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magnetic resonance imaging (mri) permits to assess tissue abnormalities in vivo and approximate histopathological changes of the multiple sclerosis (ms) disease (ganiler., 2014 ; kearney., 2014 several studies have shown that the percentage of change in brain atrophy tends to correlate with the progression of the disease (prez - miralles., 2013 ; sormani., 2014 moreover, changes in gray matter (gm) atrophy are observed independently from white matter (wm), and hence atrophy measures based on segmentation - based methods are nowadays employed as they allow classifying brain tissues separately (prez - miralles., 2013). the performance of different segmentation methods used to quantify brain atrophy or volume estimation has been evaluated deeply in the last 5 years (klauschen., 2009 ; derakhshan., 2010). however, it is well known that the presence of wm lesions can induce errors on brain tissue volume measurements (chard., 2010 ; battaglini., 2012 ; gelineau - morel., 2012) and non - rigid registration (sdika and pelletier, 2009 ; diez., 2014), if lesions are processed within the images. for instance, if wm lesion voxels are classified as wm, lesion voxels with hypointense signal intensities are added into the wm tissue distribution, increasing the probability of gm voxels with similar intensity to be misclassified also as wm (chard., 2010). in the last years, some authors have proposed different techniques to overcome these issues in ms patients by filling wm lesions with intensities similar to those of wm before performing tissue segmentation and image registration. these methods can be divided into two groups : methods which use local intensities from the surrounding neighboring voxels of lesions (sdika and pelletier, 2009 ; battaglini. 2013) and methods which use global wm intensities from the whole brain (chard., 2010). in all cases, the performance of these methods is directly related with their ability to minimize the impact of refilled voxels on original tissue distribution, not only due to the addition of these voxels into the tissue distribution, but also due to the effect on the global tissue distributions of filled images. within local methods, sdika and pelletier (2009) have proposed to refill each wm lesion voxel with the mean of its three - dimensional neighboring normal appearance white matter (nawm) voxels. (2012) have suggested refilling each wm lesion voxel with intensities derived from a histogram of nawm voxels surrounding the two - dimensional lesions. in a recent study, magon. (2013) have proposed to refill each two - dimensional lesion with the intensity from the mean of the surrounding area of the lesion. regarding global methods, (2010) have proposed a different approach by using intensities re - sampled from a global wm distribution to refill wm lesion voxels, based on the mean and standard deviation of the total nawm of the whole image. both chard. fsl - l (battaglini., 2012) runs from a computer command - line and does not provide any graphical interface that aids the process. this technique has been integrated into the latest fsl package, and therefore it depends on the whole fsl installation. in the case of leap (chard., 2010), the method runs as a stand - alone script also from the command - line and requires the installation and configuration of several external dependencies, which may be difficult to install for non - computer experts. in this paper we propose a new technique to refill wm lesions which is a compromise between global and local methods. hence, for each slice composing the three - dimensional image, we compute the mean and standard deviation of the signal intensity of nawm tissue. on the one hand, compared to local methods (battaglini., 2012 ; magon., 2013) which only make use of a limited range of voxel intensities, the fact of using global information from the whole image slice reduces the bias caused by refilled voxels on gm and wm tissue distributions, especially on images with high lesion load. on the other hand, compared to other global methods (chard., 2010), which are based on the mean signal intensity of the nawm of the three - dimensional image, our method re - computes the mean signal intensity of the nawm at each two - dimensional slice with the aim of reproducing more precisely the signal variability between mri slices, especially in low resolution images. in order to easily integrate it into current platforms, the proposed method called slf is currently available as a stand - alone program and as spm1 extension at the salem group site (http://atc.udg.edu/salem/slftoolbox). to evaluate the performance of our method, we estimate the deviation in gm and wm tissue volume between a set of healthy images and the same images where artificial wm lesions have been refilled with the proposed technique. to do so, we register wm lesion masks from diagnosed ms patients into two sets of 30 1.5 and 3 t t1-weighted (t1-w) images of healthy subjects, respectively. afterwards, we simulate realistic lesions on healthy images by replacing the signal intensities of registered lesion voxels with values similar to those of the mean gm / wm interface. brain tissue volume is computed using both fast (zhang., 2001) and spm8 (ashburner and friston, 2005) segmentation methods, in order to avoid possible correlations between the filling and segmentation processes. furthermore, we compare our results with the same images where artificial wm lesions have been segmented as normal tissue, masked - out before tissue segmentation, and refilled using also the methods proposed by chard. the first set of images is composed of 30 images of healthy subjects (matrix size : 176 208 176, voxel size : 1 1 1.25 mm), acquired on a 1.5 t vision scanner (siemens, erlangen, germany) and obtained from the open access series of imaging studies (oasis) repository2 (marcus., 2007). only images from young and middle - aged subjects are selected (age 0.13). wm lesions are masked out from the t1-w image using the provided lesion mask, in order to avoid the influence of artificial lesions on tissue distributions. the resulting image is used to estimate the probability of each voxel to be classified as csf, gm, and nawm, by segmenting tissue with a fuzzy - c - means approach (pham, 2001). the fuzzy - c - means implementation used here follows the algorithm described in pham (2001), with clusters initialized according to bezdek. moreover, input signal intensities are constrained to the mean plus three standard deviations of the signal intensity of the image, in order to avoid outlier signal intensities, such as residual parts of the eyes or neck. from the obtained tissue segmentation output, we compute the three - dimensional nawm mask from the image voxels with the highest probability to pertain to the wm cluster. finally, the lesion - filling process is achieved as follows : for each axial slice composing the three - dimensional image, we compute the mean and standard deviation of the signal intensity of nawm tissue. axial sampling is motivated because after testing the sampling procedure on the coronal, axial and sagittal planes, we found that the best results were obtained when we sampled the axial plane. this was due to the fact that using the axial plane reduced the variability of possible existing wm intensities, when compared to coronal and sagittal sampling. the fuzzy - c - means approach used to estimate the tissue probabilities is a simple method which in fact does not take into account neither spatial nor neighboring information, and hyper - intense signal intensities such as residual parts of the eyes or the neck produced in the skull - stripping process can bias significantly the clusters. the risk of adding these parts into the wm distribution is minimized in the axial plane because we are reducing it to a certain slice where lesion volume is usually lower than that in central slices. the computed mean and standard deviation values are used to generate a normal distribution with mean equal to the computed nawm mean intensity and standard deviation equal to half of the computed nawm standard deviation. standard deviation is always fixed to half of the wm mean independently of the dataset used. this value was chosen empirically with the aim of balancing the accuracy of the method with both 1.5 and 3 t images. although a specific tuning of this parameter could provide a better performance on certain cases, we decided to fix it avoiding therefore the number of parameters to tune. lesion voxel intensities from the current image slice are replaced by random values of the generated distribution. we compute the absolute percentage % difference in normalized gray matter volume (ngmv) and normalized white matter volume (nwmv) between each original and its correspondent lesion - filled images. normalized volumes are obtained as the ratio of voxels outside lesion regions segmented as gm or wm and the total number of segmented voxels, respectively. for instance, the % difference in ngmv is computed as:%=ngmvfilledngmvorigngmvorig100 where ngmvfilled and ngmvorig values refer to the computed volumes for the lesion - filled and original images, respectively. the higher the performance of the lesion - filling method, the lower the percentage difference between lesion - filled and original images. in order to analyze possible correlations between the filling process and the segmentation method employed, brain tissue volume is calculated independently on the same subjects using fast (zhang., 2001) (v.5.0.5) and spm8 (ashburner and friston, 2005) (v.4667) approaches. we compare the performance of our method with respect to other existing techniques such as the ones proposed by chard. we also add two more sets of images into the comparison : images segmented with artificial lesions and images where wm lesions have been masked out before tissue segmentation. given the small differences in ngmv and nwmv between original and lesion - filled images, the use of a standard analysis of the variance (anova) or a classic t - test is impractical here. instead, we perform a series of permutation tests to determine significant differences in tissue volume between pairs of methods (menke and martnez, 2004 ; valverde., 2014). the permutation tests return the mean and standard deviation of the fraction of times that the difference in ngmv and nwmv for a current lesion - filling method is smaller than the rest of methods with p - value 0.05. afterwards, methods are presented in 3 ranks determined by the mean and standard deviation of the best method and the distance with respect to the mean of the rest of methods (valverde., 2014). in our experiments, we set the number of comparisons between each pair of methods to n = 1000. 2 depicts the absolute mean % difference in ngmv and nwmv between the 30 original 1.5 t images and the same images with artificial lesions (none), masked - out lesions before segmentation (masked), and lesion - filled using magon. (2010) (leap), and finally our proposed algorithm slf. when fast is used, slf reports the lowest absolute mean difference in ngmv (0.16 0.14), followed by leap (0.40 0.30) and fsl - l (0.43 0.58) methods. our proposal also provides the lowest difference in nwmv (0.29 0.36), followed by fsl - l (0.81 1.28). maximum values in ngmv are found in none images, with differences up to 2.30 2.62 in ngmv and 3.85 4.81 in nwmv. when spm8 is used, slf also reports the lowest differences in ngmv (0.09 0.14), followed by leap method (0.12 0.13). our proposed method also performs better than the rest of the methods on nwmv (0.20 0.24), followed by the leap method (0.36 0.40). again, the highest differences in ngmv (1.84 1.97) and nwmv (4.82 4.58) are found in none images. table 1 shows the absolute mean difference in wm volume for all methods where lesion volume has been ranged by size intervals. table 2 presents the performance of each filling - method after running all possible pair - wise permutation tests. with a significant p - value of 0.05, all tests run on images segmented with fast show the superiority of slf over the other methods presented. on images segmented with spm8, all tests show a clear superiority of slf over the other methods on nwmv, while a similar performance of slf and leap over the other methods on ngmv. we also test the performance of our algorithm using 3 t data. as before, fig. 3 shows the absolute mean % difference in ngmv and nwmv between the 30 original 3 t images and the same images with added lesions (none), masked - out lesions before segmentation (masked), and lesion - filled methods magon, fsl - l, and leap, and our proposed approach slf. when fast is used, slf reports the lowest absolute mean % difference in ngmv (0.06 0.06), followed by leap (0.09 0.10). our method slf also performs the lowest difference in nwmv (0.09 0.09), followed again by leap (0.12 0.08). maximum values in ngmv are found in none images, with differences up to 1.40 1.56 in ngmv and 1.00 1.32 in nwmv. when spm8 is used, both leap (0.04 0.06) and slf (0.05 0.05) yield the lowest absolute % mean difference in ngmv. on nwmv, also leap (0.09 0.12) and slf (0.08 0.09) report the lowest absolute mean % difference in volume between original and lesion - filled images. again, highest differences in ngmv (1.84 1.97) and nwmv (4.82 4.58) are found in none images. table 3 shows the absolute mean difference in wm volume for all methods on ixi images, where lesion volume has been ranged by size intervals. table 4 shows the performance of each filling - method after running the permutation tests. tests run on images segmented with fast show a significant superiority of slf over the rest of the methods on nwmv, and a slightly better performance of slf with respect to leap on ngmv, although both methods are clearly superior to the rest of methods presented. when spm8 is used, tests show a similar performance of slf and leap over the rest of the methods on both nwmv and ngmv. several studies have proposed to use different filling techniques in order to reduce the effects of wm lesions on brain tissue measurements of t1-w images. up to date, only leap (chard., 2010)5 and fsl - l (battaglini., 2012)6 are publicly available methods that permit to refill t1-w images given a wm lesion mask. the lesion segmentation toolbox (lst) proposed by schmidt. (2012) also provides a lesion - filling approach based on the work of chard. (2010), but it is dependent of a flair image and an internal lesion - probability map obtained during the lesion segmentation step. in general, deviation in tissue volume between original and lesion - filled images tends to be higher on 1.5 t oasis images than on 3 t ixi images. the observed deviation is caused by differences in intensity, slice thickness and dimensionality between datasets. on ixi images, the distance between gm and wm signal intensity distributions is narrower than that of 1.5 t data. applying the lesion generation algorithm (battaglini., 2012) with identical parameters of those used with 1.5 t images creates simulated lesions whose intensity are noticeably similar to the mean wm, because the standard deviation of the generated lesion distribution is the mean between the gm and wm tissue divided by 4. however, this fact only explains the difference found on images segmented with artificial lesions. in the rest of the methods, the signal intensity of the generated lesions is not interfering with the obtained results since in all cases lesion voxels are replaced before tissue segmentation. on images where lesions have been masked before segmentation (masked), the lower deviation in tissue volume of 3 t images can be explained by the increase in the resolution of the images when compared to 1.5 t data, which reduces the effect of masked voxels in tissue distributions. the same reason can be behind the lower deviation found on all four lesion filling methods. by increasing the number of slices, differences produced by the methods on certain slices can be smoothed by tissue segmentation methods. moreover, the use of a reduced sampling space or a better tuning of the parameters involved in the wm tissue distribution generated to refill lesion voxels could increase the performance of the presented method. nevertheless, in all our experiments we decided to fix the standard deviation to 2 for simplicity. analyzing the results by dataset, on 1.5 t images from the oasis dataset, our results show that compared to the available methods, the proposed algorithm slf reduces significantly the differences in nwmv between original and filled images, independently of the brain tissue