Primary Objective. To test the hypothesis that two injections of enzyme-potentiated mosquito antigen significantly reduce the size of experimental mosquito bites in participants with LLR-MB. Design. Randomised, double-blind, placebo-controlled, parallel group comparison over 3 months. Setting. Hospital outpatient clinic. Participants. Fifty adult participants of both sexes. Interventions. Two injections of mosquito antigen or matching placebo, 6 weeks apart. Main Outcome Measures. Early (1 hour) and late (24 hours) mean square root of erythema area (SREA) following controlled mosquito bite with the second bite given at least 6 weeks following the final injection. Results. At 1 hour, mean SREA was slightly higher in the EPD group compared to placebo after adjusting for baseline values , but this was not statistically significant ; neither were the results at 24 hours  (P = 0.57). The proportion of participants experiencing a decrease in wheal size at 1 or 24 hours was similar between groups. Conclusions. EPD was not demonstrated to be effective for immediate or delayed LLR-MB. Methodological problems included a high variability in LLR-MB between subjects, suggesting that a crossover design should be used in future. The mec process , 3 and o process , convent process .−12 mmol/L, comparable to skin prick test doses) are given intradermally in combination with the enzyme beta-glucuronidase, which is used to enhance the desensitising effect of the low-dose allergen. Prospective audit has demonstrated EPD to be safer than conventional immunotherapy [Enzyme-potentiated desensitisation (EPD) is a therapeutic technique in use for 30 years –10 in whotherapy  with no Several small but poorly reported RCTs using EPD based on mixed inhalant allergens suggested effectiveness in seasonal allergic rhinitis due to grass pollen –15 and cThe objective of the study was to test the hypothesis that two injections of EPD mosquito antigen would result in a reduced erythema area after controlled mosquito bite challenge in a group of adult sufferers of LLR-MB, when compared against a similar group given placebo injections. As a pilot study, we also aimed to test the methodology and provide estimates for a sample size calculation to power a definitive study if the results proved encouraging.The study was conducted in accordance with the Declaration of Helsinki . The stu50 participants were recruited in London from a database kept by one of the authors (N. Hill) of LLR-MB sufferers who had participated in previous clinical studies of topical antibite agents. Most were employees at the London School of Hygiene and Tropical Medicine. Mosquito challenge was performed there by NH and EPD injections were performed at the Royal London Homeopathic Hospital outpatient department by S. Berkovitz. Participants were recruited from March to July 2003 and were not followed up beyond their final mosquito exposure, the last of which was performed in January 2004.In order to be candidates for inclusion, participants had to be aged between 18 and 65, of either sex, with a diagnosis of LLR-MB determined by (1) a history of large local reaction after insect bite identified as mosquito by participants, (2) mean erythema diameter (ED) of 20 mm or greater within 24 h of first experimental challenge to mosquito bite (for details see below). They were not admitted to the study if any of the following criteria were present: (1) previous treatment with EPD, (2) treatment with conventional immunotherapy in the last 5 years, (3) history of anaphylaxis or laryngeal oedema, (4) moderate-to-severe asthma , (5) currently pregnant or breast feeding, (6) severe skin disease or pigmentation making the measurement of erythema difficult (this excluded participants of Negroid or other dark skin type), (7) chronic severe urticaria or dermatographism, (8) use of regular oral antihistamine, non-steroidal anti-inflammatory drugs, oral steroids, or immunosuppressants, (9) concomitant serious illness or cardiovascular or respiratory disease requiring specialist care or drug treatment ; or (10) any other condition that in the investigators' opinion excluded the participant on grounds of safety.Aedes Aegypti mosquito heads in buffer solution, containing approximately 120 fg of salivary gland per 0.04 mL) or placebo . 0.04 mL of mosquito antigen or placebo was drawn up by the Gilman pipette and mixed with 0.01 mL of beta-glucuronidase . The resulting 0.05 mL was drawn up in an insulin syringe and administered by intradermal injection into the flexor aspect of the forearm. All EPD vials were prepared by McEwen Laboratories, Pangbourne (details of the method of preparation are available from S. Berkovitz) and administered by S. Berkovitz who had several years' experience in the technique. Participants waited one hour after the first injection and half an hour after the second injection to ensure the absence of immediate adverse effects. They were asked to not to observe the immediate reaction to maintain blinding. They were given a questionnaire to post back after 24 hours to ascertain any delayed adverse effects and to report the presence and size of any swelling at the injection site (a possible cause for unblinding) and any itching.Participants were randomised to receive two injections of mosquito antigen or placebo at an interval of six-to-eight weeks, the first no less than a week after the first mosquito exposure. Blocked randomisation was performed with random block sizes with the investigators blinded to the allocation list. Individualised EPD vials were kept refrigerated or in a thermos flask filled with iced water. EPD was supplied as sterilised individual vials containing mosquito antigen  and 24 hours (T24) after mosquito exposure. A secondary outcome measure was the degree of itching at T0 and T24. Erythema area has been used by N. Hill in previous studies of topical antipruritics for mosquito bites and has been found to be a reliable measure. The square root transformation was used to satisfy statistical assumptions .Aedes aegypti mosquitos were starved for 24 hours prior to use. Per daily period, the mosquitoes were isolated singly in small plastic tubes closed by netting to prevent escape whilst enabling them to feed through the netting.Participants were all subjected to an initial mosquito exposure to confirm their eligibility for the study and again six-to-eight weeks following the second EPD injection. The method was as follows. Female (3-4 day old) Aedes aegypti mosquito bite. A mosquito tube was secured on the midpoint of the nondominant forearm and allowed to feed for 10 minutes. At the end of this period, the feeding status of the mosquito  was noted. If a mosquito was not observed to be feeding after 5 minutes, it was replaced by another. In addition, the number of probes (the places where the mosquito penetrates the skin to find a good blood source) was recorded, since saliva could be injected even if no blood is extracted.The participants' forearms were cleaned with 70% ethanol and allowed to dry. Each subject was exposed to a single The diameter of erythema was measured with a digital calliper . Erythema length and width were defined as the maximum diameter and orthogonal diameter, respectively, (in millimetres). Erythema area (EA) was calculated as the area of an ellipse with these diameters (in millimetres squared). If there was more than one erythematous bite, the largest was used for the measurement. All calliper measurements were performed by the same person throughout the study. Diameter measurements were made at 15 minutes (T0), 1 hour (T1), 24 hours (T24).Participants were not permitted any symptomatic medication following mosquito exposure until 24 hours after exposure . They were allowed to use topically applied ice packs for symptomatic relief. Regular medication was permitted as usual.t-test (at the 5% level), a 45% reduction in erythema area after verum treatment with no change in the placebo group, with an assumed baseline mean area of 1206 mm2 and a standard deviation of 772 mm2, 33 participants were required in each group. We decided to recruit 25 participants in each group since we were not performing a definitive study. However we note that the latter sample size actually has 94% power to detect such as difference using analysis of covariance (ANCOVA), assuming a correlation of 0.7 between the pretreatment and posttreatment responses and no loss to followup. ANCOVA was used to analyse the results on an intention-to-treat basis, comparing the posttreatment wheal size adjusted for the pretreatment wheal size between EPD and placebo.With no previous studies of EPD or similar treatments in this condition on which, to base a power calculation, an approximate calculation, using ANCOVA methodology, was performed using data from a similar group of participants in a previous study of a mosquito repellent. To have an 80% chance of detecting as significant using a two-sample Randomisation to verum or placebo groups was performed by the trial statistician using block randomisation with blocks of random size. The code was posted to McEwen Laboratories in Pangbourne, the company supplying the EPD vials, and to no one else. Prior to delivery of vials to London, they labelled the identical vials with sequential numbers (1–50) according to the allocation sequence and placed them in two boxes (one for the first and one for the second injection). There was no further contact between the laboratory and any of the investigators for the duration of the trial. After enrolment and first mosquito exposure by N. Hill, S. Berkovitz allocated the next available number at each subject's first EPD injection. The code was only revealed to the researchers once recruitment and data collection were complete, and data analysis was proceeding. All study personnel and participants were not aware of treatment assignment throughout the study. However, participants may have become inadvertently unblinded because of prolonged swelling or itching at the site of injection after EPD compared to placebo at 24 hours. This was explored using a participant questionnaire filled out at 24 hours after each injection and returned by post to the investigators.Participant flow is shown in P = 0.89, ANCOVA analysis). Both groups decreased slightly compared to their baseline values. Likewise, at 24 hours after bite, mean SREA was slightly lower in the EPD group