp://www.ebi.ac.uk/imgt/hla/align.html). Despite being informative, the allele relationship within the leukemic blasts was still masked by the donor, and, even after the depletion of the donor cells, it had to be proven that the cells had been completely removed. To address this issue, we analyzed three distinguishable HLA-DPB1 alleles present in the mixed cell population of recipient and donor. In spite of the homozygous HLA-DPB1 genotype of the patient, we were able to distinguish the donor- and host-derived cell populations, as both inherited DPB1*04:01 alleles and the DPB1*17:01 allele from the father showed different length in the microsatellite-like intron regions before exon 3.A patient suffering from AML relapsed four years after allogeneic hematopoietic stem cell transplantation (HSCT) from his HLA-identical brother. Following multiple salvage therapies , HLA hapTo attribute the allele discrepancy to the patient's leukemic blasts we confirmed testing in separated cells Figures –4.  Figu+/CD56+ cells, resembling enriched leukemic blasts, confirmed the previously obtained allele ratio as depicted in −/CD56− cells were donor derived  and showed equal peak heights . By multichannel fluorescent-activated cell sorting (FACS) we aimed for the 7.5% LAIP positive cells from bone marrow; therefore, RFQ could be performed in well-defined cell populations. The RFQ analysis of the CD34 heights . As addi heights . In addi heights , proofinThe third case illustrates the detection of a loss of HLA heterozygosity prior to any treatment . A patieThe loss of HLA alleles is one of the most abundant resistance mechanisms after HLA haploidentical HSCT . Its detThe method presented herein is a robust and easy-to-use approach for testing in abundantly available peripheral blood samples. As presented, the selection of leukemic blasts by HLA-based CDC is reliable and the results are comparable to those achieved by LAIP based FACS. The availability of antisera against the majority of HLA-A and HLA-B antigens renders CDC to an optimal tool for cell enrichment and in combination with RFQ to an appropriate screening tool for HLA-LOH.RFQ analysis of HLA alleles can be used to determine evidence of LOH. The loss of HLA alleles becomes evident by a shift in the ratio of the heterozygous alleles, approximating a homozygous genotype. Testing of various HLA and non-HLA gene loci in the MHC region allow to survey the extent of the LOH. Disparities between the patients' leukemic and healthy cells prove the acquired, leukemia-specific loss of mismatched “target” HLA alleles. −/CD56− negative population, a normal HLA ratio was detected. The result of the CDC-based selection was equivalent to those achieved by leukemia-associated aberrant immune phenotype (LAIP) based FACS (Case B).Since not every allele is directly accessible for analysis, evidence may be derived from the neighboring genomic area as outlined in the second patient (Case B). Despite identical DPB1*04:01 alleles and therefore without immunological selection pressure, the DPB1 gene reflected a shift in the allele quantity caused by other HLA members in the nearby surrounding genomic area, such as the DQ, DR, or class I regions, which themselves were experimentally not directly accessible for quantification. This imbalance in the allele number throughout the HLA region was confirmed by the data for HLA-B. Specificity of HLA-LOH for leukemic cells was verified in well-characterized cell populations. With an increase of leukemic blast content the HLA-LOH became more evident in all tested samples. Conversely, in the controls, as, for example, the CD34A detectable minor proportion of the heterozygous HLA genotype, even after LAIP based FACS, might be attributed to the inherently limited distinction of leukemic blasts from healthy cells. Even if a LAIP, mirroring the patient's relapses, is available, its specificity and sensitivity are limited , 16. IndBy HLA-specific CDC, we were able to demonstrate complete removal of donor cells. A leukemia relapse, in contrast to rejection, is characterized by accumulation of leukemic, host-derived blasts. Therefore and despite accompanied regeneration of the “healthy” host-derived hematopoiesis , a suffiFurthermore, CDC might enable detection of HLA-LOH even in early stages and prior to allogeneic HSCT. HLA-LOH mutated cells are resistant to certain HLA-specific CDC, which might enable enrichment and testing of the desired cells.On the other hand, since HLA testing is often performed at first diagnosis or progression of disease, the patients' samples often exhibit high blast contents, rendering them susceptible to actual “blast typing.” In accordance with other authors we identified pretransplant HLA-LOH as a pertinent source of error for the patients' HLA typing , 6–10. ADue to the significance of HLA-LOH we intend to develop the presented approach into a routine screening method. Its implementation will assure optimal donor choice and hopefully better outcome after allogeneic HSCT.