segmentation method used to measure the tissue volume. with the same data, although our method reports the lowest mean % difference in ngmv when spm8 method is used, the permutation test clearly shows that differences between slf and leap are not relevant. on 3 t images from the ixi dataset, slf also yields the lowest mean % differences in ngmv and nwmv, when fast is used to measure tissue volume. these results are clearly significant in nwmv, but not in ngmv, although our method reports also the lowest difference among all methods. when spm8 is used, slf presents a similar performance of that of leap, and both methods tie on the results of the significance tests. compared with local methods, our algorithm performs quantitatively better than local methods on images with high lesion load (> 36 ml). the magon method incorporates all neighbor voxels surrounding a wm lesion region to compute a mean intensity which is used to refill all lesion voxels. on images with high lesion load touching gm tissue, including gm voxels can decrease refilled intensities and modify the tissue distribution of filled images. fsl - l overpasses this limitation by building an intensity distribution based only on wm voxels surrounding lesions. however, on large lesion regions, all lesion voxels will be filled with a narrow range of intensities coming from the neighboring voxels that can have a direct incidence on gm and wm tissue distributions. by contrast, lesion volume appears to affect less global methods. in our case, the intensity distribution generated to refill lesion voxels will be independent of both the size and the position of lesion. furthermore, the effect of filled voxels on the global wm tissue distribution is smoothed by the addition of intensities which try to reassemble the global nawm of the current slice. compared with global methods, there are some interesting differences between our method and leap. contrary to local methods, global methods have to deal with the skull - stripping process before processing images. leap incorporates the skull - stripping process as part of the processing pipeline. in addition by contrast, our method does not deal with skull - stripping internally, and the method requires an already skull - stripped image or a brain mask. as noted previously, the skull - stripping method employed seems to not interfere significantly in the results obtained by our method. while setting up each of the different processes involved in the proposed pipeline, we found that, at least with our data, the performance of leap decreased or failed in 1.5 t scans when the skull - stripping methods bse (shattuck., 2001) and bet (smith, 2002) were used with default options. by contrast, leap provided the best results when the optimized method proposed by popescu. (2012) was used. this fact motivated the selection of this skull - stripping method for all the experiments of the study. furthermore, on both datasets, we have also compared the differences between our method and leap estimating the mean nawm intensity used as a basis to fill lesion voxels. in most of the images, the global mean nawm intensity does not differ significantly between fityk7 on leap and our fuzzy - c - means approach. hence, we can reject the hypothesis that observed differences in 1.5 t images can be caused by the approach employed to compute the nawm tissue distribution before filling lesion voxels. however, on both lesion - filling approaches, tissue segmentation methods tended to increase the apparent mean wm tissue distribution on 1.5 t images with high lesion load (> 40 ml) due to the increase of voxels refilled with intensities higher than the actual mean wm signal intensity. this effect is clearly more visible on leap than in our method, especially when fast is used. the resolution of the oasis 1.5 t images (176 208 176 slices) is lower than that of ixi 3 t images (256 150 256 slices). on images with low number of slices, after comparing the a priori wm tissue distribution values estimated by both the leap and slf methods with the already computed wm tissue distributions obtained from healthy images, we found that as lesion size increases, global methods such as leap and slf tend to increase the differences in tissue volume with respect to original images. in both methods, we have observed that the a priori estimated mean intensity of the wm distribution tends to be higher than the actual tissue distribution as computed by fast and spm8 on healthy images. as lesion volume increases, the addition of more filled voxels with intensity higher than the actual mean tissue intensity is more prominent, causing a displacement of the mean intensity of the wm distribution returned by the segmentation methods on filled images. consequently, more voxels bordering gm / wm are segmented as gm and wm tissue volume decreases. in this scenario, the strategy followed by slf, where wm is sampled independently at each slice, is more robust to the increase of lesions size than a global estimation of the wm tissue (leap) because possible errors introduced by a particular slice are not propagated into the rest of the slices. contrary to spm8, which estimates the tissue distributions based on a gaussian mixture model approach of the whole image, fast builds a network of neighboring relations based on a markov random field approach, more sensible to changes between slices. the same reason can also be behind the better performance of our method on 3 t when fast is used. compared with 1.5 t images, the probability of intensity change between slices is less prominent on 3 t images due to a higher resolution between slices. analyzing the possible deviations in tissue volume caused by each tissue segmentation process, we obtained results which suggest that the chosen tissue segmentation method does not affect significantly the performance of our filling - method. results between the same filled images segmented with fast and spm8 differ (< 0.1%) in the worst case on both datasets and tissues. by contrast, magon, fsl - l and leap switch their rank on 1.5 t images, depending on the segmentation method used. on 3 t images, only magon and fsl - l appear to switch between ranks when fast or spm8 is used, respectively. the most important one is the lack of images of ms patients with brain tissue expert annotations. (2014) have been only provided with lesion annotations delineated by a trained expert, but not brain tissue annotations. to overpass this limitation, we have registered wm lesions from ms patients into healthy images as performed in battaglini. (2012) and double - checked that registered lesions have replaced voxels segmented as wm by fast and spm8. this strategy has a negligible impact on the performance of the filling - methods analyzed in this study, because we assure a priori that generated lesions are on wm, and moreover none of the methods use information from the artificial lesions generated. furthermore, although we tested the performance of the proposed method with two datasets with different magnetic field strengths, our results are limited to these two different scanners with particular configurations, and hence it is difficult to generalize the results to all 1.5 and 3 t scanners. in conclusion, the results of this study show that regardless of the lesion size, the slf method performs consistently well compared to other existing methods such as leap, especially on 1.5 t images. furthermore, the results obtained show that the proposed method can be an effective method for low resolution images. the skull - stripping process does not especially affect the accuracy of the method, which allows integrating it with different preprocessing pipelines. additionally, volume estimations of lesion filled images processed by our algorithm appear to be not affected by the segmentation method employed. in contrast to other approaches, slf may be installed by non - computer experts who can easily use it without any parameter tuning. slf is currently available to researchers as a stand - alone script and as an spm library extension which facilitates to incorporate the lesion filling process into the expert workflow for tissue volume segmentation. | multiple sclerosis white matter (wm) lesions can affect brain tissue volume measurements of voxel - wise segmentation methods if these lesions are included in the segmentation process. several authors have presented different techniques to improve brain tissue volume estimations by filling wm lesions before segmentation with intensities similar to those of wm. here, we propose a new method to refill wm lesions, where contrary to similar approaches, lesion voxel intensities are replaced by random values of a normal distribution generated from the mean wm signal intensity of each two - dimensional slice. we test the performance of our method by estimating the deviation in tissue volume between a set of 30 t1-w 1.5 t and 30 t1-w 3 t images of healthy subjects and the same images where : wm lesions have been previously registered and afterwards replaced their voxel intensities to those between gray matter (gm) and wm tissue. tissue volume is computed independently using fast and spm8. when compared with the state - of - the - art methods, on 1.5 t data our method yields the lowest deviation in wm between original and filled images, independently of the segmentation method used. it also performs the lowest differences in gm when fast is used and equals to the best method when spm8 is employed. on 3 t data, our method also outperforms the state - of - the - art methods when fast is used while performs similar to the best method when spm8 is used. the proposed technique is currently available to researchers as a stand - alone program and as an spm extension. |
osteonecrosis of the femoral head (onfh) is a debilitating disease.123 the etiology of the disease is unknown.45 however, it is thought to be multifactorial.678 it results in femoral head collapse in 7585% of untreated patients.91011121314 the current trend in the treatment of onfh aims to preserve the joint in the initial stages and to delay the replacement surgery in advanced cases.141516171819 recently, mesenchymal stem cells and prp have been used as an adjunct to core decompression to improve clinical success in the treatment of precollapse hips.20212223 prp was first described by whitman. in 1997,24 it contains multiple growth factors and has been shown to have positive effects on the stimulation of bones, blood vessels and the formation of chondrocytes.2425 this study describes early results of treatment onfh by replacement of the necrotic segment after multiple drilling with bone graft and prp covered by collagen sheet to augment healing process. 30 patients (40 hips) with modified ficat stages iib and iii onfh [table 1].26 underwent surgery between december 2009 and march 2014. there were 19 males and 11 females with a mean age 36.7 6.93 years (range 2048 years). the causes of osteonecrosis in this series were steroid intake (n = 15, 37.5%), post traumatic (n = 5, 12.5%), idiopathic (n = 20, 50%). in 10 patients, the procedure was performed bilaterally with average 3.5 months interval (2.84.6 months). 16 hips (40%) had stage iib and 24 hips (60%) had stage iii onfh. all the patients were assessed clinically during pre- and postoperative period according to the hhs,14 vas27 and radiologically by x - rays. mri was done preoperatively to confirm the diagnosis and every 6 months postoperatively for assessment of healing. the inclusion criteria were : (1) stage iib or iii onfh as evidenced radiologically (2) age between 20 and 50 years (3) disabling pain that interfered with daily activity. the exclusion criteria were (1) active endocrine disorder (e.g. hypothyroidism) (2) active neurological disorder that might affect the patient 's pain (e.g. peripheral neuropathy and multiple sclerosis) (3) any active disease requiring continuous use of corticosteroids (e.g., rheumatoid and systemic lupus erythematosis). modified ficat classification under general or regional anesthesia, the patient was placed on a standard operating table in a supine position with the buttock of the affected side sticks a few centimeters out of the border of the table. the skin incision began about 2 cm proximal to the tip of the greater trochanter and extended for 78 cm distally. the incision was angled about 25 with respect to the axis of the femoral shaft. after dissection of subcutaneous tissues, the fascia of the muscles was dissected in line of incision. the anterior margin of the gluteus medius was cut for about 45 cm at its insertion onto the greater trochanter. the gluteus minimus was then identified below the gluteal medius and was separately dissected, taking care to maintain about 0.5 cm of tissue distally to allow an easier reconstruction. two were placed at 11 and 2 oclock, while the third was placed at 9 oclock for the right hip and at 3 oclock for the left one. these retractors proximally and superiorly shifted the glutei and medially shift the rectus femoris and iliopsoas. the hip capsule was then tensioned by forcing the hip in flexion, adduction and external rotation and then a reversed t - shaped incision was performed. the hip was dislocated anteriorly, with care not to damage the posterior capsule [figure 1 ]. surgical approach, (a) patient positioning, (b) iliotibial band incision, (c and d) incision of anterior fibers of gluteus medius, minimus and capsule, (e and f) anterior dislocation of the hip joint, (g) repair of the gluteus medius and minimus at the end of the procedure the necrotic area of the femoral head was identified and approached through the damaged articular surface, then curetted with the removal of all necrotic bone. multiple drilling was done with a 4.5 mm drill bit for 1 - 5 cm depth. finally, the cavity was covered with collagen sheet made of porcine collagen type i membrane. it consisted of 4.8 mg / ml rat tail collagen type i gel, the diameter of the samples was 9 mm with a height of 3 mm and stored at 4c until implanted. (a biocollagen merg collagen membrane, bioteck, vicenza, italy) and fixed with fibrin glue to the articular surface [figure 2 ]. gentle reduction was done and the anterior capsule was repaired. operative technique, (a) stage iii osteonecrosis of the femoral head after curettage and multiple drilling, (b) platelet rich plasma (c) composite of platelet rich plasma and bone graft (d) femoral head after impaction of bone graft and platelet rich plasma (e and f) coverage with collagen sheet the procedure for preparation of prp consisted of 150-ml venous blood sample that was centrifuged twice for 10 and 15 min, respectively, to concentrate and produce 20 ml of prp.28 postoperatively, skin traction was applied for 3 days, with the functional training of the hip. full weight bearing started at the beginning of the 3 months and heavy physical activity up to 1 year postoperatively. they were followed up at 6 weeks, 3 months, 6 months and at 1 year then every 6 months. at each followup, clinical evaluation was done according to vas27 and hhs.14 in addition to radiological evaluation by x - rays (anteroposterior and lateral views), mri study was done every 6 months. the following tests were used : (1) the nonparametric wilcoxon test - to compare the average of the subjective pain difference (difference between preoperative and postoperative followup as determined by the visual analog score [vas ]) and the joint function (as measured by hhs comparing preoperative function with function at followup). (2) the kruskal wallis test - this test was used to study the difference between the subjective pain and hhs parameters based on the length of followup. clinical success was defined as a good or excellent hhs score, improvement in vas and no revision surgery. under general or regional anesthesia, the patient was placed on a standard operating table in a supine position with the buttock of the affected side sticks a few centimeters out of the border of the table. the skin incision began about 2 cm proximal to the tip of the greater trochanter and extended for 78 cm distally. the incision was angled about 25 with respect to the axis of the femoral shaft. after dissection of subcutaneous tissues, the fascia of the muscles was dissected in line of incision. the anterior margin of the gluteus medius was cut for about 45 cm at its insertion onto the greater trochanter. the gluteus minimus was then identified below the gluteal medius and was separately dissected, taking care to maintain about 0.5 cm of tissue distally to allow an easier reconstruction. two were placed at 11 and 2 oclock, while the third was placed