compared to the placebo group after adjusting for baseline values , but again this was not statistically significant (P = 0.57). This time both groups increased compared to their baseline values. The proportion of participants experiencing a decrease in wheal size was similar for EPD and placebo and not one likely to prove clinically useful.Primary outcomes are summarised in P = 0.92) and 24 hour: 7.02 . Although there is an imbalance (by chance) in the numbers in the subgroup analysis, the ANCOVA does take account of baseline measures.We then restricted the analysis to patients who had exhibited exposure diameters exceeding 50 mm in the baseline measures. There were 23 patients in this subgroup (8 on placebo and 15 on drug). Repeating the ANCOVA analyses for the 1 hour and 24 hour data led to no difference in the conclusions. The estimates were 1 hour: −0.54  of 6.4 mm. For the placebo participants, only 11/48 injections produced such a reaction, with a mean diameter of 8.5 mm. This might indicate some degree of unblinding, since persistent swelling might lead participants to conclude (correctly) that they had received verum. One participants' data was missing. However, only two participants complained of itching at 24 hours, both in the placebo group.Only two adverse effects occurred other than expected local reactions. Both of these occurred in the placebo group. One patient sustained myalgia lasting a few hours; the other had a mild vasovagal episode 10 minutes following the injection.The disappointing results contrast with a retrospective audit of 11 practitioners carried out by the first author prior to the study. This documented 53 patients, of whom 83% were said to have achieved a moderate or great improvement after two EPD injections of mosquito antigen. The most likely reason for the discrepancy is observer bias in everyday practice. Another possible but unlikely reason is that we gave injections at 6 weekly intervals (due to time pressure), whereas the recommended interval is 8–12 weeks. Since the native mosquito extract was not analysed or standardised for allergen content, the exact amount of administered antigen is unknown and likely to show batch-to-batch variation. Therefore, it is possible that a higher dose might have produced a better clinical effect, although by definition EPD is a “low-dose” treatment.A noteworthy feature of the controlled mosquito bites is the high variation between participants suggesting that a cross-over trial might be appropriate for future studies in this area. Also, there is relatively high intraparticipant variability in the placebo group over a few months, with erythema diameters varying by a factor of up to 2 and therefore erythema area by a factor of up to 4. Scatter plots Figures  illustraThis pilot study suggests that EPD, although safe, is not of value in preventing large local reactions to mosquito bites or the itching associated with them, and that a larger study would not be worth performing. Methodological problems to note include a high variability in LLR-MB across subjects, suggesting that a cross-over trial design should be used for further studies of this condition.


Objective techniques to assess the amelioration of vision in patients with impaired visual function are needed to standardize efficacy assessment in gene therapy trials for ocular diseases. Pupillometry has been investigated in several diseases in order to provide objective information about the visual reflex pathway and has been adopted to quantify visual impairment in patients with Leber Congenital Amaurosis (LCA). In this paper, we describe detailed methods of pupillometric analysis and a case study on three Italian patients affected by Leber Congenital Amaurosis (LCA) involved in a gene therapy clinical trial at two follow-up time-points: 1 year and 3 years after therapy administration.Pupillary light reflexes (PLR) were measured in patients who had received a unilateral subretinal injection in a clinical gene therapy trial. Pupil images were recorded simultaneously in both eyes with a commercial pupillometer and related software. A program was generated with MATLAB software in order to enable enhanced pupil detection with revision of the acquired images , and computation of the pupillometric parameters for each stimulus. Pupil detection was performed through Hough Transform and a non-parametric paired statistical test was adopted for comparison.The developed program provided correct pupil detection also for frames in which the pupil is not totally visible. Moreover, it provided an automatic computation of the pupillometric parameters for each stimulus and enabled semi-automatic revision of computerized detection, eliminating the need for the user to manually check frame by frame. With reference to the case study, the amplitude of pupillary constriction and the constriction velocity were increased in the right (treated eye) compared to the left (untreated) eye at both follow-up time-points, showing stability of the improved PLR in the treated eye.Our method streamlined the pupillometric analyses and allowed rapid statistical analysis of a range of parameters associated with PLR. The results confirm that pupillometry is a useful objective measure for the assessment of therapeutic effect of gene therapy in patients with LCA.ClinicalTrials.gov NCT00516477 Leber congenital amaurosis (LCA) is a rare ocular disease, affecting around 1 in 81,000 people -8.The severe visual impairment in most patients affected by LCA, as well as other early-onset retinal degenerations, is difficult to quantify with conventional clinical instrumentation . In the In this paper, we propose and describe the most updated version of the pupillometric analysis method which has been explored in the framework of the clinical trial NCT00516477 for evaluation of pupillary reflexes in LCA2 patients undergoing gene therapy .As a case study, we reported the results of the analysis of the three Italian patients involved in the clinical trial focusing on two post-treatment time-points at 1 year and 3 years respectively.Three patients  involved in the clinical gene therapy LCA2 trial were analysed in this study: subject 1 is a 26 year old female, subject 2 is a 26 year old male, and subject 3 is a 19 year old female . All patThe stimulation protocol was modelled after the “swinging flashlight test”, in order to identify relative afferent pupillary defects (rAPDs) . ResponsTwo tests were performed:● Test 1 - eight consecutive cycles consisting of a light stimulus presented for 0.2 seconds followed by a 1 second dark interval;● Test 2 - six consecutive cycles consisting of a light stimulus presented for 1 second followed by a 0.6 second dark interval.In both tests, the light stimulus was presented alternatively to the right and left eye and each test was repeated twice: the first time starting with the stimulation of the right eye, the second time starting with the stimulation of the left one. Each sequence of light stimulus was interleaved with a pause of at least 3 seconds in order to recover baseline pupil diameter. The tests were usually performed in the morning, after at least 8 hours of sleep.Pupil responses were recorded simultaneously in both eyes with a Procyon P2000 pupillometer and PupilFit4 software . Each eyepiece was equipped with infra-red diodes GaA1A’s type SFH485 with peak emission at 880 nm to illuminate the pupil without stimulating the eye, and an infra-red sensitive camera that captured the video images at 25 frame/s, allowing the pupil diameter of both eyes to be measured every 40 ms. Images were saved in bitmap format after real-time processing. Means, maximums and minimums of the pupil diameter for the entire test were computed by PupilFit4. Responses to individual stimuli were not computed. Therefore, a package of functions was developed in Matlab  to provide the following functionalities for each acquired frame:● pupil detection;● manual check/revision of pupil detection;● elimination of unsuitable frames (e.g. due to blinking);● computation of pupillometric features for each stimulus;● statistical analysis as described below.The offline image processing algorithm determined the pupil diameter when the results of the PupilFit4 were not reliable, that is, when there were percentage variations in the pupil diameter from previous frames higher than a user-defined threshold (10% default).The algorithm consisted of four major steps:● image thresholding - as the images were acquired under the same light condition, the darkness of the pupil is approximately the same for all the subjects (around 35 gray level), for that reason the threshold was set at the same value (40 gray level).● hole filling - holes, mainly due to light reflexes, were filled using an algorithm based on morphological reconstruction .2, equivalent to a circle with a radius of 0.8 mm.● small object removing - all connected components (objects) that had fewer than a N pixels, were removed from the image. The number N was chosen in order to eliminate objects with a surface area less than 2 mm● edge detection - morphological operations, such as dilatation and removing the interior pixel, were performed in order to leave only the boundary pixels, which should represent the pupillary border.