The risk of fetal aneuploidies is usually estimated based on high resolution ultrasound combined with biochemical determination of criterion in maternal blood, with invasive procedures offered to the population at risk. The purpose of this study was to investigate the effectiveness of a new rapid aneuploidy screening test on amniotic fluid (AF) or chorionic villus (CV) samples based on BACs-on-Beads (BoBs) technology and to compare the results with classical karyotyping by Giemsa banding (G-banding) of cultured cells in metaphase as the gold standard technique. The prenatal-BoBs kit was used to study aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes in 321 AF and 43 CV samples. G-banding of metaphase cultured cells was performed concomitantly for all prenatal samples. A microarray-based comparative genomic hybridization (aCGH) was also carried out in a subset of samples. Prenatal-BoBs results were widely confirmed by classical karyotyping. Only six karyotype findings were not identified by Prenatal-BoBs, all of them due to the known limitations of the technique. In summary, the BACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples. Birth defects are responsible for many cases of infant mortality and morbidity around the world , with abThese congenital defects are caused by chromosomal aneuploidies or monogenic disorders; nevertheless, environmental causes such as fetal infections, environmental teratogens, or micronutrient deficiencies could be hidden by the low percentages for these factors. In fact, most of these defects are due to the combined effects of environmental and genetic factors .β-hCG) and pregnancy-associated plasma protein A (PAPP-A), and the most important ultrasound marker is the measurement of nuchal translucency. In the second trimester, alpha-fetoprotein (AFP) and beta-human chorionic gonadotropin (βhCG) are also measured ..16].2 at 37°C under sterile conditions. Once a cell monolayer was obtained, it was trypsinized and DNA was extracted. DNA extraction from CV samples did not require previous cell culture. DNA was extracted using  according to the manufacturer's protocol and the concentration and purity of the extracted DNA were measured with a NanoDrop spectrophotometer .Approximately 7–10 mL of AF was cultured for 10–12 days with 5% COFollowing DNA extraction, the test and reference DNA were cohybridized to the array. Briefly, 400 ng of patient and reference DNA was labelled by random priming with Cyanine 3 and Cyanine 5 fluorescent dyes, respectively. DNA was then hybridized on the arrays  over night at 48°C. Once the arrays were washed, images were acquired using a PowerScanner microarray scanner  and the image files were analyzed with the BlueFuse microarray software package .P < 0.001).Finally, 364 Prenatal-BoBs tests were performed (321 in AF and 43 in CV samples), obtaining conclusive results in 362 cases (99.45%). Conventional G-band karyotyping was also performed and the results from cultured cells were obtained in 335 cases out of the initial 364 samples (92.03%). Karyotype failures were mainly attributed to cell culture failures, with an increased percentage of growth failure in CV samples , but we also found an abnormal result corresponding to Cat Eye microduplication syndrome (22q11). The rate of concordance with the conventional cytogenetic method was 98.51% (n = 335). Three of the five samples (1.49%) that did not show concordance corresponded to chromosome abnormalities which were not detectable by Prenatal-BoBs: two Robertsonian translocations  and one reciprocal translocation . The other two samples correspond to polymorphisms . Summarized results are shown in Using Prenatal-BoBs tests, we found normal results in 309 AF (96.26%) and 35 CV (81.40%) samples. The most frequent chromosome abnormality found was trisomy 21  and earlier anxiety relief. This fact is particularly important in the process of medical decision-making in cases of maternal anxiety, where there are fetal abnormalities found in the ultrasound examination, or if there are very few days to make a decision regarding a termination of pregnancy , 22. Howin utero . It has in utero , 22. TheCurrently, there are two main trends of opinion in this issue: either rapid aneuploidy tests should replace karyotyping for indications such as positive Down's syndrome screening, in cases of advanced maternal age, and in pregnancies without ultrasound abnormalities –23 or boAlthough rapid aneuploidy tests indicate that only 1% of all invasive prenatal samples have an undetected chromosomal abnormality, a third of these can have a significant risk of serious phenotypic consequences , and so In contrast to other rapid aneuploidy tests, Prenatal-BoBs presents some special features: it is a CE-IVD certified kit with the ability to detect nine microdeletion syndromes with a high specificity  and sensitivity  , 19. MorIn the near future, the advent of massively parallel sequencing is likely to augur a big change in clinical practice in noninvasive prenatal testing (NIPT) . NIPT foBACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples. High concordance of detected aneuploidies was observed between karyotyping and Prenatal-BoBs test.