at 9 oclock for the right hip and at 3 oclock for the left one. these retractors proximally and superiorly shifted the glutei and medially shift the rectus femoris and iliopsoas. the hip capsule was then tensioned by forcing the hip in flexion, adduction and external rotation and then a reversed t - shaped incision was performed. the hip was dislocated anteriorly, with care not to damage the posterior capsule [figure 1 ]. surgical approach, (a) patient positioning, (b) iliotibial band incision, (c and d) incision of anterior fibers of gluteus medius, minimus and capsule, (e and f) anterior dislocation of the hip joint, (g) repair of the gluteus medius and minimus at the end of the procedure the necrotic area of the femoral head was identified and approached through the damaged articular surface, then curetted with the removal of all necrotic bone. multiple drilling was done with a 4.5 mm drill bit for 1 - 5 cm depth. finally, the cavity was covered with collagen sheet made of porcine collagen type i membrane. it consisted of 4.8 mg / ml rat tail collagen type i gel, the diameter of the samples was 9 mm with a height of 3 mm and stored at 4c until implanted. (a biocollagen merg collagen membrane, bioteck, vicenza, italy) and fixed with fibrin glue to the articular surface [figure 2 ]. gentle reduction was done and the anterior capsule was repaired. operative technique, (a) stage iii osteonecrosis of the femoral head after curettage and multiple drilling, (b) platelet rich plasma (c) composite of platelet rich plasma and bone graft (d) femoral head after impaction of bone graft and platelet rich plasma (e and f) coverage with collagen sheet the procedure for preparation of prp consisted of 150-ml venous blood sample that was centrifuged twice for 10 and 15 min, respectively, to concentrate and produce 20 ml of prp.28 postoperatively, skin traction was applied for 3 days, with the functional training of the hip. full weight bearing started at the beginning of the 3 months and heavy physical activity up to 1 year postoperatively. they were followed up at 6 weeks, 3 months, 6 months and at 1 year then every 6 months. at each followup, clinical evaluation was done according to vas27 and hhs.14 in addition to radiological evaluation by x - rays (anteroposterior and lateral views), mri study was done every 6 months. the following tests were used : (1) the nonparametric wilcoxon test - to compare the average of the subjective pain difference (difference between preoperative and postoperative followup as determined by the visual analog score [vas ]) and the joint function (as measured by hhs comparing preoperative function with function at followup). (2) the kruskal wallis test - this test was used to study the difference between the subjective pain and hhs parameters based on the length of followup. clinical success was defined as a good or excellent hhs score, improvement in vas and no revision surgery. two patients were noted to have postoperative trochanteric bursitis at immediate followup and one case of deep venous thrombosis, however, these were managed nonoperatively. the average values of vas were 78 21 and 35 19 at preoperative and final followup, respectively, with an average reduction of 43 points. significant pain relief was reported in 34 hips (85%), while the rest of patients reported little or no pain relief. as regards improvement in vas, there was no significant difference between stage iib and iii onfh, risk factors, age, gender, or bilateral treatment. hhs improved from 46.0 7.8 preoperatively to 90.28 19 at the end of followup. the comparison between average scores showed statistical significant difference (p < 0.0001) [figures 3 and 4 ]. stage iib (a and b) subchondral collapse is indicated by the appearance of a crescent sign without flattening of the femoral head. the lateral radiograph shows a typical crescent sign, (c and d) preoperative magnetic resonance imaging showing avascular necrosis changes (e) immediate postoperative x - ray showing adequate curettage and filling with composite of pla telet rich plasma and bone graft, (f) 6 months followup postoperative x - ray showing healing (g) 25 months followup postoperative x - ray showing healing of lesion (a) preoperative x - ray pelvis showing both hips anteroposterior view showing bilateral stage iii osteonecrosis of the femoral head (b and c) preoperative frog lateral view of both hips showing bilateral avascular necrosis changes (d) preoperative magnetic resonance imaging showing avascular changes (e) immediate postoperative x - ray pelvis anteroposterior view showing curettage and filling with composite of platelet rich plasma, 30 months postoperative followup anteroposterior (f) and frog leg lateral views of (g, h) both hips showing healing changes (i) 30 months followup postoperative magnetic resonance imaging showing healing changes twenty seven hips (67.5%) had excellent results and nine hips (22.5%) had a good result. patients with stage iib has better improvement in hhs than stage iii, but this was statistically insignificant. radiologically, one hip at stage iib progressed to stage iii and one hip at stage iii progressed to stage iv. unchanged radiological appearance over the followup period was observed in three hips (7.5%), but with improvement in functional score and patient satisfaction. although options to halt the progression of onfh are available (e.g. core decompression, osteotomy, vascularized fibular graft and medical treatments), the results have been disappointing, with up to 40% of patients progressing to tha. conceptually, the best option is removal of the necrotic bone from the femoral head and replacement with a viable and structurally - sound bone, thus restoring vitality to the femoral head, preventing collapse of the articular surface and delaying tha.2930 of the various treatment options available to avoid tha, core decompression, as described originally by ficat.31 and later by mont.2 is one of the most commonly used surgical treatments for onfh.231 core decompression may be a suitable option for stage i or iia but the main problem is with more advanced stages, especially in young active patients. most literatures about management of onfh try to preserve the hip joint in early stages of the disease, but there is a debate on the efficacy of this treatment in advanced stages, especially with articular damage.323334 the effectiveness of core decompression alone in preventing collapse in onfh has been a major source of controversy.3536373839 a wide range of success rates has been reported for core decompression according to mont.40 63.5% of 1166 hips achieved a satisfactory clinical result after core decompression.40 a retrospective review described a technique that utilized a trephine approach to enter the area of necrosis under fluoroscopy and then inject concentrated bone - marrow directly into this area. it found excellent results in patients who were precollapse (stage i or ii). however, in patients who had already collapsed (stage iii or iv), 25 out of 44 hips required a tha.23 keizer.41 described the long term results of core decompression and placement of a nonvascularized bone graft with 44% revision rate at a mean of 4 years.41 these results were less satisfactory compared to the results of our technique who reported good to excellent results in 90% with a revision rate of 10% (four hips). on the other hand, our results may be comparable to some reported results with the use of vascularized fibular graft as reported by zhao.34 the procedure was successful in 90% at ficat iii. he also described a modified technique of tantalum rod implantation combined with vascularized iliac grafting for the treatment of onfh stage ii iv. their overall success rate of the entire group was 87.5%.34 however, contrast of the others, chen.42 reported that the use of vascularized iliac bone grafting may not be as promising as originally suggested resulting 76% required tha.42 since progenitor cells may be lacking in the lesion area, newer treatment modalities have been developed to introduce biologically active cells to the areas of necrosis in an attempt to prevent fracture and collapse by restoring the architecture of the femoral head. hernigou and beaujean23 first described a technique for injecting mesenchymal stem cells combined with standard core decompression to introduce biological active cells into an area of necrosis, 23 patients with early (precollapse) disease had excellent results, only nine of 145 hips requiring tha. however, among patients who had stage iii or greater (25 of 44) hips required tha.23 this may clarify better results we obtained because of direct attacking of the pathology and removal of all necrotic segment as it has been demonstrated that biologically active cells may not be able to survive in the necrotic lesions, in addition to the use of collagen sheet as a scaffold that may increase the repairable capacity of prp. gangji.20 reported that the addition of mesenchymal stem cell to core decompression was found to improve its results ; he observed that the level of pain was significantly decreased from 37.8 8.4 to 18.5 6.2. this was comparable to our work, in which the average values were 78 21 and 35 19 at preoperative and final followup, respectively, with an average reduction of 43 points. daltro.43 assessed the efficacy and safety of autologous bone - marrow mononuclear cells implantation in necrotic lesions with a significant postoperative increase in the hhs (98.3 2.5 points) compared to preoperative hhs (78.5 6.2 points) (p < 0.001).43 our results found significant improvement in hip function with success rate 90% [27 hips (67.5%) had excellent results and nine hips (22.5%) had a good result ] with improvement of hhs from 46.0 7.8 preoperatively to a thorough review of the literatures, we found an old technique of treatment that may be similar to ours done by merle daubign.10 who used cancellous bone graft harvested from the iliac crest, have been used to fill the defect in the femoral head after complete evacuation of the necrotic bone.10 these bone graft can be introduced through a cortical window in the femoral neck or via a trapdoor through the articular cartilage of the femoral head, after dislocating the femur head and exposing the flap from the chondral surface of the femur head. this was first performed in conjunction with an osteotomy by ganz and bchler.44 mont.45 had reported their observations with this procedure in 24 ficat stage iii and six stage iv hips. with an average followup of 56 months, 73% their patients had good to excellent results.45 our results were much better than that recorded by mont.,45 although the similarity of both techniques that can be explained by the improvement of healing and positive reparable effect of prp with collagen sheet scaffold that was used in our research. there are some limitations to our study including small sample size and short term of followup. accordingly, prospective, randomized, controlled studies with large sample size are necessary to verify the therapeutic effects of prp. however, according to our present results, we are optimistic that this novel approach may lead to a successful outcome in the treatment of advanced stages of onfh. to conclude, in young active adult, the use of prp with collagen sheet scaffold can increase the reparable capacity after adequate curettage and drilling of necrotic segment with the addition of bone graft in stage iib and iii onfh. | background : osteonecrosis of the femoral head (onfh) is a debilitating disease in orthopedics, frequently progressing to femoral head collapse and osteoarthritis. it is thought to be a multifactorial disease. onfh ultimately results in femoral head collapse in 7585% of untreated patients. total hip arthroplasty (tha) yields satisfactory results in the treatment of the end stage of the disease. however, disease typically affects males between the ages of 20 and 40 years and joint replacement is not the ideal option for younger patients. recently, mesenchymal stem cells and platelet rich plasma (prp) have been used as an adjunct to core decompression to improve clinical success in the treatment of precollapse hips.materials and methods : a prospective study of 40 hips in 30 patients was done. there were 19 males and 11 females with a mean age 36.7 6.93 years. the indication for the operation was restricted primarily to modified ficat stages iib and iii. 16 hips (40%) had stage iib and 24 hips (60%) had stage iii onfh. the period of follow up ranged between 3650 months with a mean 41.4 3.53 months. all patients were assessed clinically during pre- and post - operative period according to the harris hip score (hhs), visual analog score (vas) and radiologically by x - rays. magnetic resonance imaging (mri) was done preoperatively to confirm the diagnosis and every 6 months postoperatively for assessment of healing. the operative procedure include removal of necrotic area with drilling then the cavity was filled with a composite of bone graft mixed with prp.results:the mean hhs improved from 46.0 7.8 preoperatively to 90.28 19 at the end of followup (p < 0.0001). the mean values of vas were 78 21 and 35 19 at preoperatively period and final followup, respectively, with an average reduction of 43 points.conclusion:we found that the use of prp with collagen sheet can increase the reparable capacity after drilling of necrotic segment in stage iib and iii onfh. |
peyronie 's disease (pd) is a relatively common condition and affects 3.2 to 8.9% of the male population. pd is characterized by severe fibrotic processes in the tunica albuginea, which hinders expansion of the tunica albuginea and results in penile curvature. recent studies suggest that localized inflammatory processes and aberrant wound healing in the tunica albuginea following minor trauma to the penis during sexual intercourse are responsible for the plaque development. however, the available medical treatment options, including oral, intralesional injection, and topical therapies, have failed to demonstrate conclusive effects, and surgical treatment is currently the only efficacious treatment for pd [6 - 8 ]. it is necessary to understand the pathophysiologic mechanisms of pd at the cellular and molecular levels to develop targeted and curative treatment modalities for pd. overexpression of profibrotic cytokines is known to play an important role in the development of fibrotic plaque, and transforming growth factor-1 (tgf-1) is the most extensively studied as a candidate fibrogenic cytokine. we and other investigators have reported that the expression and activity of the tgf-1 and smad transcription factors are significantly increased in human pd plaque and in fibroblasts derived from patients with pd [9 - 11 ]. moreover, we observed that local injection of adenovirus encoding the tgf-1 gene (ad - tgf-1) into the tunica albuginea of rats results in histologic and morphological changes in the tunica that are similar to those found in human pd plaques, which further supports a causative role of tgf-1 in the pathogenesis of pd. therefore, targeted inhibition of the tgf- signaling pathway might be a valuable therapeutic strategy for the treatment of pd. recently, small - molecule inhibitor of tgf- type i receptor, activin receptor - like kinase 5 (alk5) inhibitor, was shown to decrease fibrosis in kidney, lung, and liver [13 - 15 ]. we also recently found that local injection of an alk5 inhibitor, 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1h - imidazol-2-yl)methyl)benzamide (in-1130), into the tunica albuginea induced the regression of tunical fibrosis and corrected penile curvature in a rat model of pd induced by repeated intratunical injection of ad - tgf-1. in the present study, we determined the effectiveness of in-1130 on the tgf-1-induced activation of the smad transcription factors and extracellular matrix production in fibroblasts isolated from human pd plaque. plaque tissue was transferred into sterile vials containing hank 's balanced salt solution (invitrogen, carlsbad, ca, usa). after washing three times in phosphate - buffered saline (pbs), the tissue was minced into 1 mm segments and incubated in dulbecco 's modified eagle medium (dmem) supplemented with 0.06% collagenase a (sigma - aldrich co., st. after centrifugation (400 g 5 minutes) and washing in fresh culture medium, the cells and tissue fragments were collected and placed in 100 mm cell culture dishes (bd, franklin lakes, nj, usa) under standard conditions using dmem supplemented with 10% fetal calf serum, penicillin (100 u / ml), and streptomycin (100 g / ml). the study protocol was