● pupil detection and diameter measurement - generalized Hough transform was implemented to detect the position and the diameter of the pupil, modelled as a circular shape.Figure A user-friendly Graphical User Interface was developed to check the results of the automatic pupil detection. It shows the image of the eye and the shape of the pupil detected by the off-line processing. If the user is satisfied by the automatic pupil detection, he/she just has to click the mouse, otherwise he/she can adjust the position and diameter of the detected shape by using the mouse. If the pupil could not be detected, not even manually, for instance due to blinking, the user could evaluate whether to accept a linear interpolation of the diameter between neighbor frames not affected by noise or, in extremis, to discard the stimulus.According to the experience of previous studies ,7,19, pu·Baseline Diameter (BD) computed as the average of the pupil diameter 100 ms prior to the light stimulus;·Minimum Amplitude (MA) defined as the lowest pupil diameter after a light stimulus;Response Amplitude (RA) computed as the difference between BD and MA;·Percent Constriction (PC) defined as the ratio between MA and BD expressed as percentage;·Response Time (RT) defined as the difference between the time corresponding to the minimum amplitude and the time corresponding to the stimulus;·Constriction Velocity (CV) computed as the ratio between RA and RT.Figure A non-parametric statistical paired test (Wilcoxon signed rank test) was performed to compare:● the pupillometric parameters obtained when the right eye was stimulated with those obtained when the left eye was stimulated;● the pupillometric parameters obtained at the first time-point (1 year after treatment) with those obtained at the second time-point (3 years after treatment).The developed MATLAB package provided an automatic pupil detection, which was correct also for frames in which the pupil was not totally visible (up to 30% of area not visible), for instance when the eyelashes obscured the pupil, as shown in Figure The results related to the comparison between the two eyes are reported in Tables At 1 year follow-up, Response Amplitude (RA) and Constriction Velocity (CV) were higher when the injected (right) eye was stimulated compared to when the uninjected (left) eye was stimulated. These differences were statistically significant in all three subjects (with the exception of CV in patient 2). The asymmetry between left and right eye persisted through 3 years, the latest time-point tested, even if the differences of RA were statistically significant only in subjects 1 and 2 and the differences of CV were statistically significant only in subject 1. In Test 2, RA and CV were higher when the injected (right) eye was stimulated instead of the uninjected (left) eye at both follow-up time-points, even if the differences were not statistically significant. Results of Test 2 are not reported in details as no statistical difference was found for any parameter.The results of the comparison between the two follow-up time-points were reported in Tables Moreover, the analysis was performed also by distinguishing PLR according to the light intensity stimulus. The asymmetry between the two eyes with respect to RA, was statically significant in subject 1 at all three intensity levels, in subject 2 only at the highest one, in subject 3 only at the lower one.In this study, we describe the application of pupillometric analysis for evaluation of pupillary reflexes in patients undergoing gene therapy. This is clinically relevant because the pupillometry appeared to be the only objective ophthalmologic test able to detect an amelioration of visual function in treated LCA patients. The results showed that the patients had a higher PLR when the treated eye was stimulated rather than when the control one was stimulated. These findings are especially noteworthy as before treatment the treated eye had the worst responses . The absPupillometry has been widely investigated in patients with diabetes to provide information about autonomic neuropathy -24, showThe comparison of our results with the findings of other clinical gene therapy trial using pupillometric analysis  is limitAs regards software implementation, the developed MATLAB package improved the on-line pupil detection and automatically computed the pupillometric parameters for each stimulus. We believe that a check by the user is required in this field, as it would be improper that a result derived from an incorrect automatic detection might affect the decision whether or not an expensive therapy should be further investigated or transferred to clinical practice. A manual standardized protocol had been developed to check the results of the online pupil detection but the procedure required about 60 minutes for each pupillometric time-series. For that reason, a Grafical User Interface was developed to enable a semi-automatic revision of computerized detection, eliminating the need of this time-consuming procedure in which the user should manually check frame by frame using other imaging software. We tested that the same results  were achieved but in about 5 minutes  for each pupillometric time-series.Other objective techniques, such as fMRI, have been investigated to provide objective information about the improvement in retinal and visual function of LCA2 patients treated by gene therapy . fMRI haThis study presented some limitations, which are described in the following paragraphs. The implemented algorithm is designed to improve the real-time processing results provided by PupilFil4, and is not intended to be a method for real-time pupil measurement, such as those proposed by Zhu  or by IsAn interesting further development could extend to using pupillometric data as a measure of the ANS activity, for instance in conjunction with the Heart Rate Variability, another non-invasive marker of the ANS activity, widely investigated by the authors (PM and LP) both in healthy subjects  and in pIn conclusion, the results shown in this paper confirm that pupillometry is a useful objective measure to assess the effect of gene therapy in LCA patients. The developed program streamlined the analyses and allowed rapid statistical analysis of a range of parameters associated with PLR. The methods could be suitable in randomized clinical trials in order to assess the efficacy of gene therapy in LCA patients with no/low risk of patients’ and experimenters’ bias. With reference to the case study, the pupillometric analysis provided further support for the persistence over the time (3 years) of a significant improvement of retinal/visual function in the treated eyes.The authors declare that they have no competing interests.PM conceived the study and developed the method for off-line processing and data analysis, LP revised them. JB provided the set-up of the hardware for the pupillometry measurement. FT, SR, JB and FS designed the clinical protocol. FT performed the pupillometric test and acted as user of the MATLAB software. PM drafted the manuscript, LP, FT, SR and JB participated to drafting of the manuscript. FT, SR and FS recruited the subjects. All authors read, reviewed and approved the final manuscript.


Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.Withaferin A (WFA) is a steroidal lactone present in  Withania somnifera a plant that has been used in Indian (Ayurvedic) medicine for centuries. WFA has been employed in the treatment of a wide range of diseases including skin disease, diabetes, arthritis, and epilepsy Withaferin A (WFA) is a steroidal lactone isolated from Vimentin is a member of the large family of IF proteins which are encoded by more than seventy genes It has been shown that a normal cytoplasmic organization of VIF plays an important role in determining the shape and motility of mesenchymal cells such as fibroblasts. For example, the induction of vimentin expression and its assembly into VIF in MCF7 breast epithelial cells, which normally express only keratin IF, causes a rapid change to a mesenchymal cell shape. This shape transition is accompanied by a dramatic increase in cell motility, thereby mimicking the behavioral changes seen during the EMT Since there are no known small molecule inhibitors that specifically interfere with IF structure and function, it is important to determine how WFA interacts with VIF and to determine the specificity of these interactions with respect to altering VIF functions. This is especially important as WFA has recently been proposed as an anti-tumor agent. Even though a number of recent papers have reported the use of WFA as an inhibitor of VIF function, little is known regarding the mechanisms responsible for its effects on IF or the alterations in cell physiology that accompany WFA treatment. In this study we investigate the effects of short and long-term WFA treatment on VIF and its effects on cell shape, motility and proliferation. We have also determined the specificity of WFA for VIF by testing its affects on other types of IF, such as those comprised of the keratins, peripherin, and neurofilaments, as well as the microtubule and microfilament cytoskeletal systems.The effects of withaferin A (WFA) on vimentin IF (VIF) networks were determined in human fibroblasts (BJ-5ta) by immunofluorescence. In controls, VIF networks extend throughout the cytoplasm, from the nucleus to peripheral regions . After aPrevious studies from our laboratory have demonstrated that changes in the organization of VIF networks within fibroblasts affect cell shape, motility and the formation of lamellipodia The effects of WFA on BJ-5ta motility were determined using time-lapse imaging. The average rate of cell migration is significantly slower during the first 240 min following the addition of 2 μM WFA , which is located in the 2B region of the central rod domain The phosphorylation state of VIF has been shown to affect their assembly states, cytoplasmic organization, and their dynamic properties in vitro studies clearly show that WFA binds to vimentin in vitro. First, we analyzed the steps in assembly by sedimentation velocity ultracentrifugation in the presence and the absence of WFA  and 6.2 S (Na3PO4-buffer).Our results show that cysteine-328 is not required for the WFA induced reorganization of VIF in cells. However, e of WFA . We did The rate and extent of the assembly of VIF in the presence of WFA was observed by electron microscopy of negatively stained samples and viscometry as previously described Different mammalian cells express one or more types of IF proteins. Since the proposed binding site for WFA is in a domain that is highly conserved in all cytoskeletal IF We also determined the effects of WFA on the type III IF comprised of peripherin found mainly in peripheral neurons Most cancers arise from epithelial cells, which in their normal differentiated states express keratin (KIF), but not VIF. However, vimentin expression is up-regulated in, and is indeed one of the hallmarks of, the epithelial-mesenchymal transition (EMT) that accompanies the transformation of pre-metastatic to metastatic cells We also examined the effects of long-term treatment of cells with WFA as this would be required in cancer treatment. To this end, BJ-5ta cells were treated with 2 μM WFA for 24 hrs. Following this period of exposure, 76.1%±3.2% of the cells die as determined by Trypan blue exclusion. We also determined that apoptosis is the most likely cause of cell death as 69.0±2.2% of the cells treated with WFA are positive