Dejian Mind-Body Intervention (DMBI), a traditional Chinese Shaolin healing approach, on human frontal brain activity/functions, including patients with autism who are well documented to have frontal lobe problems. This study aims to compare the effects of DMBI with a conventional behavioural/cognitive intervention (CI) on enhancing the executive functions and memory of a nine-year-old boy with low-functioning autism (KY) and to explore possible underlying neural mechanism using EEG theta cordance. At post-one-month DMBI, KY's inhibitory control, cognitive flexibility, and memory functioning have significantly improved from “severely-to-moderately impaired” to “within-normal” range. This improvement was not observed from previous 12-month CI. Furthermore, KY showed increased cordance gradually extending from the anterior to the posterior brain region, suggesting possible neural mechanism underlying his cognitive improvement. These findings have implicated potential applicability of DMBI as a rehabilitation program for patients with severe frontal lobe and/or memory disorders.There has been increasing empirical evidence for the enhancing effects of  Qi). Methods that facilitate the flow of Qi in restoring balance in the body (including the brain) are believed to be effective in improving all-round functioning in autism. Autism is a neurodevelopmental disorder with clinical manifestations similar to those of frontal lobe/executive dysfunctions, including inflexible thinking, disinhibited emotional reactivity, repetitive or socially inappropriate acts. Structural and functional abnormalities in the frontal lobes of such individuals are well documented –4. ConveDejian Mind-Body Intervention (DMBI) was developed based upon traditional Chinese Shaolin healing practice (Chanyi) by the last and first authors. Chanyi is a unique healing approach to strengthen the mind power and bodily wellness by unblocking Qi and clearing bodily orifices . DMBI comprises four interconnected components of Chan practice, mind-body exercises, dietary monitoring, and opening the orifices [Chanyi herbal nasal drop [Chanyi approach by the last author and some other monks in the temple. These cases included patients with neurological  and psychiatric disorders , and physical health problems  [orifices . Severalorifices . A controrifices . Anothersal drop . An adolsal drop . In addiipation) .In the present study, we explored DMBI's effect on improving the cognitive functions of a child with low-functioning autism, who showed minimal improvement after one year of conventional intervention (CI). In addition, we examined the underlying possible neural mechanism associated with this improvement by using the EEG cordance measure.Qigong) since age 2, for his delayed sensory, language, motor, and social developments, with limited effect. Given KY's frequent temper tantrums  and his very brief attention and memory span, his parents brought him to the Integrative Neuropsychological Rehabilitation Center (INRC) at age 7 for conventional neuropsychological intervention.KY is a 9-year-5-month-old right-handed boy with mental retardation and autism. He had received one year of physiotherapy at age 1.5, and five years of alternative treatment  , a well-KY was assessed at pre- and post-12-month CI and post-1-month and post-8-month DMBI, where the post-CI also represented the pre-DMBI assessment. All assessments were administered by the clinical psychologist who did not provide the DMBI for KY. As suggested by Jacobson et al. , KY's prCI. KY was trained on impulse control and attention by the second author, a clinical psychologist, for 30 minutes per week for a year. This involved functional communication training with behavioral modification techniques, substituting his temper outbursts with socially appropriate expressions  and 30-minute daily home practices of a computerized attention and impulse control training program developed by the INRC. After the 12-month CI, KY's duration of temper tantrum reduced from >20 minutes to <5 minutes. However, no improvement was shown in his inhibitory control and memory functioning, which remained to be moderately to severely impaired Figures . His motChan practice was not applicable to KY given his limited intelligence. KY was treated with DMBI by the first author for 15-minute weekly sessions during the first month, and 15-minute monthly sessions in the subsequent seven months, plus home application of three of the four integrated components in DMBI. KY was recommended to take seven categories of food everyday and with one to three kinds from each category. The seven categories are (1) grains , (2) vegetables , (3) fruits , (4) beans , (5) mushrooms , (6) nuts , and (7) roots . The amount of food was not fixed