approved by the institutional review board of our university. for the characterization of primary cultured cells, after serial washes with pbs, the cells were fixed in 4% paraformaldehyde for 10 minutes at 4 and in 100% methanol for 10 minutes at 4. individual chambers were incubated with antibody to vimentin (1:500 ; a fibroblast marker, sigma - aldrich co.), smooth muscle -actin (1:500 ; a myofibroblast marker, sigma - aldrich co.), desmin (1:500 ; a smooth muscle cell marker, abcam, cambridge, uk), or platelet / endothelial cell adhesion molecule (pecam-1, an endothelial cell marker ; chemicon, temecula, ca, usa ; 1:500) for 1 hour at room temperature. after serial washes with pbs, the chambers were incubated with fluorescein isothiocyanate (fitc)-conjugated goat anti - mouse immunoglobulin (igg) (1:1,000 ; zymed laboratories, south san francisco, ca, usa) or fitc - conjugated goat anti - hamster igg (1:1,000 ; jackson immunoreseach laboratories inc., west grove, pa, usa) for 2 hours at room temperature. for the nuclei labeling, in-1130, an alk5 inhibitor, was provided by sk chemicals (seongnam, korea). after serum starvation for 24 hours, the fibroblasts were pretreated for 1 hour with 10 m in-1130 (molecular weight, 518). for detection of the p - smad2, total smad2, p - smad3, and total smad3 proteins, the fibroblasts were treated with 10 ng / ml tgf-1 (r&d systems inc., minneapolis, mn, usa) for 1 hour, and for detection of the plasminogen activator inhibitor-1 (pai-1), fibronectin, collagen i, and collagen iv proteins, the cells were incubated with tgf-1 for 24 hours. cytosolic and nuclear proteins were extracted by using a commercially available kit (ne - per nuclear and cytoplasmic extraction kit ; pierce biotechnology, rockford, il, usa). briefly, after serial washes with pbs, the cells were harvested and centrifuged for 5 minutes at 3,000 rpm (4). cytoplasmic extraction reagent i on ice for 10 minutes, and then 5.5 l cytoplasmic extraction reagent ii was added to the sample and incubated on ice for 1 minute. after centrifugation for 5 minutes at 13,000 rpm (4), the supernatant (cytosolic extract) was transferred to a tube on ice. pelleted nuclei were resuspended in 100 l nuclear extraction reagent on ice. after serial vortexes for 15 seconds every 10 minutes on ice for 40 minutes, the samples were centrifuged for 10 minutes at 13,000 rpm (4) and the supernatant (nuclear extract) was transferred to a tube on ice. equal amounts of each cytosolic and nuclear protein fraction (80 g / lane) were separated by 10% sodium dodecylsulfate - polyacrylamide gel electrophoresis and immunoblotting. after protein transfer, nitrocellulose membranes were incubated with antibody against p - smad2, total smad2 (which also recognizes smad3), p - smad3 (1:300 ; cell signaling, technology inc., danvers, ma, respectively), total smad3 (1:300 ; zymed laboratories inc.), hsp90 (1:300 ; a cytosol marker, abcam plc), or lamin (1:50 ; a nuclear marker, abcam plc). equal amounts of protein from whole - cell extracts (80 g / lane) were separated by 10% sodium dodecylsulfate - polyacrylamide gel electrophoresis and immunoblotting. after protein transfer, nitrocellulose membranes were incubated with antibody against pai-1 (1:600 ; abcam plc), fibronectin (1:300 ; abcam plc), collagen i (1:300 ; abcam plc), collagen iv (1:300 ; abcam plc), or -actin (1:6,000 ; abcam plc). after serum starvation for 24 hours, the cells were pretreated for 1 hour with 10 m in-1130 and were then treated with 10 ng / ml tgf-1 (r&d systems inc.) for 1 hour. individual chambers were processed as described above and were then incubated with antibody to total smad2 (which also recognizes smad3, 1:500 ; cell signaling inc.) for 3 hours at room temperature. after several washes with pbs, the chambers were incubated with fitc - conjugated goat anti - mouse igg (1:1,000 ; zymed laboratories inc.) for 2 hours at room temperature. for the nuclei labeling, digital images were captured with an apotome microscope (zeiss, gttingen, germany), and fluorescent intensity was measured for every nucleus in the field and averaged for the field with an image analyzer system (national institutes of health image j, bethesda, md, usa). statistical analysis was performed by using one - way anova followed by student - newman - keuls post - hoc tests. plaque tissue was transferred into sterile vials containing hank 's balanced salt solution (invitrogen, carlsbad, ca, usa). after washing three times in phosphate - buffered saline (pbs), the tissue was minced into 1 mm segments and incubated in dulbecco 's modified eagle medium (dmem) supplemented with 0.06% collagenase a (sigma - aldrich co., st. after centrifugation (400 g 5 minutes) and washing in fresh culture medium, the cells and tissue fragments were collected and placed in 100 mm cell culture dishes (bd, franklin lakes, nj, usa) under standard conditions using dmem supplemented with 10% fetal calf serum, penicillin (100 u / ml), and streptomycin (100 g / ml). the study protocol was approved by the institutional review board of our university. for the characterization of primary cultured cells, after serial washes with pbs, the cells were fixed in 4% paraformaldehyde for 10 minutes at 4 and in 100% methanol for 10 minutes at 4. individual chambers were incubated with antibody to vimentin (1:500 ; a fibroblast marker, sigma - aldrich co.), smooth muscle -actin (1:500 ; a myofibroblast marker, sigma - aldrich co.), desmin (1:500 ; a smooth muscle cell marker, abcam, cambridge, uk), or platelet / endothelial cell adhesion molecule (pecam-1, an endothelial cell marker ; chemicon, temecula, ca, usa ; 1:500) for 1 hour at room temperature. after serial washes with pbs, the chambers were incubated with fluorescein isothiocyanate (fitc)-conjugated goat anti - mouse immunoglobulin (igg) (1:1,000 ; zymed laboratories, south san francisco, ca, usa) or fitc - conjugated goat anti - hamster igg (1:1,000 ; jackson immunoreseach laboratories inc., west grove, pa, usa) for 2 hours at room temperature. for the nuclei labeling, in-1130, an alk5 inhibitor, was provided by sk chemicals (seongnam, korea). after serum starvation for 24 hours, the fibroblasts were pretreated for 1 hour with 10 m in-1130 (molecular weight, 518). for detection of the p - smad2, total smad2, p - smad3, and total smad3 proteins, the fibroblasts were treated with 10 ng / ml tgf-1 (r&d systems inc., minneapolis, mn, usa) for 1 hour, and for detection of the plasminogen activator inhibitor-1 (pai-1), fibronectin, collagen i, and collagen iv proteins, the cells were incubated with tgf-1 for 24 hours. cytosolic and nuclear proteins were extracted by using a commercially available kit (ne - per nuclear and cytoplasmic extraction kit ; pierce biotechnology, rockford, il, usa). briefly, after serial washes with pbs, the cells were harvested and centrifuged for 5 minutes at 3,000 rpm (4). cytoplasmic extraction reagent i on ice for 10 minutes, and then 5.5 l cytoplasmic extraction reagent ii was added to the sample and incubated on ice for 1 minute. after centrifugation for 5 minutes at 13,000 rpm (4), the supernatant (cytosolic extract) was transferred to a tube on ice. after serial vortexes for 15 seconds every 10 minutes on ice for 40 minutes, the samples were centrifuged for 10 minutes at 13,000 rpm (4) and the supernatant (nuclear extract) was transferred to a tube on ice. equal amounts of each cytosolic and nuclear protein fraction (80 g / lane) were separated by 10% sodium dodecylsulfate - polyacrylamide gel electrophoresis and immunoblotting. after protein transfer, nitrocellulose membranes were incubated with antibody against p - smad2, total smad2 (which also recognizes smad3), p - smad3 (1:300 ; cell signaling, technology inc., danvers, ma, respectively), total smad3 (1:300 ; zymed laboratories inc.), hsp90 (1:300 ; a cytosol marker, abcam plc), or lamin (1:50 ; a nuclear marker, abcam plc). equal amounts of protein from whole - cell extracts (80 g / lane) were separated by 10% sodium dodecylsulfate - polyacrylamide gel electrophoresis and immunoblotting. after protein transfer, nitrocellulose membranes were incubated with antibody against pai-1 (1:600 ; abcam plc), fibronectin (1:300 ; abcam plc), collagen i (1:300 ; abcam plc), collagen iv (1:300 ; abcam plc), or -actin (1:6,000 ; abcam plc). the fibroblasts were cultured on sterile cover glass until nearly confluent. after serum starvation for 24 hours, the cells were pretreated for 1 hour with 10 m in-1130 and were then treated with 10 ng / ml tgf-1 (r&d systems inc.) for 1 hour. individual chambers were processed as described above and were then incubated with antibody to total smad2 (which also recognizes smad3, 1:500 ; cell signaling inc.) for 3 hours at room temperature. after several washes with pbs, the chambers were incubated with fitc - conjugated goat anti - mouse igg (1:1,000 ; zymed laboratories inc.) for 2 hours at room temperature. for the nuclei labeling, medium containing dapi (vector laboratories inc.) was applied to the chamber. digital images were captured with an apotome microscope (zeiss, gttingen, germany), and fluorescent intensity was measured for every nucleus in the field and averaged for the field with an image analyzer system (national institutes of health image j, bethesda, md, usa). statistical analysis was performed by using one - way anova followed by student - newman - keuls post - hoc tests. more than 95% of primary cultured cells showed positive staining for fibroblast marker (vimentin) but did not reveal positive staining for antibody to desmin (smooth muscle cell marker) or pecam-1 (endothelial cell marker) (fig. 1). similar to the result from a previous study, about 20% of the primary cultured cells stained positive for antibody to smooth muscle -actin (myofibroblast marker) (fig. 1). we determined the ability of in-1130 to inhibit tgf-1-induced smad2 and smad3 phosphorylation. at 1 hour after treatment with recombinant tgf-1 protein, we observed a profound increase in phosphorylation of smad2 and smad3 in both cytosolic and nuclear fractions. tgf-1 is known to be involved in the nuclear shuttling of smad2 and smad3, i.e., the translocation of smad proteins from the cytoplasm to the nucleus. therefore, we asked whether alk5 activity is necessary for tgf-1-induced nuclear translocation of smad2/3. fluorescent immunocytochemistry of fibroblasts with antibody against total smad2 (which also recognizes smad3) and nuclei labeling with dapi revealed that treatment with in-1130 significantly reduced tgf-1-induced nuclear accumulation of smad proteins (fig., we examined the effect of in-1130 on tgf-1-induced pai-1, fibronectin, collagen i, and collagen iv expression in fibroblasts. in-1130 did not affect the basal production of extracellular matrix proteins in fibroblasts not stimulated with tgf-1 (fig. more than 95% of primary cultured cells showed positive staining for fibroblast marker (vimentin) but did not reveal positive staining for antibody to desmin (smooth muscle cell marker) or pecam-1 (endothelial cell marker) (fig. 1). similar to the result from a previous study, about 20% of the primary cultured cells stained positive for antibody to smooth muscle -actin (myofibroblast marker) (fig. we determined the ability of in-1130 to inhibit tgf-1-induced smad2 and smad3 phosphorylation. at 1 hour after treatment with recombinant tgf-1 protein, we observed a profound increase in phosphorylation of smad2 and smad3 in both cytosolic and nuclear fractions. tgf-1 is known to be involved in the nuclear shuttling of smad2 and smad3, i.e., the translocation of smad proteins from the cytoplasm to the nucleus. therefore, we asked whether alk5 activity is necessary for tgf-1-induced nuclear translocation of smad2/3. fluorescent immunocytochemistry of fibroblasts with antibody against total smad2 (which also recognizes smad3) and nuclei labeling with dapi revealed that treatment with in-1130 significantly reduced tgf-1-induced nuclear accumulation of smad proteins (fig. to evaluate the effect of alk5 inhibition on extracellular matrix production, we examined the effect of in-1130 on tgf-1-induced pai-1, fibronectin, collagen i, and collagen iv expression in fibroblasts. in-1130 did not affect the basal production of extracellular matrix proteins in fibroblasts not stimulated with tgf-1 (fig. in this study, we showed that a small - molecule inhibitor of alk5, in-1130, successfully blocked tgf-1-induced signaling, i.e., phosphorylation and nuclear translocation of smad2 and smad3, and inhibited extracellular matrix production in fibroblasts derived from human pd plaque. tgf--mediated fibrotic responses begin by activating the receptor - associated smads, including smad2 and smad3. alk5 is a tgf- type i receptor specifically involved in the activation of tgf-. tgf- induces the phosphorylation of serine / threonine residues of alk5 and then phosphorylates the major downstream signaling molecules smad2 and smad3. phosphorylated smad2 and smad3 form a heteromeric complex with smad4 and translocate into the nucleus. when translocated, phospho - smad2 and phospho - smad3 regulate the transcription of tgf--responsive genes and induce tissue fibrosis previous studies revealed an activation of smad2 or smad3 in the kidneys of diabetic animals, and targeted deletion of the smad3 gene attenuated diabetes - induced renal fibrosis, which supports a major role of smad2 and smad3 in renal fibrosis. the activation of smad2 and smad3 in human pd plaque or in fibroblasts isolated from a pd patient as shown by us and other investigators indicates that inhibition of the tgf- pathway may be a promising therapeutic strategy for pd. the phosphorylation of smad2 or smad3 is necessary for nuclear translocation and tgf--mediated fibrosis. in the present study, in-1130 substantially inhibited tgf-1-induced smad2 and smad3 phosphorylation and the nuclear shuttling of smad proteins. the alk5 kinase activity affects nuclear translocation and accumulation of smad transcriptional factors [24 - 26 ], which is responsible for activating the profibrotic genes that are involved in the production and deposition of extracellular matrix protein. therefore, we examined whether in-1130 can prevent the stimulatory effect of tgf-1 on the production of extracellular matrix markers. a previous study of newborn foreskin fibroblasts and dermal fibroblasts showed that an alk5 inhibitor, sb431542, inhibited the tgf-1-induced expression of extracellular matrix protein. similarly, in-1130 also significantly reduced tgf-1-induced production of pai-1, fibronectin, collagen i, and collagen iv, which was comparable to the basal levels. in the present study, we determined the antifibrotic effect of a selective alk5 inhibitor, in-1130, in human pd fibroblasts in vitro. the current results showing the efficacy of in-1130 in primary cultured fibroblasts derived from human pd and our recent results in a pd animal model in vivo suggest that alk5 inhibition may represent a promising option for treating this condition. a small - molecule inhibitor of alk5, in-1130, successfully blocked tgf-1-induced smad2/3 activation and extracellular matrix production in primary fibroblasts derived from human pd plaque. overexpression of tgf- and activation of smad transcriptional factors play a crucial role in the pathogenesis of pd. thus, inhibition of tgf- signaling pathway will be a promising therapeutic strategy for treating pd. | purposetransforming growth factor-1 (tgf-1) is the key fibrogenic cytokine associated with peyronie 's disease (pd). the aim of this study was to determine the antifibrotic effect of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1h - imidazol-2-yl) methyl)benzamide (in-1130), a small - molecule inhibitor of the tgf- type i receptor activin receptor - like kinase 5 (alk5), in fibroblasts isolated from human pd plaque.materials and methodsplaque tissue from a patient with pd was used for primary fibroblast culture, and we then characterized primary cultured cells. fibroblasts were pretreated with in-1130 (10 m) and then stimulated with tgf-1 protein (10 ng / ml). we determined the inhibitory effect of in-1130 on tgf-1-induced phosphorylation of smad2 and smad3 or the nuclear translocation of smad proteins in fibroblasts. western blot analyses for plasminogen activator inhibitor-1, fibronectin, collagen i, and collagen iv were performed to evaluate effect of in-1130 on the production of extracellular matrix proteins.resultsthe treatment of fibroblasts with tgf-1 significantly increased phosphorylation of smad2 and smad3 and induced translocation of smad proteins from the cytoplasm to the nucleus. pretreatment with in-1130 substantially inhibited tgf-1-induced phosphorylation of smad2 and smad3 and nuclear accumulation of smad proteins. the tgf-1-induced production of extracellular matrix proteins was also significantly inhibited by treatment with in-1130 and returned to basal levels.conclusionsoverexpression of tgf- and activation of smad transcriptional factors are known to play a crucial role in the pathogenesis of pd. thus, inhibition of the tgf- signaling pathway by alk5 inhibitor may represent a promising therapeutic strategy for treating pd. |