for annexin V Cells treated with lower concentrations of WFA (0.5 μM and 1 μM) for 24 hrs remain viable and contain less extensively altered VIF networks as indicated by a slight retraction of VIF away from the cell periphery compare  G.Based upon these findings, we also determined the rate of BJ-5ta cell proliferation by monitoring the number of cells every 3 days over a nine-day period. The rate of cell proliferation is similar between treated cells and controls during the first 3 days of WFA treatment at a concentration of 1.0 μM WFA. However there is a dramatic decrease in growth rate after day 3 in the WFA treated cells . These dSince IF interact extensively with microtubules (MT), and actin/microfilaments (MF) Withaferin A causes the redistribution of VIF in fibroblasts, from their normal arrays extending throughout the cytoplasm, into perinuclear aggregates. Reorganization of VIF into juxtanuclear aggregates has also been reported in cells exposed to either microtubule depolymerizing agents such colchicine In vitro studies suggest that WFA binds covalently to cysteine-328 in vitro. However we could not detect any alteration in the assembly kinetics of vimentin nor in the formation of their tetramer building blocks. Overall our results demonstrate that there are no detectable changes in the polymerization of vimentin subunits into 10 nm IF in the presence of WFA. Although it would be interesting to determine whether WFA binds to VIF in cells, this would require a method for labeling and localizing this small molecule in fixed cells or in vivo. Unfortunately this methodology is not yet available. Overall our data suggest that WFA may interact with other factors involved in regulating IF organization such as kinases or phosphatases. Support for this comes from the finding that WFA treatment is associated with a significant increase in phosphorylation of vimentin at serine-38. This particular residue is targeted by at least seven different protein kinases including PKA, PKC, Rho-Kinase, and Akt The changes in VIF networks in the presence of WFA are accompanied by alterations in the organization of MT and MF. This is not surprising as VIF and MTs have been shown to interact either directly In addition to VIF, WFA also alters the organization of peripherin, keratin, and neurofilament IF. Previous studies have demonstrated that the distribution of networks comprised of glial fibrillary acid protein (GFAP), another type III IF, is also altered during WFA treatment Prior to metastasis epithelial derived tumor cells undergo an epithelial to mesenchymal transition (EMT). One of the hallmarks of the EMT is an induction of vimentin expression 2 incubator at 37°C as described Human BJ-5ta fibroblasts  and A549  cells were maintained in DMEM  supplemented with 10% fetal calf serum . SW13-1HF5 For cell based experiments withaferin A (WFA) was purchased from ChromaDex  and dissolved in DMSO  at a stock concentration of 10 mM. WFA was added to culture medium at different concentrations for varying time intervals. Controls consisted of adding DMSO to culture medium at the same dilutions.Construction of wild-type vimentin expression plasmid is described elsewhere Vimentin IF were stained with either mouse monoclonal anti-vimentin V9  or in Cells grown on glass coverslips were rinsed with phosphate buffered saline (PBS) and fixed in methanol at −20°C for 5 min. For Time-lapse observations of live cells were made using the Nikon Eclipse TE2000-E microscope equipped with the Perfect Focus system  and an INU stage incubator system . Images were captured using a CoolSnapEZ camera  at 5 min intervals through a 10x objective.The rate of cell motility was determined by tracking the position of the center of the nucleus over time using Metamorph v7.0 . The distance between the positions were determined and divided by the interval between the time points and then averaged. The motility of control cells was determined for one hour in normal growth medium. Cell motility was monitored for 4 hrs in 2 μM WFA; the medium was refreshed and motility was monitored for an additional 16 hrs.For quantification of cell shape, fixed cells were imaged using a Zeiss AxioImager.Z1 equipped with a 40x phase contrast objective and a Zeiss AxioCam MRm camera . Cell perimeter and area were determined by tracing the cell perimeter using Zeiss AxioVision software . Cell shape was expressed using form factor (FF), 4π*(area)/(perimeter)∧2, as described previously .2.The induction of cellular senescence by treatment with WFA was determined by staining for β-galactosidase activity according to previously published protocols Cells were stained for annexin V externalization using the Annexin V Apoptosis Assay  according to the manufacturer's instructions and counted by FACS.Recombinant human vimentin, solubilized in 8 M urea, was renatured by overnight dialysis into either 5 mM Tris-HCl (pH 8.4) or 2 mM sodium phosphate (pH 7.5) containing 1 mM DTT as described Quantitative results are presented as mean ± standard deviation from three separate experiments. P-values were determined by a two-tailed Students t-test with unequal variances. Results were considered significant when the p-value was equal or less than 0.05.


To develop a reliable magnetic resonance elastography (MRE)-based method for measuring regional brain stiffness.First, simulation studies were used to demonstrate how stiffness measurements can be biased by changes in brain morphometry, such as those due to atrophy. Adaptive postprocessing methods were created that significantly reduce the spatial extent of edge artifacts and eliminate atrophy-related bias. Second, a pipeline for regional brain stiffness measurement was developed and evaluated for test-retest reliability in 10 healthy control subjects. This technique indicates high test-retest repeatability with a typical coefficient of variation of less than 1% for global brain stiffness and less than 2% for the lobes of the brain and the cerebellum. Furthermore, this study reveals that the brain possesses a characteristic topography of mechanical properties, and also that lobar stiffness measurements tend to correlate with one another within an individual.The methods presented in this work are resistant to noise- and edge-related biases that are common in the field of brain MRE, demonstrate high test-retest reliability, and provide independent regional stiffness measurements. This pipeline will allow future investigations to measure changes to the brain’s mechanical properties and how they relate to the characteristic topographies that are typical of many neurologic diseases. Magnetic resonance elastography (MRE) is a technique for performing noninvasive and quantitative “palpation” by measuring tissue stiffness . MRE is Clinically, MRE is most often used to quantify liver disease severity from the early stages of fibrosis to cirrhosis . More reThe purpose of this work was to develop a new pipeline to address two significant limitations in the field, and to assess test-retest reliability of this technique in a cohort of 10 healthy control subjects. First, we used simulation experiments to demonstrate how atrophy can produce a systematic bias in MRE-based stiffness measurements. This issue is of critical importance when using MRE to study neurodegenerative diseases, where the disease group is expected to have smaller brain volumes on average compared to the control group. We then developed methods to remove this bias using novel, adaptive postprocessing techniques. Second, since diseases of the brain have characteristic topographies, it was critical to develop MRE as a tool to measure regional brain stiffness. Recent publications have presented regional brain property estimates ,10. We rTo test postprocessing methods in a 3D object, we created simulated wave images (with no attenuation) masked by spherical shells of varying thickness. These images have the form u=sin(2πρ/λ-2πtf), where u is the displacement field, ρ is the distance from the point source, λ is the shear wavelength, t is time and f is the shear wave frequency. The shear waves were generated to simulate an object with a true stiffness of 3 kPa at 60 Hz with 3 mm isotropic image resolution. We simulated 3 motion encoding directions  by generating 3 wave images with the shear waves propagating from point sources of varying location outside the spherical shell masks. The wave images were masked with spherical shells of varying thicknesses, beginning with a 15 voxel thickness down to a 9 voxel thickness in increments of 2 voxels.3, and the Poisson's ratio was 0.49995. The density of the CSF was set to 1000 kg/m3 with a speed of sound of 1540 m/s. The models were solved using a frequency-response analysis at 60 Hz using quadratic Lagrange elements and a direct solver (UMFPACK). The FEM solves for the in-plane (x and y) components of the motion in the tissue. The inversion algorithms described below are designed for 3D vector displacement data. To process the 2D FEM data, it was assumed that the z component of the motion was zero everywhere and that all derivatives in the z direction were zero.2D FEMs of brains simulating progressive atrophy were constructed in COMSOL Multiphysics . An axial MR image from a normal subject was segmented to create a geometry consisting of brain tissue (not distinguishing gray and white matter) and cerebrospinal fluid (CSF). The FEM was a fluid-structure interaction model involving the plane strain mode of the Structural Mechanics Module  for the brain tissue and the pressure acoustics mode of the Acoustics Module for the CSF. Four geometries were constructed, one based on the original brain segmentation, and three others with progressive serial erosions of the brain geometry to represent atrophy. The mesh statistics for the four geometries are summarized in in vivo data. Second, the curl images are smoothed to make the stiffness estimate more resistant to noise. We use a filter of the form (1-x2)2(1-y2)2(1-z2)2 where x, y and z are linearly spaced from -1 to 1 over the chosen window size [ A number of methods exist for calculating stiffness from the acquired wave images. The approach we use in the brain can be summarized by three steps. First, we calculate the first temporal harmonic of the vector curl of the acquired wave images. Applying the inversion algorithm to the first temporal harmonic ensures that the shear waves only