and it was recommended that the child ate up to 80% full in each meal. More importantly, KY has abstained from ginger, garlic, green onion, spicy foods, and seafood, and reduced 70% intake of meat since DMBI. These foods are believed to be hard to digest by people with illnesses and easily generate excessive heat inside the body and block the orifices. KY's dietary change was recorded by his parents on a questionnaire about the types of food taken. According to his mother's report, KY's dietary has met the daily recommended guideline during the intervention.KY applied intranasally a specially formulated herbal remedy at the dosage of 10 mL twice daily to clear his nasal cavity. The herbal remedy was manufactured following the product safety guidelines of the Hong Kong Department of Health for heavy metal, pesticides residue, and microbiological substances. The remedy was manufactured by the Hong Kong Institute of Biotechnology that is a Chinese Medicine Manufacturer meeting the Good Manufacturing Practice standards and is owned by the Chinese University of Hong Kong. The herbal nasal drop has been produced for research purpose only, and not for commercial purpose. The formula is currently under testing and modifying for safety, effectiveness, and patent application. Some major ingredients include Herba Artemisiae Annuae and Rhizoma Coptidis. KY's mother performed Nose-Bridge Massage for KY to unblock his nasal cavity, by softly and slowly moving her index fingers up and down each side of KY's nose bridge for 36 times every night. KY also practiced Tranquil Stand, that is, standing squarely and relaxingly on both feet and raising the hands gently in front of his abdomen. Instead of practising every day, KY was only willing to practice once to twice weekly, for <5 seconds at the beginning to one minute after one year of practice. Quantitative EEG data were recorded from 19 electrodes based on the International 10–20 system during eyes-open resting state before and after 1-month and 8-month DMBI. The EEG signal was digitized at 256 Hz with a low pass filter of 30 Hz, and impedances below 10 kΩ. At least one minute of artefact-free data were selected and computed into cordance indices using a three-step algorithm .Cordance has been proposed as an indirect measure of brain perfusion, where higher perfusion implicates higher metabolism. Cordance values were grouped topographically into anterior , centrotemporal , and posterior  regions to provide a measure of mean cordance intensity for each region. Theta (4–7 Hz) cordance intensity was used in this study given its association with cerebral perfusion , 25, andThis represents the proportion of electrodes showing concordance  or discordance  along the two dimensions of absolute and relative power . The eleCompared with KY's severely to moderately impaired inhibitory control at post-12-month CI , KY showKY demonstrated significant memory enhancement from the “severely impaired” to the “low-average to average” level after receiving DMBI . KY's prAt pre-DMBI, KY showed suppressed theta cordance value in the right hemisphere and bilateral anterior regions . At postFurthermore, KY's global brain state became more concordant (suggesting higher perfusion) when the intervention progressed, as indicated by more and more electrodes turned from discordant  into concordant  state. At pre-DMBI, KY had 47% of electrodes (9 out of 19 electrodes placing in quadrant I & III) displaying discordance , which iThis study revealed the positive effects of DMBI on inhibitory control and memory of a child with low-functioning autism, from impaired level to normal level after 8-month-DMBI. Given KY's minimal improvement after 12-month CI, his enhancement at post-8-month-DMBI is unlikely to be due to maturation or spontaneous response to any treatment. In addition, the EEG measures provided insights into the possible neural mechanism that may be associated with the improvement of executive and memory functions. That is, the increased anterior cordance and the increasingly concordant global brain state at post-8-month-DMBI may suggest a change of neural activities. Given the well-documented involvement of frontal-temporal/parietal connections in attentional and memory processing , the incThe majority of conventional intervention for individuals with low-functioning autism is behavioral or educational based, which are time and labor intensive  and withThe present study has provided encouraging findings on the potential effects of DMBI on improving the cognitive functions and altering the neural activity of a child with low-functioning autism. It implicates the possiblity of applying DMBI as a rehabilitation program for patients with severe brain disorders.No competing financial interests exist.