recent advances in radiation oncology and technology, such as 3d - conformal radiotherapy (3d - crt) and intensity - modulated radiation therapy (imrt), have improved the dose conformality of radiation treatment planning (rtp). especially in the head and neck area, highly conformal rtp is very useful for escalating the tumor dose without increasing normal tissue injury, and precise identification of the target volume in rtp is essential. functional or biological imaging by positron emission tomography (pet) is expected to provide more useful information than anatomical imaging alone so that more appropriate rtp can be performed [1, 2 ]. f - fluorodeoxyglucose (fdg)-pet has recently been used to verify the target volume in rtp for various malignancies, especially non - small - cell lung cancer, head and neck cancer, etc. [36 ] ; however, it has not been well established how fdg - pet can be utilized for actual treatment planning for many different types of malignancies, and various approaches for the suitable use of pet for rtp have been suggested. in the present study, we evaluated the efficacy of fdg - pet for defining the gross tumor volume (gtv) of head and neck cancer to establish rtp using functional imaging. we analyzed the data for 53 patients with histologically proven primary squamous cell carcinoma of the head and neck treated with radiotherapy in nara medical university hospital between february 2006 and august 2009. all patients underwent routine contrast - enhanced (ce)-ct and fdg - pet for staging and defining the gtv. pet images were acquired about 60 min after intravenous administration of 3 mbq / kg fdg. if fdg - pet could not be performed before rtp, patients were excluded from this study. plain ct simulation for rtp of patients in the supine position, immobilized with a head - rest and thermoplastic mask, was also performed. rtp of the head and neck cancer was performed based on ct simulation using both ce - ct and fdg - pet images with a visual method [5, 7 ], according to the institute definition of the target volume for pet - based rtp. the definition is as follows : ce - ct - based gtv (ct - gtv) for rtp is defined using conventional ce - ct images alone. pet - based gtv (pet - gtv) for rtp is defined using both ce - ct and fdg - pet images. the ce - ct volume corresponding to a positive fdg - pet image was regarded as the pet - gtv, and rtp was performed using the gtv, with inflammatory fdg accumulation being excluded by a nuclear radiologist. if pet images were regarded as inappropriate for rtp due to false - positive or false - negative, ct - gtv was used predominantly for rtp. in the present study, pet - gtv was compared with ct - gtv to evaluate the importance of fdg - pet information on target definition in rtp. the sensitivity of ce - ct alone for identifying primary lesions, and that of the combination of ce - ct and fdg - pet was calculated, comparing the histologically proven lesions as the standard of reference ; however, the specificity was not assessed because of the bias of the patients in this study, i.e. exclusively patients with histologically proven squamous cell carcinoma of the head and neck. statistical significance of the difference in sensitivity was assessed by the mcnemer test (statmate iv for windows v4.01 ; atms, tokyo). the pet - gtv for each case was then compared with the ct - gtv in order to evaluate the importance of the fdg - pet information in identifying lymph node metastases for rtp. however, the sensitivity and the specificity of pet - gtv and ct - gtv were not assessed for lymph nodes, because most of the lymph nodes had not been histologically studied in these cases, although every primary squamous cell carcinoma was histologically confirmed. in addition to the above study, ct - gtv delineation and pet - gtv delineation in 19 cases (13 patients with tongue cancer and 6 patients with oropharyngeal cancer) were performed by four radiation oncologists independently to evaluate the difference in volume due to interobserver variability in the gtv delineation. the statistical significance of the difference in gtv was assessed by the wilcoxon signed - ranks test (statmate iv for windows v4.01 ; atms, tokyo). patient characteristics for the 53 cases are shown in table 1. of the 53 primary tumor sites, 51 lesions showed positive accumulation of fdg ; the sensitivity of pet - gtv for identifying the primary site was 96% (table 2 and figures 15). the margins of the tumors on pet and pet / ct images were relatively ill - defined and not always clear. the pet - gtv was smaller in some cases and larger in other cases than the ct - gtv. in two cases (tongue cancer and gingiva cancer), it was difficult to determine the gtv by pet (table 2 and fig. the primary tumor lesion was not evident on plain ct (a) and ce - ct (d - f) images due to artifacts induced by artificial teeth, but the lesion was evident on fdg - pet (b) and fdg - pet / ct (c) images (arrows). the primary tumor lesion was not evident on plain ct (a) and ce - ct (d f) images due to artifacts induced by artificial teeth. fdg - pet (b) and fdg - pet / ct (c) images showed positive fdg accumulation (arrows), but it was difficult to determine the gtv because the accumulation was not specific to the tumor. table 1.patient characteristicscharacteristicsnumber of patientsgender male39 female14stagei0ii13iii13iva27ivb0tumor site oral cavity31 tongue14 gingiva8 buccal mucosa4 mouth floor4 others1 pharynx13 oropharynx8 hypopharynx5 nasal cavity / paranasal sinus7 others2total53 table 2.sensitivity (%) of ct - gtv and pet - gtv for identifying primary tumorsct - gtvsensitivitypet - gtvsensitivitynpositive%positive%oral cavity3124772994 tongue1410711393 gingiva8675788 bucca441004100 mouth floor441004100 others1001100pharynx13107713100 oropharynx85638100 hypopharynx551005100nasal / para771007100others221002100total5343815196nasal / para = nasal cavity / paranasal sinus a case of squamous cell carcinoma of the tongue. the primary tumor lesion was not evident on plain ct (a) and ce - ct (d - f) images due to artifacts induced by artificial teeth, but the lesion was evident on fdg - pet (b) and fdg - pet / ct (c) images (arrows). the primary tumor lesion was not evident on plain ct (a) and ce - ct (d f) images due to artifacts induced by artificial teeth. fdg - pet (b) and fdg - pet / ct (c) images showed positive fdg accumulation (arrows), but it was difficult to determine the gtv because the accumulation was not specific to the tumor. patient characteristics sensitivity (%) of ct - gtv and pet - gtv for identifying primary tumors nasal / para = nasal cavity / paranasal sinus in contrast, the gtv of 10 cases was not clear (that of 43 cases was evident) on ce - ct images ; the sensitivity of ct - gtv for identifying the primary tumor site was 81% (table 2) and was significantly lower than that of pet - gtv (p < 0.01). in 43 cases, no significant difference was found between pet - gtv and ct - gtv when they were compared to identify the localization of the primary lesions. in 14 patients with tongue cancer, 10 tumors were evident on ce - ct images, but 4 primary lesions were not clear and 3 of these were due to artifacts induced by bones, teeth, or artificial teeth ; sensitivity of ct - gtv to a primary site on the tongue was 71% and that of pet - gtv was 93% (table 2, figures 1 and 2). in 8 patients with gingival cancer, 6 tumors were evident, but 2 primary lesions were not clear due to artifacts ; sensitivity of ct - gtv to the primary site was 75% and that of pet - gtv was 88% (table 2, figures 3 and 4). in 8 patients with oropharyngeal cancer, 3 primary lesions were not obvious ; sensitivity of ct - gtv to the primary site was 63% and that of pet - gtv was 100% (table 2 and fig. the primary tumor lesion was not evident on plain ct (a) and ce - ct (d f) images regardless of slight artifact, but the lesion was evident on fdg - pet (b) and fdg - pet / ct (c) images (arrows). the primary tumor lesion was not evident on plain ct (a) and ce - ct (d f) images due to artifacts induced by artificial teeth, but the lesion was evident on fdg - pet (b) and fdg - pet / ct (c) images (arrows). the primary tumor lesion on plain ct (a) and ce - ct (d f) images was not as obvious as on fdg - pet (b) and fdg - pet / ct (c) images (arrows). the primary tumor lesion was not evident on plain ct (a) and ce - ct (d f) images regardless of slight artifact, but the lesion was evident on fdg - pet (b) and fdg - pet / ct (c) images (arrows). the primary tumor lesion was not evident on plain ct (a) and ce - ct (d f) images due to artifacts induced by artificial teeth, but the lesion was evident on fdg - pet (b) and fdg - pet / ct (c) images (arrows). the primary tumor lesion on plain ct (a) and ce - ct (d f) images was not as obvious as on fdg - pet (b) and fdg - pet / ct (c) images (arrows). lymph node metastases were suggested in 41 cases (77%) by ce - ct and in 32 cases (60%) by fdg - pet. neither ce - ct nor fdg - pet showed evident metastases in 11 cases (21%). the pet - gtv differed from the ct - gtv in 11 cases (21%) when we evaluated lymph nodes for rtp. swollen but fdg - negative lymph nodes were not regarded as the pet - gtv according to the definition of the target volume for pet - based rtp. in 10 cases (19%), lymph nodes were regarded as the ct - gtv but they were fdg - negative. in contrast, in only one case, lymph nodes were not swollen significantly, but showed evident accumulation of fdg. when both the primary lesions and the lymph nodes were evaluated for rtp, the pet - gtv differed from the ct - gtv in primary lesions, lymph nodes, or both in 19 cases (36%) and no significant difference was found between the pet - gtv and the ct - gtv in the other 34 cases (64%). in comparison of the ct - gtv and the pet - gtv as delineated by 4 radiation oncologists, pet - gtv delineation was successfully performed in all 19 cases, but ct - gtv delineation was not performed in 4 cases by any radiation oncologists due to unclear tumor images (fig. we evaluated the differences in the ct - gtv and the pet - gtv in the other 15 cases. the ct - gtv was larger than the pet - gtv in 10 of 15 cases, but the difference was not significant (p = 0.12). the standard deviation (sd) for the ct - gtv was larger than that for the pet - gtv in 10 cases, and the difference was statistically significant (p < 0.01). 6.comparison of gross tumor volume (gtv) delineated by four radiation oncologists (mean + standard deviation) : it was difficult to delineate ct - gtv in four cases (no. 3, 4, 6 and 14). in the other 15 cases, the mean ct - gtv was larger than pet - gtv in 10 cases, and the standard deviation of ct - gtv was larger than that of pet - gtv in 10 cases. comparison of gross tumor volume (gtv) delineated by four radiation oncologists (mean + standard deviation) : it was difficult to delineate ct - gtv in four cases (no. the mean ct - gtv was larger than pet - gtv in 10 cases, and the standard deviation of ct - gtv was larger than that of pet - gtv in 10 cases. fdg - pet has been used as a very useful imaging modality for detecting malignant primary lesions and lymph node metastases. very high sensitivity of the primary lesions, and the high sensitivity and specificity of lymph node metastases of the head and neck rtp using fdg - pet has recently been performed in anticipation of more appropriate target planning [5, 6 ]. many studies of lung cancer and head and neck cancer have been reported during the past decade [57, 1017 ] ; however, the efficacy of fdg - pet for rtp for various malignancies has not been well established. recently, troost. concluded that pet can characterize tumors for radiotherapy, which is a promising prospect, but unresolved issues remain and the applications are not yet ready for introduction into routine clinical practice. suggested that fdg - pet may be important for gtv definition, but the choice of a segmentation tool for target - volume definition based on pet images is not trivial and the absolute pet volume is dependent on the segmentation method. several different threshold techniques for delineating tumors on pet have been used, and the choice of technique leads to large differences in target volume [5, 10 ]. in our institute, rtp is performed based on ct simulation using both ce - ct and fdg - pet or fdg - pet / ct images with a visual method [5, 7 ], and fusion of fdg - pet images and ct simulation is not performed. the institute definition of the target volume for pet - based rtp has been used for the past five years. pet - based gtv for rtp is defined using both ce - ct and fdg - pet images. when pet images are regarded as inappropriate for rtp due to false negatives or false positives, the present study has shown the utility of fdg - pet for rtp of head and neck cancer in patients with histologically proven squamous cell carcinoma. the addition of fdg - pet image information to the ce - ct image resulted in significant changes to the gtv for rtp in 19 cases (36%). the sensitivity of pet - gtv for the 53 primary tumor sites was 96% ; however, that of ct - gtv was 81% ; in patients with oropharyngeal cancer and those with tongue cancer, the sensitivity of ct - gtv was no more than 63% and 71% respectively, due to false negatives for artifacts, etc. changes in the gtv using fdg - pet have been reported in several studies of head and neck cancer [5, 1113 ], varying from 11% to 93%, and the pet - gtv was smaller in some cases and larger in other cases than the ct - gtv. these results suggest that the additional use of fdg - pet is more effective for rtp than ce - ct alone and this will be recommended for defining the gtv for rt ; however, the specificity for the identification of lesions could not be evaluated in this study because the cases were limited to patients with histologically proven cancer who had been referred to the department of radiation oncology for rt. target delineation, or automated tumor contouring for rtp by fdg - pet, has often been demonstrated [5, 6, 10 ], but tumor contouring by pet was outside the scope of the present study. target delineation for rtp by defining the percentage of the maximum standardized uptake value (suv) of fdg has been reported [12, 13, 21 ] ; however, this method may have limitations. tumor delineation using an suv of 2.5 has been considered insufficient in other studies [20, 23 ]. visual comparison of fdg - pet images and ct simulation [5, 7 ] was utilized in this study, and the visual correlation of fdg - pet and ct simulation yielded higher sensitivity for the identification of primary lesions of the head and neck cancer regardless of exact tumor contouring, as suggested above. it has often been indicated that interobserver variability in target volume delineation of both head and neck cancer and lung cancer is reduced by using pet [2428 ] ; however, breen. reported that the addition of pet - ct to primary site gtv delineation of head and neck cancer did not change the gtv defined by expert observers and ce - ct would be more reliable than pet - ct. the present study suggests that the interobserver difference in target delineation will be decreased by using fdg - pet due to its excellent ability to identify primary gross tumors of the head and neck. in conclusion, the results of this study have shown that fdg - pet is effective for defining the gtv in rtp for squamous cell carcinoma of the head and neck, and the sensitivity of fdg - pet for defining the primary tumor is higher than that of ce - ct alone. pet - gtv evaluated by both ce - ct and fdg - pet images is indicated to be preferable to ct - gtv by ce - ct alone, but further studies will be necessary to establish the standard use of fdg - pet for rtp. in particular, it would be desirable to perform more accurate gtv delineation by using other methods different from those mentioned above. this study was partially supported by a grant - in - aid from the ministry of education, science, sports, and culture of japan (no. 22791216). | we analyzed the data for 53 patients with histologically proven primary squamous cell carcinoma of the head and neck treated with radiotherapy between february 2006 and august 2009. all patients underwent contrast - enhanced (ce)-ct and 18f - fluorodeoxyglucose (fdg)-pet before radiation therapy planning (rtp) to define the gross tumor volume (gtv). the pet - based gtv (pet - gtv) for rtp was defined using both ce - ct images and fdg - pet images. the ce - ct tumor volume corresponding to a fdg - pet image was regarded as the pet - gtv. the ce - ct - based gtv (ct - gtv) for rtp was defined using ce - ct images alone. additionally, ct - gtv delineation and pet - gtv delineation were performed by four radiation oncologists independently in 19 cases. all four oncologists did both methods. of these, pet - gtv delineation was successfully performed in all 19 cases, but ct - gtv delineation was not performed in 4 cases. in the other 15 cases, the mean ct - gtv was larger than the pet - gtv in 10 cases, and the standard deviation of the ct - gtv was larger than that of the pet - gtv in 10 cases. sensitivity of pet - gtv for identifying the primary tumor was 96%, but that of ct - gtv was 81% (p < 0.01). in patients with oropharyngeal cancer and tongue cancer, the sensitivity of ct - gtv was 63% and 71%, respectively. when both the primary lesions and the lymph nodes were evaluated for rtp, pet - gtv differed from ct - gtv in 19 cases (36%). these results suggested that fdg - pet is effective for defining gtv in rtp for squamous cell carcinoma of the head and neck, and pet - gtv evaluated by both ce - ct and fdg - pet images is preferable to ct - gtv by ce - ct alone. |