contain displacements at the frequency of interest, while calculating the curl removes the effects of longitudinal waves that would otherwise produce artifacts in the inversion results . For tradow size . Typicaldow size . These e Adaptive MRE postprocessing follows the 3 major steps described above: 1) calculate the first temporal harmonic of the curl of the wave data; 2) smooth the curl images; and 3) calculate stiffness by direct inversion. However, after an ROI has been defined for analysis, adaptive methods use unique convolution kernels for edge voxels in steps 1 and 2 in order to reduce edge artifacts in the final elastogram. To calculate the curl, central difference estimates of the partial derivatives are used for any voxels whose convolution kernel lies completely within the ROI mask. For voxels that lie along the edge of the mask, derivatives are calculated using a nearest neighbor estimate. The smoothing filter can likewise be made adaptive by creating a unique filter for each voxel that sets any elements outside the mask to 0, and then normalizes the filter to the sum of its non-zero elements. To evaluate the impact of these postprocessing methods on edge-related bias, we measured stiffness in these simulations as a function of ROI size. The ROI began as a full mask of the object of interest, and then was serially eroded by 1 voxel from all edges up to 3 times. Erosion was performed with a 6-connected neighborhood for the 3D spherical shell simulations, and with a 4-connected neighborhood for the 2D FEM simulations. This study was approved by the Mayo Clinic Institutional Review Board. All subjects were scanned after obtaining informed written consent. We scanned 10 volunteers  without known neurological disease 3 times each on one day to assess test-retest reliability. The subjects were removed from the scanner table, and the MRE apparatus was disassembled and reassembled between each MRE exam. MRE data were collected using a modified single-shot, spin-echo EPI pulse sequence . Shear waves of 60 Hz were introduced using a pneumatic active driver (located outside of the scanner room) and a soft, pillow-like passive driver placed under the subject’s head as previously described [ For each of the x, y and z motion encoding directions, complex-valued phase difference images for each phase offset were calculated by taking the product of the complex-valued MR images with positive motion encoding and the complex conjugate of the images with negative motion encoding. These resulting images have a magnitude equal to the product of the magnitudes of the positive- and negative-encoded images, and phase equal to the difference of the phases of the positive- and negative-encoded images. To minimize slice-to-slice phase discontinuities, constant and slowly varying phase in the acquisition plane was removed by first filtering the complex-valued phase difference images with a 2D lowpass filter (3x3 rectangular window function in k-space) and then calculating the difference between the phase of the original complex-valued images and the phase of the low pass filtered images . A brain mask and atlas regions for each subject were obtained using a separately acquired 3D IR-SPGR T1 weighted image . This image was segmented to calculate gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) content for each voxel as previously described . A lobarGM/WM  and the cerebellum. Each ROI was generated as the intersection between the brain mask and the warped atlas region. To calculate regional stiffness, the displacement data were first masked by the ROI for reasons which will be discussed later. Adaptive methods were used for calculating the curl and smoothing the masked data. As above, elastograms were calculated for each component of the curl using a direct inversion algorithm, which were then combined into one elastogram using a weighted average as above. Finally, the complex modulus was converted to shear stiffness and the stiffness was then summarized as the median over the ROI excluding 1 voxel from the edge of the ROI.The ROIs investigated included global (whole brain excluding cerebellum), frontal lobes, occipital lobes, parietal lobes, temporal lobes, deep  In the final step of the pipeline, brain stiffness was corrected for potential bias due to noise. To begin this process, we calculated SNR maps (one for each component of the curl) as previously described . Brieflyin vivo data. Based on simulated data, the relationship between the true stiffness (μtruth), stiffness calculated from noise-free data (μ∞), and stiffness calculated at a particular SNR (μSNR) is shown schematically in ∞) due to discretization errors [m) and the measured SNR. The approach is summarized as follows: Based on simulation experiments, low SNR is known to lead to underestimated stiffness measurements using a direct inversion technique when calculating stiffness as the median over an ROI. We implemented an SNR correction algorithm that uses simulated data with known SNR to correct for this bias. As in the spherical shell-masked simulations above, these simulations have the form u=sin(2πρ/λ-2πtf), where u is the displacement field, ρ is the distance from the point source, λ is the shear wavelength, t is time and f is the shear wave frequency. The point source was placed outside the FOV, and the resolution and number of time offsets were set to mimic the n errors . Stiffnesim) as the measured stiffness (μm).Initialize the stiffness of the simulation  by taking the median of this elastogram excluding 3 voxels from each edge.Apply the smoothing filter described above and calculate an elastogram using the direct inversion algorithm. Calculate the stiffness in the noise-free simulation  by taking the median of this elastogram excluding 3 voxels from each edge.Apply the smoothing filter and calculate an elastogram using the direct inversion algorithm. Calculate the stiffness in the noise-added simulation (μ∞/μSNR).Calculate a correction factor (CF) as the ratio of the stiffness calculated in the noise-free simulation to the stiffness calculated in the simulation with SNR equal to the measured SNR (CF= μm - μ∞), and increment the simulated stiffness μsim by Δμ.Calculate the difference between the measured stiffness multiplied by this correction factor and the stiffness calculated from the noise-free simulation (Δμ=CF*μRepeat steps 2 through 7 until Δμ is less than the tolerance (0.001 kPa for this work).sim).Report the corrected stiffness as the current simulated stiffness  and noise-added (μn) data for each of the 30 exams (10 volunteers with 3 repeated exams), and the error in the inversion was calculated as (μ0- μn)/μ0*100. Ideally the errors would be centered about 0 and have no relationship with the measured SNR. Therefore, the algorithm was tested by a paired Wilcoxon rank sum test between μ0 and μn, and by a Spearman rank correlation between the inversion error and the measured SNR within each ROI.The SNR correction algorithm was evaluated by testing the resistance of the stiffness measurements to additional artificial noise. Each MRE exam was reconstructed twice . For the noise-added images, Gaussian noise with zero mean was added to the data in k-space prior to wave image calculation. The standard deviation of this noise was chosen to cut the median SNR in the global ROI to 50% of the original SNR. Stiffness in each region was then calculated using the original (μ0) and noise-added (μn) data as defined above. The summary statistics tested include the mode . SNR within the brain is not normally distributed, as seen in the example histogram in post hoc comparisons were made with paired t-tests. Correlations between regions were tested by Pearson correlation. Test-retest repeatability in the 10 subjects was assessed by coefficient of variation (CV). To determine if stiffness in any region was different from any other region, we performed an ANOVA for repeated measures. Subsequent Example images of the spherical shell simulation are shown in This edge-related bias was also evaluated in the FEM simulations to test the adaptive methods in a more realistic geometry. The left column of 0) and stiffness calculated with the noise-added data (μn), as well as a significant relationship between inversion error and SNR . One more ROI had only a significant relationship between inversion error and SNR . However, these errors are small in absolute terms as the largest average inversion error is 0.29%, or equivalently in units of stiffness, 0.0093 kPa. Error was minimized by using the median of the most likely SNRs as the summary metric. Using the median to summarize SNR led to under-corrected stiffness values , and using the mode resulted in over-corrected stiffness values . The inversion error as a function of region both with and without SNR correction is shown in 0 and μn. The SNR correction algorithm was also cross-validated in an elderly cohort, which is summarized in Based on the above results we developed a pipeline to measure regional brain stiffness, which is summarized in GM/WM). The curl wave image is shown in the top-right panel, and the elastogram after 1 erosion is shown in the bottom-right.Example images for the global ROI are shown in GM/WM and cerebellum regions also indicate strong test-retest reliability with a median CV no greater than 1.98% and a maximum CV no greater than 4.48%. The repeatability of regional brain stiffness measurements is summarized in Post hoc comparisons indicated that stiffness in the parietal lobes and cerebellum were both significantly different from all other regions. In addition, frontal lobe stiffness versus deep GM/WM stiffness met trend level significance (p<0.1). We also calculated a correlation matrix to demonstrate the relationship of stiffness between regions of the brain within an individual. The results indicate that stiffness within the four lobes of the brain track together (5 out of 6 correlations are significant at the p<0.05 level), but stiffness within the deep GM/WM and the cerebellum are independent of the rest of the brain (1 out of 9 correlations significant at the p<0.05 level).We performed ANOVA for repeated measures, which indicated significant differences both between individuals (p<0.01) and between brain regions (p<0.001). In this work, we presented novel MRE postprocessing methods to measure regional brain stiffness. These techniques are resistant to