Bone marrow-derived fibrocytes reportedly play important roles in the pathogenesis of idiopathic pulmonary fibrosis. Pirfenidone is an anti-fibrotic agent; however, its effects on fibrocytes have not been investigated. The aim of this study was to investigate whether pirfenidone inhibits fibrocyte pool size in the lungs of bleomycin-treated mice.Bleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice, and pirfenidone (300 mg/kg/day) was orally administered daily for 2 wk. The lungs were removed, and single-cell suspensions were subjected to fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes, which were defined as CD45 and collagen-I double-positive cells. Immunohistochemistry was performed on the lung specimens to quantify fibrocytes. Chemokines in the lung digests were measured with enzyme-linked immunosorbent assay. The effect of pirfenidone on alveolar macrophages was evaluated with bronchoalveolar lavage (BAL). In a therapeutic setting, pirfenidone administration was initiated 10 days after bleomycin treatment. For chemotaxis assay, lung fibrocytes were isolated with immunomagnetic selection  after culture and allowed to migrate toward chemokines in the presence or absence of pirfenidone. Moreover, the effect of pirfenidone on the expression of chemokine receptors on fibrocytes was evaluated.P = 0.0097). Increased chemokine (CC motif) ligand-2 (CCL2) and CCL12 production in bleomycin-treated mouse lungs was significantly attenuated by pirfenidone . Pirfenidone also attenuated macrophage counts stimulated by bleomycin in BAL fluid. Fibrocyte migration toward CCL2 and chemokine (CC motif) receptor-2 expression on fibrocytes was significantly inhibited by pirfenidone in vitro.Pirfenidone significantly ameliorated bleomycin-induced pulmonary fibrosis as assessed with quantitative histology and collagen measurement. Fibrocyte pool size in bleomycin-treated mice lungs was attenuated from 26.5% to 13.7% by pirfenidone on FACS analysis. This outcome was also observed in a therapeutic setting. Immunohistochemistry revealed that fibrocytes were significantly decreased by pirfenidone administration compared with those in bleomycin-treated mice (in vivo, and fibrocyte migration was inhibited by pirfenidone in vitro. Fibrocyte inhibition is considered a mechanism of anti-fibrotic action of pirfenidone.Pirfenidone attenuated the fibrocyte pool size in bleomycin-treated mouse lungs via attenuation of CCL2 and CCL12 production  Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by progressive scarring of the lung parenchyma . AlthougAmong these possible sources of lung fibroblasts, bone marrow-derived circulating fibrocytes are mesenchymal progenitor cells that express markers compatible with leukocytes, hematopoietic progenitor cells, and fibroblasts . FibrocyAlthough no well-accepted medical therapy for patients with IPF has yet been established , vigorouin vitro. This study is the first to demonstrate the effect of pirfenidone on fibrocytes, which are now considered essential to the pathogenesis of IPF. Findings from our preliminary studies were reported in abstract form at a meeting of the American Thoracic Society [In this study, we hypothesised that pirfenidone elicits its pharmacological effects by inhibiting fibrocytes. To test this hypothesis, we administered pirfenidone to bleomycin (BLM)-treated mice and examined the effect of pirfenidone on fibrocytes using fluorescence-activated cell sorter (FACS) analysis. We also investigated the effect of pirfenidone on chemokine production in BLM-treated mice lungs. Moreover, we examined the effect of pirfenidone on cultured fibrocyte migration and chemokine receptor expression  Society .Detailed materials and methods are described in Additional file Nine-week-old female C57BL/6 mice  were used in all experiments. They were randomised into various groups before the initiation of the experimental protocols, which were approved by the animal care and use committee of Nippon Medical School .Osmotic pumps  containing 200 μL saline with or without BLM  were implanted subcutaneously . BLM wasLung samples were fixed in 10% formalin buffer  for histological examination. Paraffin Sections 2- to 4-μm thick were cut from fixed lungs, stained with hematoxylin and eosin (HE) and Masson trichrome, and examined with a microscope.Lungs harvested on day 28 were used for collagen assay. Total lung collagen was determined using a Sircol Collagen Assay kit  according to the manufacturer’s instructions.On day 14 of BLM treatment, the lungs of the mice were removed and minced to obtain single-cell suspensions for FACS analysis. This time point was chosen according to previous studies that investigated