overall, one fifth of the female population experience this discomfort up to 10 days after natural vaginal delivery (nvd) (1). perineal trauma has been reported in 63% of vaginal deliveries, 15% of episiotomies, 46% of spontaneous ruptures, and 2% of the spontaneous ruptures accompanied by episiotomies. therefore, a large number of women experience perineal trauma after delivery (2). in a study in canada, perineal pain was reported in 97% of the women with episiotomy, 95% of those with types i and ii ruptures, and 75% of the women with healthy perineum. however, 71%, 60%, and 38% decrease was observed in these measures during the first week, respectively (3). the pain can reduce mobility, cause discomfort in urination and defecation, has negative effects on breast feeding, interferes with the women 's taking care of themselves or their babies, and leads to maternal depression and fatigue. moreover, perineal pain and long - lasting pain during the puerperium can have long term effects, such as painful intercourse, up to more than 18 months after the delivery (4). (2001) conducted a randomized clinical trial in australia in order to investigate the effect of perineal massage in the second stage of labor on the labor outcomes. in that study, the two groups were similar regarding the rate of healthy perineum, types i and ii ruptures, and episiotomy. however, the rate of type iii rupture was lower in the massage group compared to the control group (1.7% vs. 3.6%) (5). pain is the most important reason for treatment and various complementary methods as well as traditional medicine have been advised for pain relief. the more intense the pain lead to the higher number of using treatment methods (6). due to the fact that, regardless of the incidence of rupture, most of the women experience the perineal pain in the first days of the puerperium, pain relief interventions can be used for them. these interventions include pharmacological options, such as paracetamol, non - steroidal anti - inflammatory drugs (aspirin and naproxen and etc.), and opioids as well as non - pharmacological methods, such as subcutaneous electrical nerve stimulation, massage, and using local ice packs (7). in addition, cupping therapy is a type of physical therapy which is highly important in treatment of diseases, particularly painful syndromes (8). cupping therapy removes the extra fluids, loosens and moves up the connective tissue joints, directs the blood flow toward the skin and muscles, stimulates the peripheral nervous system, and reduces pain (9). cupping was performed by who trained in cupping and clinical setting which has been used in the treatment of pain in different diseases (10). cupping therapy is an ancient medical technique from europe, asia, and the middle east culture. each of the different techniques of glass creates a suction cup on the painful area. dry or fire cupping is used for normal skin (intact skin), while in the so - called wet or bloody skin (hijama), the skin is cut. cupping is applied to increase local blood and lymph circulation and to relieve painful muscle tension (11, 12). acupressure is also another branch of acupuncture. in this method, fingers are used for pressing the key points on the skin in order to stimulate and induce the body 's natural self - healing capabilities (13). in the present study, bl23 point (shenshu) which is located 1.5 cuns lateral to the lower border of the spinous process of the second lumbar vertebra was employed. due to the location of this point, the cups can be correctly placed on a flat space and acupressure can be appropriately applied. this point has also been used in treatment of pain syndromes such as low back and knee pain, genital pain, gynecological disorders such as infertility, irregular menstruation, chronic vaginal discharges, and insomnia (14). furthermore, acupressure leads to improvement in blood circulation and energy flow, balance between ying yang symbols, secretion of neurotransmitters, activation of opioids system, and removal of lactic acid and carbon monoxide accumulated in the body during muscle contraction. thus, it maintains the body 's natural function and reduces the pain (15). considering the high frequency of the women suffering from this pain in iran and other countries around the world and due to the limited number of clinical trials on cupping therapy, the present study aimed to link this science to the traditional medicine using the reliable scientific articles as well as the instructions of traditional medicine experts. the present randomized clinical trial was conducted in the postpartum unit of hafez hospital affiliated of shiraz university of medical sciences in 2012. according to the sample size formula and statistical consultation, a 150-subject sample size (50 cases in each group) however, 19 and 13 women were excluded from the study due to the pregnancy complications and demographic characteristics, respectively. finally, 150 women were selected using purposeful random sampling and divided into three groups of cupping therapy, acupressure, and control. the inclusion criteria were the age between 18 and 40 years, having at least middle school education, not suffering from any serious physical and mental disorders such as vertebral fractures, disk hernia, acute inflammation, and deep venous thrombosis, living in shiraz, being willing to take part in the study, and signing written informed consents. after at least 48 hr of delivery, cupping intervention was performed in the postpartum care unit of the hospital. it was conducted by researchers, but for 3 weeks under the supervision of a professional adviser, necessary training was performed and cupping therapy was approved by the researchers. cupping therapy the patients were laid in prone position and cupping was performed as follows : 34 glasses with diameters from 75 mm to 120 mm for obese and lean subjects were held inverted over bl23 point. a glass cup was utilized to create suction over a painful area. as the air inside of the cups was cooled, vacuums were created, drawing up the skin within each cup (12). the glasses were removed after 10 to 20 min depending on the color of the circular so - called cupping marks, which range from slightly rose to dark pink. it was hypothesized that specific changes in the local tissue structures occur as a result of local negative pressure in the cups used which stretches the nerve and muscle, thereby increasing blood and lymphatic circulation and causing autohemolysis and relieving painful muscle tension (16). on the other group, pressure points- bl 23 with his thumb, in counter - clockwise direction for 5 min. in the second 5 min, in fact, this process took about 20 min, twice in the hospital and once after discharge. the third intervention was coordinated by calling mothers to refer to the hospital (17, 18). the short - form of mcgill pain questionnaire was completed in the three study groups before and immediately, 24 hr, and 2 weeks after the intervention. this questionnaire is one of the most reliable pain assessment tools allowing the patients to express their perception of pain using appropriate words. the short - form of mcgill pain questionnaire consists of 11 items in the sensory dimension and 4 items in the emotional dimension and the patients have to identify their pain quality through 4 options of none, mild, average, and severe. in the study conducted by bagheri. (2007) on 78 patients who had undergone open surgery of lower extremity fractures at imam hossein hospital, shahrood, iran, the reliability of this questionnaire was reported as 98%. the reliability and validity of the questionnaire in the study of bagheri. repeated measures anova was used for comparing the changes in the mean score of pain before and after the intervention regardless of the effect of group and time. in addition, chi - square test was utilized in order to compare the demographic features among the three study groups. strengths of this study include the following ; first, the study employed bl 23 point (shen shu) through acupuncture and acupressure to relieve back pain and perineal pain, but few studies in this field are available. second, a study of cupping therapy and its impact has been done on chronic pain. but so far, in obstetrics and diseases of women no study has been performed in the last 30 years. this research project was approved by the local ethics committee of shiraz university of medical sciences and written informed consents were obtained from all the participants. the research in iranian registry of clinical trial has been registered with registration number irct : 2013072611944n2. apparently, placebo could be used in order to avoid bias. however, it was not employed in the current study due to the transparent nature of its performance. thus, further placebo - controlled clinical trials are needed to be conducted in order to determine the accuracy of the obtained results. the inclusion criteria were the age between 18 and 40 years, having at least middle school education, not suffering from any serious physical and mental disorders such as vertebral fractures, disk hernia, acute inflammation, and deep venous thrombosis, living in shiraz, being willing to take part in the study, and signing written informed consents. after at least 48 hr of delivery, cupping intervention was performed in the postpartum care unit of the hospital. it was conducted by researchers, but for 3 weeks under the supervision of a professional adviser, necessary training was performed and cupping therapy was approved by the researchers. cupping therapy the patients were laid in prone position and cupping was performed as follows : 34 glasses with diameters from 75 mm to 120 mm for obese and lean subjects were held inverted over bl23 point. as the air inside of the cups was cooled, vacuums were created, drawing up the skin within each cup (12). the glasses were removed after 10 to 20 min depending on the color of the circular so - called cupping marks, which range from slightly rose to dark pink. it was hypothesized that specific changes in the local tissue structures occur as a result of local negative pressure in the cups used which stretches the nerve and muscle, thereby increasing blood and lymphatic circulation and causing autohemolysis and relieving painful muscle tension (16). on the other group, pressure points- bl 23 with his thumb, in counter - clockwise direction for 5 min. in the second 5 min, in fact, this process took about 20 min, twice in the hospital and once after discharge. the third intervention was coordinated by calling mothers to refer to the hospital (17, 18). the short - form of mcgill pain questionnaire was completed in the three study groups before and immediately, 24 hr, and 2 weeks after the intervention. this questionnaire is one of the most reliable pain assessment tools allowing the patients to express their perception of pain using appropriate words. the short - form of mcgill pain questionnaire consists of 11 items in the sensory dimension and 4 items in the emotional dimension and the patients have to identify their pain quality through 4 options of none, mild, average, and severe. in the study conducted by bagheri. (2007) on 78 patients who had undergone open surgery of lower extremity fractures at imam hossein hospital, shahrood, iran, the reliability of this questionnaire was reported as 98%. the reliability and validity of the questionnaire in the study of bagheri. was also the basis for the present study (19). repeated measures anova was used for comparing the changes in the mean score of pain before and after the intervention regardless of the effect of group and time. in addition, chi - square test was utilized in order to compare the demographic features among the three study groups. strengths of this study include the following ; first, the study employed bl 23 point (shen shu) through acupuncture and acupressure to relieve back pain and perineal pain, but few studies in this field are available. second, a study of cupping therapy and its impact has been done on chronic pain. but so far, in obstetrics and diseases of women no study has been performed in the last 30 years. this research project was approved by the local ethics committee of shiraz university of medical sciences and written informed consents were obtained from all the participants. the research in iranian registry of clinical trial has been registered with registration number irct : 2013072611944n2. it was not employed in the current study due to the transparent nature of its performance. thus, further placebo - controlled clinical trials are needed to be conducted in order to determine the accuracy of the obtained results. the results of the present study showed that the three groups were similar regarding the demographic characteristics, such as the mothers ' age (p=0.064). moreover, according to the short - form of mcgill pain questionnaire, the means of perineal pain intensity before the intervention were 37.56.8, 35.68.1, and 34.78.8 in the cupping therapy, acupressure, and control groups, respectively. the results of repeated measures anova showed no significant difference among the three groups in this regard (p=0.1). after the intervention, the means of perineal pain intensity were 11.16.1, 22.66.6, and 26.47.0 in the cupping therapy, acupressure, and control groups, respectively and repeated measures anova showed that the difference between the intervention groups and the control group was statistically significant (p<0.001). also, significant changes were shown in pain intensity 24 hr and 2 weeks after the intervention (p<0.01). thus, both cupping therapy and acupressure played an important role in reducing the perineal pain, with cupping therapy being more effective. in spite of the fact that no difference was found among the three groups before the intervention, cupping therapy and acupressure were quite effective in reducing the sensory dimension of the perineal pain immediately, 24 hr, and 2 weeks after the intervention (p<0.001). the results of short - form of mcgill pain questionnaire (smpq) for the intervention and control groups (msd) the present study was the first clinical trial on the gynecological diseases in iran and around the world. this study aimed to investigate the therapeutic effects of cupping therapy and stimulation of body points by application of pressure on the intensity of postpartum perineal pain. according to the study results, the mean intensity of postpartum perineal pain in the cupping therapy group, compared to the control group, reduced and the difference was statistically significant (p=0.01). the results of a study by emerich. (2014) on 12 patients (6 neck pain patients and 6 healthy subjects) indicated that cupping therapy led to increased pressure pain thresholds. this result is compatible with our findings (20). also, in the study by markowski. (2014), the effect of cupping therapy was investigated on twenty - one patients who reported back pain for at least 8 weeks and the study