noise- and edge-related biases that are common in the field, and reveal new insights into the topographical distribution of the mechanical properties of the brain in healthy control subjects.The simulation data in this work provide a causal link between brain geometry and biased stiffness measurements when the ROI does not properly account for edge artifacts in the elastogram. To fairly compare brain stiffness between subjects in varying disease states, the processing methods used must be resistant to changes in geometry such as those presented in this work. As mentioned earlier, this bias due to brain geometry is particularly important when investigating diseases of the brain that are known to cause changes in brain morphometry (such as brain atrophy due to neurodegenerative disease). A correlation between brain volume and MRE-based stiffness measurements has been previously reported ,19, and With these findings in mind, we developed a novel MRE pipeline to measure regional stiffness within the brain. This pipeline uses adaptive processing methods to significantly reduce the edge artifacts discussed above, thus saving most edge voxels. Retaining as many edge voxels as possible may be of critical importance for detecting biological signals. For example, amyloid is deposited in the cortex in Alzheimer’s disease and white matter injury occurs most frequently in the periventricular regions, both of which are along the surface of the brain. th and 90th percentiles. Note that the median stiffness does not change with increasing ROI size (meaning there is no systematic bias with ROI size), but the range of the stiffness measurements decreases with increasing ROI size (meaning the precision of stiffness measurement improves as the number of voxels in the ROI increases).These developed adaptive methods also improved the reliability of regional brain stiffness measurement. This finding can be explained by two factors. First, due to wave attenuation, shear wave amplitude is strongest at the outer edge of the brain and decreases as the waves propagate toward the center the brain. Therefore, the average SNR within any ROI will be increased by retaining these voxels near the edge that possess the largest shear wave amplitude. Second, increasing the number of samples in an ROI will improve test-retest reliability. This concept is demonstrated in simulation results that are summarized in In this work, we found a median global brain stiffness of 2.99 kPa at 60 Hz (range: 2.83 to 3.23 kPa) in young to middle aged normal volunteers, which is in close agreement with the most recent brain MRE literature. Johnson et al. scanned 3 young volunteers to evaluate their newly developed mulitshot, spiral EPI pulse sequence and reported an average shear modulus of 2.43+1.21i kPa in white matter at 50 Hz . This coThis work has provided new insights into the topographical distribution of stiffness within the brain. Based on these results, we observed that on average (considering the 4 lobes of the brain), stiffness is greatest in the occipital lobes, followed by temporal, then frontal (though stiffness was similar in these 3 regions) and finally the parietal lobes. Whether lobar variation in stiffness is explained by fundamental brain architectural differences at the lobar level is the subject of future investigation. Perhaps more importantly, these methods will allow us to investigate disease-related changes in brain stiffness on a regional basis to demonstrate that brain stiffness is not only sensitive to biological processes but also offers specificity , as has been indicated by preliminary work on Alzheimer’s disease .In conclusion, we used two types of simulations to demonstrate how atrophy can bias MRE-based stiffness measurements toward an underestimate of the true stiffness. We then developed adaptive techniques to reduce these edge artifacts. Using the simulations to test these methods, we demonstrated that the edge-related bias can be eliminated by eroding the ROI by only 1 voxel from the brain’s surface. Finally, we used these methods to develop a pipeline to measure regional brain stiffness in 10 healthy control subjects. These results indicate high test-retest reliability, and also that brain stiffness follows a characteristic topography.


Mode of delivery may affect the risk of asthma but the findings have not been consistent and factors shared by siblings may confound the associations in previous studies.The association between mode of delivery and dispensed inhaled corticosteroid (ICS) (a marker of asthma) was examined in a register based national cohort (n=199 837). A cohort analysis of all first born children aged 2-5 and 6-9 years was performed. An age-matched sibling-pair analysis was also performed to account for shared genetic and environmental risk factors.Analyses of first-borns demonstrated that elective caesarean section was associated with an increased risk of dispensed ICS in both 2-5 (adjusted odds ratio (aOR)=1.19, 95% confidence interval (CI) 1.09-1.29) and 6-9  age groups. In the sibling-pair analysis, the increased risk associated with elective caesarean section was confirmed in 2-5 year olds  but not in 6-9 year olds . Emergency caesarean section and vacuum extraction had some association with dispensed ICS in the analyses of first-borns but these associations were not confirmed in the sibling-pair analyses.Confounding by familial factors affects the association between mode of delivery and dispensed ICS. Despite this confounding, there was some evidence that elective caesarean section contributed to a modestly increased risk of dispensed ICS but only up to five years of age. A moderate 20%) increase in asthma prevalence has been described in children delivered by caesarean section,2. A num0% increaMost previous studies have used a simple dichotomised categorisation of deliveries as being vaginal or caesarean, not taking into account the important differences between elective and emergency caesarean sections, and unaided and assisted vaginal deliveries. It has been hypothesed that the increased risk of asthma in children born by caesarean section is due to a lack of microbial exposure during labour, via the vagina, resulting in delayed and altered colonization of the infant gut,8. AlterSome of the inconsistencies in previous studies may be due to insufficient control of confounding, differences in study size, age at outcome assessment and outcome measures.In this study, we used a Swedish national cohort to overcome some of these obstacles to determine if mode of delivery affects the risk of dispensed ICS, as an indicator of asthma, whilst taking into account relevant socio-demographic and perinatal confounders. The large study population has enabled us to both detect very small differences, and to examine these effects in an aged-matched sibling pair analysis, to account for shared genetic and environmental exposures in the family. A recent and similar Swedish registry-based study did not detect any association between elective caesarean section and asthma in sibling-pairs aged 9 and 12 years. In contrast, association by indication seemed to explain an increased risk for asthma related to emergency caesarean section[This study was based on information from the national health data bases held by the Swedish National Board of Health and Welfare and Statistics Sweden. Cross-linkage between registers was performed using each individual’s unique personal identification number. This study was approved by the regional ethics committee in Stockholm.The catchment population for this study were all full term , singleton children born in Sweden between 1/1/1999 and 31/12/2006, according to the Swedish Medical Birth Register. From this population we selected children who were residents in Sweden on December 2009 , were offspring of two native Swedish parents, had no major malformations reported at birth by the attending paediatrician or cerebral palsy according to the Hospital Discharge Register. Children with improbable combinations of birth weight and gestational age (<-6SD and >3SD) were excluded, as were children with both instrumental vaginal delivery and caesarean section delivery recorded.Information about parity, maternal age (at childbirth), measured maternal BMI in early pregnancy, mode of delivery, smoking habits in early pregnancy, maternal diseases and pregnancy complications was collected from Swedish Medical Birth Register. From the same register, perinatal data about sex, gestational age, birth weight, and low Apgar scores (<7 at five minutes) of the offspring were collected. Birth weight and gestational age were used to create the indicator of small -for-gestation-age  and large-for-gestational-age . Mode ofData on parental education was defined as the highest formal education attained. Educational level was categorised by years of education into <10 years, 10-12 years, 13-14 years and >14 years. Social welfare benefits received were collected from the Income and Enumeration survey for 2008 and dichotomised into any versus none.We created three proxy indicators for asthma based on data of retrieved prescriptions from the Swedish Prescribed Drug Register, which contains all drugs prescribed and dispensed in Swedish pharmacies, excluding drug treatment in hospital care. The indicators “dispensed inhaled corticosteroid (ICS)” was defined as the purchase of at least one prescription of inhaled steroids, either by itself  or in combination with other agents (ATC codes R03AK06 or R03AK07) while dispensed ICS at least twice was defined as purchase of at least two prescriptions during the same calendar year with these definitions.. Finally, an indicator labeled “any asthma medication“ was defined as the purchase of at least one prescription during a calendar year with an ATC-code that starts with R03 (any medication for obstructive lung disease).In the cohort of firstborns, ICS during 2009 was used as our primary outcome variable, while dispensed ICS from all four years (2006 to 2009) was used to create age-equalised comparisons for the sibling-pair analysis. Parental ICS was defined as receipt of an ICS during 2009 for all analyses and was used as a proxy for parental asthma.county variable with different levels of retrieval of prescribed ICS was created to adjust the analysis to these regional differences.There were considerable regional differences in the purchase of asthma