fibrocyte accumulation approximately 14 days after BLM treatment . The numLung tissue sections were incubated with anti-mouse CD45 monoclonal antibody followed by Alexa Fluor 488-conjugated anti-rat immunoglobulin G . Subsequently, these sections were incubated with rabbit anti-mouse polyclonal Col-I IgG and biotin-conjugated anti-rabbit IgG as the secondary antibody followed by Alexa Fluor 594-conjugated streptavidin (Life Technologies). Sections were then stained with 4′,6-diamidino-2-phenylindole and mounted with Vectashield .Chemokine (CC motif) ligand-2 (CCL2), CCL12, and chemokine (CXC motif) ligand-12 (CXCL12) levels were measured in lung homogenates using specific ELISA kits from R&D Systems Inc. .Tissue sections were incubated with anti-mouse CCL2 monoclonal antibody overnight at 4°C. They were then incubated with biotin-conjugated anti-rat IgG2b as the secondary antibody and stained with horseradish peroxidase-conjugated streptavidin followed by staining with 3, 3′-diaminobenzidine.Tracheas were cannulated and BAL was performed with 1 mL of 0.1 mM ethylenediaminetetraacetic acid/phosphate-buffered saline. After the cell number in the BAL fluid was counted, the cells were cytospun onto glass slides and stained with Diff-Quick  for differential cell counting.6 cells/mL in Dulbecco’s modified Eagle medium containing 0.1% bovine serum albumin and allowed to migrate toward various concentrations of CCL2 and CCL12 with or without pirfenidone (100 μg/mL) at 37°C in a moist 5% CO2/95% air atmosphere incubator. After the cells were cultured overnight, migration was assessed by counting the number of cells in 10 high-power fields per well with a light microscope.Murine fibrocytes were isolated from the lungs according to previously published methods . FACS anIsolated fibrocytes were cultured in the presence or absence of pirfenidone (100 μg/mL) for 48 h. Then, total RNA was extracted using an ISOGEN with Spin Column  and converted to complementary DNA as described elsewhere . Real-tipost hoc correction. An unpaired two-tailed Student’s t test was used for single comparisons. Data were analysed using JMP 9 software version 9.0.3 . Differences were considered statistically significant if P values were less than 0.05.The animal experiment involved at least 6 mice in each treatment group unless otherwise stated. Comparisons among multiple groups were analysed using one-way analysis of variance with Tukey-Kramer P < 0.0001). Moreover, collagen content was quantified in the lungs to evaluate the anti-fibrotic effects of pirfenidone. As shown in Figure P = 0.0012).As an initial assessment, histological analysis of HE- and Masson trichrome-stained lung sections was performed to examine whether the fibrotic response in mice receiving BLM was ameliorated by pirfenidone administration. Lung histological data obtained on day 28 of BLM administration showed focal fibroplasia with destruction of the alveolar architecture and interstitial fibrosis of the alveolar wall in the group receiving BLM Figure C and 1D Several studies have reported that bone marrow-derived circulating fibrocytes migrate into the lungs after injury and contribute to fibrosis in BLM-treated mice ,17,22. TP = 0.0097).To confirm the increase in fibrocytes in BLM-treated mice and its subsequent decrease after pirfenidone administration, we performed immunohistochemical staining for CD45 and Col-I to count the number of fibrocytes in lung sections on day 14 of BLM treatment with or without pirfenidone. Increased numbers of fibrocytes expressing CD45 . CXCL12 and CCL12 production in the lungs of BLM-treated mice was also stimulated compared with that in saline-treated mice. CCL12 production was also significantly attenuated by pirfenidone as shown in Figure As shown in Figure Immunohistochemical staining for CCL2 was performed to determine the cellular sources of CCL2 on day 14 of BLM treatment with or without pirfenidone. Increased immunoreactivity for CCL2 was observed on alveolar epithelial cells, terminal and respiratory bronchiolar epithelial cells, and macrophages Figure A–C. TreaA potential profibrotic role of activated alveolar macrophages and their mediators has recently been reported in the pathogenesis of IPF ,25. In sPirfenidone reportedly inhibits fibrosis in the lungs of BLM-treated mice even when administered during the fibrotic phase in this model . To examin vitro chemotaxis assays using a Boyden chamber system. Fibrocytes were cultured as described in Materials and methods, and CD45-positive cells were selected using magnetic beads. FACS analysis revealed that the percentages of CD45 and Col-I double-positive cells in the lung culture after magnetic selection was 