revealed reduced pain and tenderness, and improved range of motion for patients with low back pain (lbp). the results showed that prompt reduction of pain and muscle tenderness and improving the range of motion have improved functional movement. this is compatible with our results, revealing the effectiveness of cupping therapy in reducing pain (21). kim. (2011) searched fourteen databases and in seven of them, all the inclusion criteria were analyzed. the positive effects of cupping was shown in cancer pain (p<0.05) and trigeminal neuralgia (p<0.01) compared with anticancer drugs and analgesics in two studies. the favorable effects of cupping on brachialgia pain compared with usual care (p=0.03) or a heating pad (p < 0.001) have been reported. however, they advised more detailed studies are needed to confirm the effectiveness of cupping which is used to treat pain (22). in a study conducted in iran, cupping therapy was introduced as an effective method in sedation to reduce lower back pain after childbirth (23). dry cupping therapy is based on the discharge principle, i.e. moving the waste materials from one place to another. dry cupping therapy is employed in treatment of various disorders, including excessive menstrual bleeding, edema, scrotal hernia, sciatica, hydrocele, and nose bleeding (22). in the acupressure group, the mean intensity of perineal pain reduced and the differences among intervention and control groups were statistically significant. this method is based on the assumption that special human body channels, called meridians, adjust the energy flow and imbalance in this flow results in incidence of diseases. however, acupressure results in opening of the channels as well as balance in the energy flow, and consequently it restores the human health (24). this might have caused significant reduction in the intensity of perineal pain in the acupressure group in this study. in a study by hsieh. (2004), the researchers used two groups of acupressure (69 patients) and physical therapy (77 patients) ; after the intervention, acupressure as an effective alternative medicine was introduced to reduce back pain (25). studied acupressure using lavender oil applied on patients with back pain. before the intervention, vas scores for the intervention and control groups were 6.38 and 5.70 of 10, respectively (p=0.24). a week after the application of acupressure, the intervention group had a 39% pain reduction (p=0.0001) ; also, walking time improved (p=0.05) (26). the study of visual pain scale was used to measure pain, but in our study mcgill questionnaire was administered. the study was conducted in iran, using acupressure at the sp6 point (27) and jian jing - gall bladder meridian point (gb-21) (28) to reduce the pain of labor, duration of labor and reducing the rate of cesarean ; also, a mechanism to reduce maternal anxiety was effective in increasing maternal fetal attachment (29). the mean of the sensory dimension of perineal pain intensity in the cupping therapy group decreased and the differences were statistically significant. cupping therapy directs the blood flow towards the skin and muscles and stimulates the peripheral nervous system. it also stimulates the autonomic nervous system through mediating the immune as well as neurohormone systems and eventually reduces the patients ' pain (30). on the other hand, the mean of the sensory dimension of perineal pain intensity in the acupressure group decreased.. acupressure plays a key role in releasing endogenous endorphins and preventing the transfer of pain signals to hypothalamus through replacement of the pressure messages in the pain control valves (31). this causes the patient not to feel lonely and to be emotionally supported by the therapist. however, the lack of a significant difference might be due to the patient 's discomfort by the therapist 's finger pressure as well as the postpartum pains while breast feeding. the mean of the emotional dimension of perineal pain intensity decreased in the cupping therapy group. however, the mean of the emotional dimension of perineal pain intensity increased after 2 weeks compared to 24 hr after the intervention which might be due to the disruption of the patients ' relationship with the therapist. in fact, the psychological effects of cupping therapy which result from the continuous nature of treatment as well as the close physical and psychological relationships between the cupping therapists and the patients play a critical role in the emotional dimension of pain intensity (32). in the acupressure group, the mean of the emotional dimension of perineal pain intensity decreased but the differences were not statistically significant compared to the cupping therapy group. moreover, the continuous presence of the researcher beside the patients during the intervention and the follow - up has been of great help in improving the patients ' emotional status. (2012) conducted a clinical trial on 60 primiparous women in kamali hospital, karaj, iran in order to investigate the effect of lavender extract on reduction of the perineal pain. a significant difference was found between the two groups regarding the pain criteria 4 hr (p= 0.002) and 5 days (p<0.001) after episiotomy. however, no significant difference was observed between the two groups after 12 hr (p=0.066) (33). using the plant extracts for treating the diseases has always been important in traditional medicine. 2012) performed a research in brazil in order to assess the effect of using local ice packs for various time periods on perineal pain resulting from trauma during delivery. according to the results, pain reduced for above 50% in 72.8% and for 3050% in 21.9% of the subjects. in addition, all the study participants were satisfied with using the ice packs (34). moreover, santos. (2012) carried out a clinical trial in australia in order to study the effect of low - level laser therapy on improvement of perineal pain resulting from episiotomy in primiparous women. laser therapy was directly performed on three perineal points after episiotomy suturing for three sessions. then, the intensity of perineal pain was assessed using a scale containing 10 scores. the study results revealed a significant reduction in the intensity of pain in the second and third sessions of laser therapy compared to the first session (p=0.003 and p=0.001, respectively) (35). application of acupuncture on the right ankle could mobilize the blocked energy flow, affect the genital organs ' function, and reduce the pain. it seems that in case of acupressure point is easily accessible to the researcher, the intervention will be accompanied by a higher success rate. up to now, the findings of various studies have supported the use of traditional medicine in treatment of diseases and no complications have been reported in this regard to affect its efficiency and safety. in this study, the intensity of pain decreased in both intervention groups, but a significant reduction of pain was found in the cupping therapy group. therefore, both cupping therapy and acupressure can be considered as effective methods for reduction of postpartum perineal pain in primiparous women. the findings of the present study showed that postpartum pain reduced in both intervention groups, but a significant reduction was found in the cupping therapy group. therefore, both cupping therapy and acupressure can be considered as effective methods for reduction of postpartum anxiety in primiparous women. of course, further studies are required to be conducted on the issue in order to confirm the obtained results. | background : perineal pain is a major morbidity in the first few days after delivery. this study aimed to investigate the effect of dry cupping therapy and acupressure at bl23 point on the intensity of postpartum perineal pain based on the short - form of mcgill pain questionnaire (smpq).methods : the present clinical trial was conducted on 150 subjects in 3 groups of 50 cases. after at least 48 hr of delivery, cupping therapy was performed for 1520 min up to 3 times a week (once a day) and acupressure was performed for 1520 min based on clockwise model. the short - form of mcgill pain questionnaire was completed both before and after the intervention. the spss statistical software was used to analyze the data using repeated measures anova. besides, p<0.05 was considered statistically significant.results:in the cupping therapy group, mean of the perineal pain intensity reduced from 37.56.8 before the intervention to 11.16.1, 6.94.7, and 3.83.6 immediately, 24 hr, and 2 weeks after the intervention, respectively. the results of study showed that the differences between the intervention and control groups were statistically significant (p<0.01). mean difference of the perineal pain intensity in the acupressure group reached from 35.68.1 before the intervention to 10.45.5 two weeks after the intervention, so the variation between intervention and control groups was statistically significant.conclusion:the study findings showed that cupping therapy and acupressure reduced perineal pain. therefore, they may be considered as effective treatments for reducing pain intensity of allowing delivery. |
high blood pressure is associated with adverse morphological and functional changes in the cardiovascular and renal system, including left ventricular hypertrophy (lvh), microalbuminuria and progressive renal and heart disease. the disproportional accumulation of fibrous tissue is the major characteristic of the adverse structural remodelling of cardiac tissue in hypertensives promoting systolic and diastolic dysfunction [25 ]. transforming growth factor 1 (tgf1) is a multifunctional cytokine and its gene has been found in position 19q13.2, 19q13.1 on chromosome 19 and it has 7 exons and the length of the whole gene is 17.52 kb. tgf1 overproduction acts on cardiomyocytes as well as on cardiac fibroblasts inducing cardiac fibrosis and hypertrophy [6, 7 ]. in addition, the reduction in circulating tgf1 through a block of the renin - angiotensin system (ras) was reported to be associated with an improvement of renal function and a reversion in lvh [8, 9 ]. moreover, recent experimental data indicate that blockade of the tgf, through a novel orally specific inhibitor of the tgf receptor 1, results in significant improvement of deleterious cardiac remodelling after infarction. eight single nucleotide polymorphism (snps) have been described in tgf1 gene and related to its production and to hypertension and cardiovascular disease [11, 12 ]. two snps are located at positions 29 and 74 of the translated sequence of tgf1 and give rise to amino acid substitutions at positions 10 (leupro) and 25 (arg pro) in the signal peptide of tgf1, respectively [12, 13 ]. the t29c was reported to influence steady - state concentrations of tgf1 mrna in peripheral blood mononuclear cells and serum levels of tgf1, and the g74c was found to be related to tgf1 production in peripheral blood leukocytes [13, 14 ]. in addition, the arg allele was associated with risk of hypertension in the normotensives [12, 15 ] and with myocardial infarction [12, 16 ] compared to the pro allele. on the contrary, no studies have been addressed the evaluation of the role of t29 c polymorphism of tgf1 gene on left ventricular geometry and function in hypertensive patients [14, 16 ]. the aim of this study was to investigate the relationship between t29c tgf1 gene polymorphism (rs1800470), lvh and clinical severity of hypertension. in particular, circulating tgf1, procollagen type iii levels, microalbuminuria, left ventricular geometry and function were evaluated in hypertensive patients and related to genotype profile. subjects eligible for the study were screened at the antihypertensive center of the department of internal medicine, university of palermo (italy). subjects under antihypertensive treatment or with a casual blood pressure (sbp) 140 mmhg and/or with casual diastolic blood pressure (dbp) 90 mmhg obtained with a standard sphygmomanometer after 5 minute of rest at three independent occasions with patients sitting were considered hypertensives. exclusion criteria included secondary hypertension, endocrinal disease and diabetes mellitus, cardiovascular diseases (defined as myocardial infarction and recent stroke within previous 6 months, heart failure), severe chronic renal failure, alcoholism and psychiatric problems. 433 hypertensive subjects fulfilled the inclusion criteria and they were grouped according to the presence or absence of lvh, following standard echocardiographic criteria. in particular all the hypertensives with g / mfor men and 47 g / mfor women were considered to have lvh. accordingly, 198 hypertensives with lvh and 235 without lvh were recognized and studied. in addition 94.4% (187/198 pts) of lvh hypertensives and 94.9% (223/235 pts) of no - lvh hypertensives were under antihypertensive treatment at the beginning of the study. the percent of antihypertensive drugs utilized, such as duration of treatment, was not significantly different in all hypertensive groups (table 1), also when subgrouped according to t / c genotypes (table 2). peripheral venous blood was collected in edta from all the patients and stored at 70c. the pcr approach was used to analyze snp in the coding regions of tgf1. the polymorphism is on exon 1 at + 869 from the beginning of the transcription and generate an amino acid substitution in position 10 (leu pro). pcr was performed on purified dna obtained using the genelute blood genomic dna kit by sigma : which provided sequence - specific oligonucleotide primers forward 5-ttccctcgaggccctccta -3 reverse 5-gccgcagcttggacaggat-3 briefly, pcr reactions were carried out in a total volume of 50-l containing approximately 5 l of genomic dna(0.1 g/l), 2 l of forward and reverse primers (100 ng/l), 5 l of 10 reaction buffer (160 mm (nh4)2so4, 670 mm tris - hcl (ph 8,8 at 25c), 15 mm mgcl2, 0, 1% tween 20), 4 l of 2 mm dntps (invitrogen), 4 l of dmso, 0, 1u of taq polymerase. amplification was carried out in a robocycler using cycle parameters of 3 minutes and 30 seconds at 95c (initial denaturation), 35 cycles of 95c for 45 seconds (denaturation), 62c for 30 seconds (primer annealing) and a final extension for 10 minutes at 72c. all pcr products were resolved on 2% agarose gel with 3 l of ethidium bromide (figure 1). pcr reactions in 50 l were directly sequenced by mwg (the full name is eurofins mwg operon, see http://www.eurofinsdna.com/home.html) and from sequence electropherograms was analysed the presence of single - nucleotide polymorphisms t869c in all the subjects (figure 2). the distribution of snp tgf1 gene between both hypertensive groups were reported in table 1. patients underwent a general analytical laboratory parameters profile including bun, creatinine and clearance, glycaemia, electrolytes (serum sodium, potassium, chloride), cholesterol by routine laboratory methods. peripheral venous blood was obtained from each patient and the sera were isolated and stored at 70c. tgf 1 levels were determined by using a solid - phase specific sandwich elisa technique (r&d systems, inc. the interassay and intra - assay variations for determining tgf1 were 8% and 6%, respectively. the sensitivity, hence minum level of detection of tgf1 by sandwich elisa, was 5 pg / ml. to determine amino - terminal piiip, blood samples were taken from each patient and stored at 40c until manipulation. piiip were determined by using a specific radioimmunoassay (orion diagnostic finland), as previously described. the sensitivity of piiip was 1 ng / ml, the intra - assay variations ranged from 1.7% to 4.3% and interassay variations from 3.2% to 5.3%. to eliminate the intra - individual day - to - day variability of uae, three consecutive 24-hour urine collections were used. in addition, to assess the completeness of a 24 hours urine collection, measurements of urinary rate of clearance of creatinine were evaluated. uae was measured by radioimmuno - assay (limit of detection, 0.1 mg / dl ; inter - assay coefficient 3.5%). microalbuminuric patients were defined as a level of uae 20 and < 300 mg/24 hours. all patients underwent an echocardiography examination m and b - mode, by a computerized echocardiography (esaote, italy) for the determination of following parameters : left ventricular telediastolic internal diameter (lvidd), interventricular septum (ivstd), and posterior wall thickness (pwtd). the relative wall thickness (rwt) by formula [(pwtd / lvidd) 2 ] was also calculated. ejection fraction from left ventricular end - diastolic and end - systolic volumes was measured from the apical four