medications reflecting regional differences in prevalence of asthma as well as differences in access to care and prescription patterns. A four-category Two related forms of analysis were performed. In the first, all first born children were included, and unconditional logistic regression models were used to assess the relationship between the different modes of delivery and risk of dispensed ICS by the child during 2009. Model 1 was adjusted for year of birth and sex only, in Model 2 we added other socio-demographic confounders  and parental dispensed ICS, in Model 3 we added perinatal risk factors associated with mode of delivery.In a second analysis, the effect of mode of delivery was assessed within sibling pairs discordant for mode of delivery and asthma medication, using conditional logistic regression (children grouped according to sharing the same mother). In this analysis we have adjusted for the potential confounders that might vary between the two pregnancies, but not for confounders that vary between mothers only. Model 1 was adjusted for sex, in Model 2 we added maternal age and parity and Model 3 was also adjusted for SGA, LGA, preeclampsia, maternal body mass index, maternal diabetes and gestational diabetes and neonatal respiratory distress, Sibling-pairs were selected on the basis of having the widest age gap possible, whilst still being able to match siblings on age. In this analysis sib age was equalised by taking ICS dispensing at the age of the youngest sib in the analysis in 2009 from the calendar year 2006 to 2008.For all forms of analysis, potential non-linear effects of maternal BMI were assessed using the “fracpoly” command within Stata . Effect modification, by gender, parity, maternal smoking, parental asthma (dispensed ICS) was assessed using Wald tests. As the nature of wheeze changes with age, the exposure to asthma medications was classified into two discrete age groups, 2-5 and 6-9 years of age.Altogether 13 179 (12.7%) boys and 9934 (9.2%) girls in the age two to nine years had purchased any asthma medication in 2009, and of these 7180 (6.9%) boys and 4722 (4.6%) girls had retrieved at least one prescription of ICS.Dispensed asthma medication increased gradually with increasing maternal BMI Table . Childree.g. hypertension and diabetes were more common in those who were delivered by elective caesarean section compared with those who were delivered by emergency caesarean section. In contrast, low Apgar was overrepresented after emergency caesarean section and vacuum extraction.Table Children delivered by non-instrumental vaginal delivery had the lowest rates of asthma medication in all age groups Figure . ChildreWhen compared to vaginal delivery without assistance, both elective and emergency caesarean sections were associated with a modest increase in risk of any dispensed ICS (at least one prescription) in the 2-5 year olds after adjustment for socio-demographic confounders Table , model 2Delivery by vacuum extraction was associated with a slightly increased risk of dispensed ICS, in the 6-9-years olds , after adjustment for confounders and a borderline increased risk in the 2-5 year olds . Interaction analyses showed that the effect of mode of delivery was similar in boys and girls.We have also assessed the associations between mode of delivery and at least two dispensed prescriptions of ICS as the outcome. The findings differed only slightly from the analyses based on at least one dispensed prescription of ICS  to define current symptoms of asthma. We have previously used a similar definition as has aHowever, dispensed medication as a proxy for asthma is affected by a number of factors at different levels such as awareness of the symptoms by the child and the parents, severity of asthma, health seeking behavior, diagnostic criteria by the doctor, attitude to medication and its related costs. If one sibling has asthma, other siblings are more likely to be diagnosed and get a prescription for an asthma medication than children in families where the symptoms of asthma are less familiar. Sharing of medication may occur in families where several members suffer from asthma. When asthma is diagnosed compliance with medication probably differ in different families, and this will have an effect on subsequent prescriptions. Many of these family effects could contribute to a negative outcome and some of these such as sharing of medication could weaken the associations also in the sibling analyses. Using purchase of ICS may imply that we have missed individuals (parents or children) with undiagnosed or mild asthma. Therefore, residual confounding may explain some of the reduced effect sizes in the sibling analysis.In young children, dispensed ICS is a proxy for respiratory illness, but not always for asthma. as diagnosis of asthma is very difficult in preschool children. By the age of 6 years, the prevalence of transient viral wheeze has largely passed, and a diagnosis of asthma is much easier to confirm, making this a reasonable time point to divide the cohort of children. Variability in prescription pattern could dilute the association between asthma medication and mode of delivery in young children. For thiAn association between caesarean section and asthma could also be weakened or even missed, if potential effects are restricted to specific genetic variants or subgroups of asthma. We do not have direct measures for atopy in this study. Therefore, we cannot exclude that mode of delivery could affect allergic induced symptoms. Moreover, we have no information in the registers concerning postnatal exposures. However, a recent birth cohort study failed to identify modification of the association of between mode of delivery and childhood asthma by a range of key postnatal exposures, including breast-feeding behavior and age at day care entry.We cannot exclude the risk of some misclassification of the caesarean sections. “Elective” caesarean sections were not necessarily optional. We considered a caesarean section to be emergent if the operation was made after the onset of labour. Therefore, caesarean sections due to emergencies such as fetal distress or a severe maternal complication were defined as elective if the caesarean sections were performed without preceding labour. Although this would have resulted in some misclassification between emergency and elective caesarean sections, as this reason for cesarean section is relatively uncommon, we consider it to be an unlikely source of a major bias in this study. The major indications in Sweden for elective caesarean sections after 36 weeks are breech presentation, previous caesarean section, cephalopelvic disproportion and psychosocial factors. They accounted for more than 80% of all elective caesarean sections between 1996 and 2007.To conclude, elective caesarean section contributes to a modestly increased risk of asthma medication, but only up to five years of age. The associations between emergency caesarean section or vacuum extraction and asthma medication seen in the firstborns appear to be due to residual confounding by familial factors.All authors declare that they have no competing interests.LB contributed to the design of the study, conducted the literature review, prepared the first draft and revised the final version of the manuscript. CE contributed to the design of the study, conducted the data analyses and revised the final version of the manuscript. AL designed the study’s analytic strategy, performed the sibling analyses and revised the manuscript. AH designed the study and the analytic strategy, performed register linkages, created the dataset, performed some statistical analyses, directed the implementation including quality assurance and control and contributed to all parts of the manuscript. All authors read and approved the final manuscript.The association between mode of delivery and use of inhaled cortisone at least twice during 2009. Crude and adjusted odds ratios after non-conditional logistic regression. Click here for fileAssociation between mode of delivery and inhaled cortisone at least once in discordant sib-pairs. Click here for file


Juvenile dermatomyositis (JDM) is a rare, often chronic, systemic autoimmune disease of childhood, characterized by inflammation of the microvasculature of the skeletal muscle and skin. Prominent clinical features include significant exercise intolerance, muscle weakness, and fatigue. Despite pharmacological improvements, these clinical features continue to affect patients with JDM, even when the disease is in remission. Exercise training is increasingly utilized as a non-pharmacological intervention in the clinical management of (adult) patients with chronic inflammatory conditions; however no randomized controlled trials (RCT) have been performed in JDM. In the current study, the efficacy and feasibility of an exercise training program in patients with JDM will be examined.n = 30) will include 8–18 year olds diagnosed with JDM. The intervention consists of an individually tailored 12-weeks home-based exercise training program in which interval training on a treadmill is alternated with strength training during each session. The program is based on previous literature and designed with a defined frequency, intensity, time, and type of exercise (FITT principles). Primary outcome measures include aerobic exercise capacity, isometric muscle strength, and perception of fatigue. The study methodology has been conceived according to the standards of the CONSORT guidelines. The current study will be a multi-center  RCT, with the control group also entering the training arm directly after completion of the initial protocol. Randomization is stratified according to age and gender.Subjects : NTR 3184. The pediatric forms of inflammatory idiopathic myopathies (IIM) embody a group of rare systemic autoimmune conditions, characterized by chronic skeletal muscle inflammation with an age of onset of <18 years. Despite being a rare condition, juvenile dermatomyositis (JDM) is the most common type of pediatric IIM, representing about 85% of IIM diagnoses in childhood . PromineExercise training is increasingly utilized as a non-pharmacological intervention in the clinical management of patients with chronic inflammatory conditions. As opposed to typically developing healthy children, those with chronic illness are often restricted in their participation in physical