75.8% .To explore more thoroughly the mechanisms through which pirfenidone ameliorates fibrocyte accumulation in the lungs of mice with BLM-induced pulmonary fibrosis, we investigated CCR2 expression on cultured fibrocytes. Isolated fibrocytes were cultured in the presence or absence of pirfenidone, and mRNA expression of CCR2 in fibrocytes was evaluated with quantitative real-time reverse transcriptase PCR. As shown in Figure in vitro.The aim of this study was to determine the role of pirfenidone in the suppression of fibrocyte accumulation in the lungs in response to systemic administration of BLM infused with osmotic pumps. We used both ELISA and immunohistochemical analysis to confirm that pirfenidone decreased fibrocyte pool size in BLM-treated mice lungs via attenuation of both CCL2 and CCL12 production. CCL2 expression was localised to alveolar epithelial cells, bronchiolar epithelial cells, and macrophages in the lungs of BLM-treated mice, and expression was attenuated by pirfenidone administration. Moreover, pirfenidone attenuated both fibrocyte migration toward CCL2 and CCR2 expression on fibrocytes IPF is a relentlessly progressive and fatal disorder of unknown aetiology with a median survival of 3–5 yr . Recent in vitro and in vivo studies have proven that the beneficial effects of pirfenidone are mediated by its anti-fibrotic and anti-inflammatory properties [Pirfenidone is among the most promising of the limited therapies available for IPF. Pirfenidone has been shown to reduce the annual decline of vital capacity in IPF patients ,10. Seveoperties ,36,37; hoperties ,23. In aIn this study, pirfenidone clearly inhibited CCL2 production stimulated by BLM treatment. Several studies have demonstrated increased CCL2 in the circulation and BAL fluid of IPF patients ,39, and in vitro[in vitro chemotaxis assay using a Boyden chamber system. Our results revealed that CCL2 stimulated fibrocyte migration, and pirfenidone attenuated the migration with statistical significance. Although the detailed mechanisms through which pirfenidone attenuates fibrocyte migration have yet to be studied, the results of the chemotaxis assay may partially explain the decrease in fibrocyte pool size after pirfenidone administration in BLM-treated mice lungs.Fibrocytes derived from bone marrow are known to migrate to sites of tissue injury chemotactically, and CCL2 has been demonstrated to stimulate human fibrocyte migration in vitro,40. BecaCCL12 is also the ligand for the CCR2 receptor in the mouse and is reportedly responsible for fibrocyte recruitment and enhanced fibrotic response . In thisin vivo are considered essential actions of pirfenidone in humans.The biological axis of the receptor CXCR4 and its ligand CXCL12 also plays an important role in the homing of bone marrow-derived progenitor cells , and a dIn the current study, the inhibition of BLM-induced fibrosis by pirfenidone was incomplete despite significant inhibition of fibrocytes by pirfenidone. Although it is postulated that just over half of the lung fibroblast population can be accounted for by fibrocytes and EMT in BLM-induced pulmonary fibrosis models , residenIn the current study, we examined fibrocytes on day 14 of BLM treatment. In BLM-treated mice, marked lung oedema was present at day 10, and lung hydroxyproline levels started to increase on day 10 and tended to increase further by day 28 . BecauseIn conclusion, we clearly demonstrated that pirfenidone decreased fibrocyte pool size in BLM-treated mice lungs via the attenuation of CCL2 and CCL12 production. Inhibition of fibrocyte migration into lung tissue is considered a mechanism of anti-fibrotic action of pirfenidone. This study is the first to investigate the effects of pirfenidone on fibrocytes, which are currently considered essential to the pathogenesis of IPF.The authors declare that they have no competing interests.MI, KK, and KM: participated in the design of the study and analysis and interpretation of data, performed the statistical analysis, drafted the manuscript, and revised the manuscript critically for important intellectual content. AA participated in the design of the study and analysis and interpretation of data, performed the statistical analysis, drafted the manuscript, revised the manuscript critically for important intellectual content, and acquired funding. NK, YM, HH, TN, KF, and YS revised the manuscript critically for important intellectual content. AG: Participated in the design of the study, revised the manuscript critically for important intellectual content, and acquired funding. All authors read and approved the final manuscript.Detailed description of Materials and methods section.Click here for file