activity as a consequence of real or perceived limitations imposed by their condition. The chronic condition itself often causes hypoactivity leading to a deconditioning effect and a reduction in functional ability, which ultimately results in a downward spiral of further hypoactivity and deteriorating health . FurtherStudies in patients with adult and juvenile dermatomyositis suggest that exercise training is safe since it has not been associated with any increases in the degree of inflammation after a single bout of exercise in JDM , or afteWhile there is limited evidence to support the role of exercise training in the management of patients with JDM, definitive evidence is still lacking. In fact, to our knowledge, no randomized controlled trials (RCT) of exercise training in JDM have been performed, which may be secondary to limited patient availability and other feasibility issues.As such, we designed a RCT to study the feasibility of an individually tailored 12-week home-based exercise training program, and to assess its efficacy in children and adolescents with JDM. The wash-out effects of this program after another 12 weeks will also be examined.The current study is a multi-center (4 sites) randomized controlled trial, with the control group also entering the training arm directly following completion of the initial protocol. After randomization 1:1), subjects in both groups will perform a test battery of exercise tests, questionnaires, and other measurements (see study outcomes) (T0 measurement). Thereafter, subjects in the intervention group will receive an exercise training regimen, while the subjects in the control group will continue to receive standard care over a 12-week period. Subsequently, subjects in both groups will repeat the battery of tests from T0 (T1 measurement). Subjects in the intervention group will then receive individual advice based on their T1 results and educated on the physical activity guidelines for children  in order:1, subjeAll patients diagnosed with JDM by a pediatric rheumatologist/immunologist according to the Bohan and Peter criteria between the ages of 8 and 18 years from 4 outpatient clinics in the Netherlands will be assessed for eligibility. The 4 outpatient clinics include the Wilhelmina Children’s Hospital University Medical Center (UMC) in Utrecht, the Beatrix Children’s Hospital UMC in Groningen, the Sophia Children’s Hospital Erasmus Medical Center in Rotterdam, and the St. Radboud Children’s Hospital UMC in Nijmegen.Patients will be excluded for eligibility upon (1) a medical status that contraindicates any form of exercise testing; (2) an insufficient understanding of the Dutch language in the patient and/or the parents/caregivers; (3) the presence of medical events that might intervene with the outcome of testing and/or the trial (such as a planned operation) and/or (4) a negative advise from the rheumatologist and/or physiotherapist based on resent relapse or no limitations in exercise tolerance (in other words: the patient was either not good enough or too good). All eligible patients will be asked to participate in the study. All parents/caregivers as well as subjects >12 years of age years must provide informed consent before enrolment in this study, which is approved by the Medical Ethics Committee of the UMC Utrecht, the Netherlands.The flow of the subjects in the study will be depicted in the CONSORT Flowchart format . The reaThe intervention comprises of an individually tailored 12-week home-based exercise training program, combining interval training on the treadmill  with strength training. Both types of training are selected based on recommendations for patients with chronic conditions , as wellThe aim of the interval training is to improve the aerobic exercise capacity as it has previously been shown to increase peak oxygen uptake in healthy children and adolescents . MoreoveThe aim of the strength training program is to improve the muscle strength of proximal muscle groups, since these tend to be most affected in JDM. Along with the recommendation that strength training in youth should include large muscle groups , the folInterval training on the treadmill and strength training will be performed in an alternating fashion during each session.In order to improve aerobic exercise capacity and muscle strength in children and adolescents, a training frequency of 3-4  and 2-3 peak) among children : the absolute VO2VAT divided by body mass; O2 pulse [ml·beat-1]; and endurance time [min]: the time from the start to the end of the protocol.The outcome measures related to aerobic exercise capacity that will be assessed include: VOIsometric muscle strength of the proximal muscle groups in the lower extremities will be measured on both the right and left sides with a calibrated CITEC hand-held dynamometer . Both the extensors of the knee joint and the flexors of the hip joint will be assessed with 3 consecutive measurements according to the ‘break method’, wherein the examiner gradually overcomes the muscle force and stops at the moment the extremity gives way [ives way . The higives way .Perception of fatigue will be measured with the Dutch translated PedsQL Multidimensional Fatigue Scale. This scale, designed to measure fatigue in pediatric patients, reflects 3 subscales: general fatigue, sleep/rest fatigue, and cognitive fatigue [ fatigue -37.Muscle soreness will be measured using a 10-cm Visual Analogue Scale (VAS). Muscle function will be assessed via Subscale 8 (Strength) of the Bruininks-Osteretsky Test of Motor Proficiency, Second Edition (BOT-2), which include 5 items that are related to the strength training program of the subjects [muscle function in our subjects [Functional sub maximal aerobic exercise capacity will be assessed by examining walking distance at the 6-min walk test (6MWT) [physical activity enjoyment using the Physical Activity Enjoyment Scale (PACES) [Quality of life will be measured with the patient form of the Dutch translated PedsQL Generic Core Scale [Functional ability will be measured with the Childhood Health Assessment Questionnaire (CHAQ), a disease specific questionnaire that measures the grade of disability in performing activities of daily living as observed by the parents [physical activity will be monitored via accelerometry  [subjects . The Chisubjects . Functiot (6MWT) . This wi (PACES) . Qualityre Scale ,42,43. F parents -46. The Actical)  and a BoActical) ,49 over 2peak/kg. The mean ± standard deviation VO2peak/kg of patients with JDM (in remission) is 33 ± 8 ml·kg-1·min-1[2peak/kg by at least 22%, which is the weighted mean improvement in exercise training studies in adults with inflammatory myositis [n = 5) chance of missing observations and/or drop-outs, the total sample size increase to 27 patients. For simplicity in the randomization process, we want to include 30 patients.A power calculation was performed based on previously published data relating to one of the primary outcome measures: VOg-1·min-1. The aimmyositis . Given aData will be checked for normal distribution. Three different types of analysis will be performed.To examine the efficacy of the intervention, the outcome measures of the intervention group and control group will be compared at each of time points T0 and T1 using mixed linear regression analysis with group (intervention/control) as independent variable and the outcome measures see Table as separT-test.To examine the efficacy of the intervention in the control group, changes in outcome measures between T0 and T1  will be compared with the changes in outcome measures between T1 and T2 (intervention) using a paired samples Repeated measures ANOVA analyzes will be performed to examine whether the effects of the intervention are maintained after 12 weeks. Specifically, the outcome measures of both groups will be compared between post-12-week usual care after intervention vs. post-intervention and post-12-week usual care after intervention vs. pre-intervention.Results will be presented as linear regression coefficients representing mean group differences with their corresponding 95% confidence intervals (CI). Statistical significance will be defined as cases in which the 95% CI does not include the null value. Nominal variables will be analyzed using chi-square analysis. All results will be analyzed using the modified intention to treat approach which means that all subjects will be included in the groups to which they are randomized, and the researchers make efforts to obtain outcome data for all subjects, even if they do not complete the full intervention .e.g. due to illness or holiday), the subject will be asked to resume the exercise training program where he/she was ended.Feasibility will be assessed by examining the amount of training sessions each subject performed during the intervention. Every week, the subject has to start the exercise training program of a new week, independent whether the proposed amount of training sessions of the previous week is reached. When the subject is not able to train for at least 7 consecutive days (This paper presents the design of a randomized controlled study that will provide evidence on the efficacy and feasibility of an individually tailored 12-week home-based exercise training program for children and adolescents with JDM. The wash-out effects of this program after another 12 weeks will also be examined.Among the many strength of this study is first, the fact that this will be the first RCT to assess the effects of exercise training in patients with JDM. Second, evidence from literature is used to design the treadmill training as well as the strength training program. Third, the focus of the intervention, as well as the primary outcome measures, corresponds to the major clinical concerns of patients with JDM. Finally, and perhaps most importantly, the intervention is a home-based program, which minimizes the load for the patients and their parents while likely stimulating the patient’s desire and motivation to continue exercising upon completion of the intervention.The study methodology has been conceived according to the standards of the CONSORT guidelines .The authors declare that they have no competing interests.All authors contributed in designing, drafting, and revising the manuscript, and they all have given approval of the final version. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/13/108/prepub