chamber view, using the ellipsoidal single - plane algorithm. mean ejection fraction was automatically calculated by the echocardiographic processing system. in our laboratory the ejection fraction calculated over five consecutive beats permitted optimal reproducibility and accuracy. lv relaxation and filling were evaluated by pulsed - wave doppler interrogation of the lv inflow tract from the apical four - chamber view, with the sample volume placed at the tips of the mitral valve. after a stable signal of the transmitral flow velocity was obtained, the doppler cursor was moved toward the lv outflow tract in the apical five - chamber view for recording both mitral and aortic signals, including the closing click of the aortic valve and the opening click of the mitral valve. doppler signals were recorded at high speed (80120 mm / s) with the subjects in held expiration. isovolumic relaxation time (ivrt) was calculated as the time from the closure click of the aortic valve to the opening click of the mitral valve. when either the closing or opening click was not identified, the time from the end of the aortic flow to the onset of mitral flow from the continuous wave interrogation of the lv inflow - outflow tract was used. peak early transmitral flow velocity (e), peak late transmitral flow velocity (a), and the deceleration time of e velocity (dte) were measured at the tips of mitral leaflets at the maximum amplitude of e velocity. dte was measured as the time from peak e velocity to the time when e wave descent intercept the zero line. no sample size was calculated for the lack of any information about the main goal of the study. comparisons among groups were performed by kruskall - wallis test as non parametric analysis of variance. contingency tables were analyzed by the q test or the fisher exact test when possible. pairwise comparison between frequencies were made by z - test after chi - square statistical significant value. a two tailed p value <.05 was considered significant. logistic regression analysis, according to hosmer and lemeshow method has been used to investigate association between tt or tc plus cc genotypes and both laboratory and clinical measurements. the distribution of t29c genotypes in both hypertensive groups has been reported in table 1. the prevalence of tc (p =.0434), and tc plus cc (p =.0466) genotypes, was significantly higher in hypertensives with lvh than hypertensives without lvh (64.7% versus 49.8%, and 84.9% versus 61.4%, respectively). genotype frequency distribution in the two groups of hypertensives occurred according to hardy - weinberg proportions. since genotype frequency distribution was not significant different in hypertensives without lvh, only hypertensives with lvh were further subdivided into three groups, according to t / c genotypes. the association between genotypes and clinical characteristics in the three groups of hypertensives with lvh have been reported in table 2. all the groups were homogeneous regarding to age, bmi, whr and blood pressure. lvh hypertensives with tc or cc genotype were characterized by a significant higher prevalence of subjects with microalbuminuria (p <.05 tc and cc versus tt). no significant difference in the prevalence and duration of antihypertensive drugs utilized was observed among the groups (tables 1 and 2). urinary albumin excretion, circulating tgf1, piiip, lvm and lvm / hlevels were significantly (p <.05) higher and left ventricular ejection fraction values were significantly (p <.05) lower in lvh hypertensives with tc or cc genotype than those detectable in lvh hypertensives homozygous for allele t (tables 3 and 4). this analysis indicated an association between higher levels of piiip and tc or cc genotypes, even if adjusted for lvm / h and urinary albumin excretion values (table 5). to our knowledge this is the first study to investigate the impact of tgf1 leu pro at codon 10 polymorphism (rs1800470) on left ventricular geometry and function in hypertensive patients. our data indicate a higher prevalence of tc and cc leupro polymorphism in hypertensives with lvh than hypertensives without lvh, associated to some unfavorable clinical characteristics of hypertension. in fact, lvh hypertensive subjects with tc or cc genotype were characterized by a higher prevalence of subjects with microalbuminuria, higher value of lvm and lower left ventricular ejection fraction. in our opinion, this association does n't reflect unknown differences in population ancestry between the two hypertensive groups. we consider the probability of false positive inference attributable to population studied rather small because the two hypertensive groups were recruited from an ethnically homogenous population. in this context recent results from xu. revealed a genetic association of tgf1 + 915 arg pro at codon 25 polymorphism with lvh in a chinese hypertensive population, while the codon 10 genotypes did not show any association to lvh. even if in this study we have analyzed only tgf1 leu pro at codon 10 polymorphism, our recent unpublished data indicated no association between tgf1 + 915 arg pro at codon 25 polymorphism and hypertension in a caucasian hypertensive population. these contrasting data may be explained by ethnicity of two hypertensive populations considered (caucasian and chinese). although changes in the heart caused by hypertension are well known, the effective mechanisms are not entirely clarified. despite the role of hemodynamic effects and growth factors have been largely reported, other metabolic and inflammatory factors have to be also considered. literature data and the results of our previous studies indicate an overproduction of circulating tgf1 in hypertensives. moreover, hypertensives with target organ damage (tod) have higher circulating levels of tgf1 than hypertensives without tod. this finding seems to attribute an important role to tgf1 overproduction in the pathophysiology of essential hypertension and its sequelae [24, 25 ]. the tgf1 overproduction in hypertension may be explained by the effects of various factors, such as elevated angiotensin ii, increased fluid shear stress and a differential expression of tgf1 linked to dna polymorphisms in the promoter. plasma concentrations of active and also of acid - activable tgf1 is predominantly under genetic control (heritability estimate 0.54). up regulation of tgf1 system in monocytes of hypertensive patients and association of tgf1 gene polymorphism with risk of hypertension suggests that quantitative difference in tgf1 production may determine the intensity of the process of vascular remodelling and therefore influence overall susceptibility to the development of hypertension. on the other hand experimental data indicate that abnormalities in responsiveness to tgf1 overproduction, as evidenced by collagen formation, may represent a pathophysiological molecular mechanism in hypertension [27, 28 ]. the results from ectim study suggest that tgf1 arg25-pro polymorphism might be associated with hypertension but does not address the issue of quantitative phenotypes related to tgf1 production in relation to hypertension. on the contrary no study has been addressed the evaluation of the role of leu - to - pro polymorphism at codon 10 to explain whether the substitution has functional and biological importance, or it could affect protein transport. in view of this we have now shown that the serum concentration of tgf1 was significantly different in lvh hypertensives subgrouped according to t29 c polymorphism. it is possible that the t29 c polymorphism of the tgf1 gene is linked to some other genes that are actually responsible for the development of hypertension. moreover, the significant change in circulating tgf1 values among lvh hypertensive groups indicated that tc or cc genotypes could be able to induce quantitative change in the production of the cytokine and it might affect also the function of signal peptide, perhaps influencing intracellular trafficking or export efficiency of preprotein. first, lvh hypertensive subjects with tc or cc genotypes were characterized by a higher value of microalbuminuria, lvm and by a systolic left ventricular dysfunction. this might indicate that hypertension in these subjects has to be considered more severe. another aspect of our study that seems to be interesting is related to the higher collagen production in lvh hypertensive subjects with tc or cc genotype than in those homozygous for allele t. this finding is further supported by analysis of logistic regression indicating that piiip may be considered the most important marker associated to t and c alleles. in fact, analysis of odd ratio indicates that the risk of association with t or c allele increases eight fold for each piiip quintile variation. in our opinion this finding might further support our previous results [5, 23 ] indicating that overproduction in circulating tgf1 may contribute to the progression of renal and cardiovascular damage in obese and/or hypertensive subjects. in particular, the present study emphasizes the unfavourable effects of tgf1 overproduction on left ventricular geometry and function that might be mediated by an higher collagen production [5, 24, 29 ]. in view of this the association of tc or cc with a higher clinical severity of hypertension seems to indicate that tgf1 is a susceptibility locus for hypertension. a potential limit of this study was to have computed no a priori evaluation of the error and consequently the power of our statistical analysis. however this involves considerations only about the sample size. accordingly the negative results of our study has needed further evaluations. in conclusion our data are attractive to indicate that lvh hypertensive subjects with tc or cc genotypes (leu pro polymorphism, rs 1800470) might be considered a particular subset of lvh hypertensives with a more severity of hypertension. however, the present results require verifications in other populations, since it is well known that hypertension is under the control of many genes that contribute modest individual effects, and tgf1 may act in concert with other hypertension susceptibility loci. | the distribution of the t29c tgf1 gene polymorphism was analyzed in 198 hypertensives with left ventricular hypertrophy (lvh) and in 235 hypertensives without lvh. circulating tgf1 levels, procollagen type iii levels, microalbuminuria, and left ventricular geometry and function were evaluated in all the hypertensives with lvh subgrouped according to t29c tgf1 gene polymorphism. circulating tgf1 was evaluated by elisa technique, procollagen type iii by a specific radioimmunoassay, microalbuminuria by radioimmunoassay, and left ventricular geometry and function by echocardiography. all groups were comparable for gender, age, and sex. regarding t29c tgf1 gene polymorphism, prevalence of tc or cc genotypes was significantly (p <.05) higher in hypertensives with lvh than hypertensives without lvh tc and cc lvh hypertensives were characterized by a higher prevalence of subjects with microalbuminuria (p <.05 tc and cc versus tt), by increased levels of tgf1, procollagen type iii, urinary albumin excretion, lvm, lvm / h2.7, and lower values of left ventricular ejection fraction (p <.05 tc and cc versus tt). our data suggest that t29c tgf1 gene polymorphism was associated with clinical characteristics adequate to recognize a subset of lvh hypertensives with a higher severity of hypertension. |
compared with the synovial joints of the limbs, the temporomandibular joint (tmj) develops relatively late. by the eighth week of development in humans (orahilly s stage 23), the joint cavity of the elbows, hips, and knees are already visible, while the tmj has only mesenchymal condensation of the mandibular condyle, articular disc, and squamous part of the temporal bone, and no evidence of its cavity., by approximately week nine, the condylar chondrification begins in the center of the condylar blastemal. the inferior joint cavity starts to develop at the end of the ninth week, as the density of the mesenchymal cells in the central region decreases because of their differentiation into fibroblasts, constituting the primordium of the articular disc. as the density of the ectomesenchyme decreases, small adjacent spaces between the cells coalesce, forming the inferior joint cavity between the future articular disc and the mandibular condyle. in the 10 week, these newly differentiated fibroblasts become more compact, forming collagen fibers and the articular disc, but there is still no sign of the superior joint cavity. in the eleventh week, the organization of the superior joint cavity begins between the squamous part of the temporal bone and the articular disc. more detailed studies of the tmj development, as well as of their condylar cartilage, articular disc and the matrix proteins on the fetus development were researched through immunohistochemistry and in situ hybridization. morphologically, the synovial membrane consists of two layers : an inner cell layer called the intima and a support layer called the vascular subintima, which mixes with the fibrous capsule. the intima consists of cells embedded in an amorphous, fiber - free matrix with an approximate thickness of one to four cells. the subintima consists of loose connective tissue with blood vessels, spread - out fibroblasts, macrophages, mastocytes, adipose cells, and some elastic fibers that prevent membrane folding. the intima contains macrophage - like type a cells with phagocytic ability, and fibroblast - like type b cells that synthesize proteins, glycoproteins, and proteoglycans. immunohistochemical techniques allowed to detect macrophage - like type a cells and fibroblast - like type b cells by using anti - macrophage and anti - fibroblast antibodies, respectively. grabowski., nozawa - inoue. and suzuki. successfully marked macrophage - like type a cells with the antibodies ox6, edi, cd68, and cd31. on the other hand, the laminin antibodies mab67, vcam-1, lumican, fibromodulin, udpgd, and hsp25 mark fibroblast - like type b cells, and have been used in studies of the synovial intima. although a number of groups have studied about the synovial membrane of the tmj of children, adults, and animals models, there is little in the literature regarding the development of this structure in human fetuses. understanding the morphology of fetal development is important not only to better understand the embryological steps that culminate with the individual s anatomic constitution but also to elucidate the intrinsic mechanisms that may be involved in congenital anomalies and postnatal pathologies. this study aims to improve the anatomic and histologic knowledge of the synovial membrane by determining when the synovial meml.o. carvalho de moraesbrane begins to form during morphogenesis and the chronological occurrence and dynamics of cell types present in the synovial membrane. it also aims to analyze the morphological differences that appear during the development of the superior and inferior articular cavities. we studied twenty one human fetuses from the institute of embryology of the university complutense of madrid and associao de fundo incentivo pesquisa. the specimens ranged from 40 mm to 100 mm greatest length (gl) with the following ages : three nine - week fetuses ; four ten - week fetuses ; five eleven - week fetuses ; four twelve - week fetuses ; five thirteen - week fetuses ; post - conception age was determined by measuring gl and external and internal criteria. all specimens were from ectopic pregnancies or spontaneous abortions, and there were no signs of malformation. all fetuses were fixed in 10% formalin and separated into groups studied by light microscopy and immunohistochemistry. all samples were decalcified in edta for 21 days and rinsed with tap water for 10 min. they were then fixed in 10% formaldehyde for 24 h. next, they were dehydrated as follows : one 24-h immersion in 50% ethanol ; two 6-h immersions in 70% ethanol ; and two 6-h immersions in absolute ethanol. finally, the samples were cleared by a 2-h treatment with xylol and fixed in paraffin. semi - serial, frontal, and sagittal 4 m cuts of the tmj were made by the microtome leica, model rm2035. the sections, which included three spatial planes, were stained with hematoxylin - eosin (he), azocarmine, and masson s trichrome stain. the following antibodies were used : cd68 (santa cruz biotechnology, santa cruz, ca, usa) ; monoclonal anti - human igg1 produced in mouse and diluted to 1:100 and hsp27 (santa cruz biotechnology) ; monoclonal anti - human igg1 produced in mouse and diluted to 1:150 using the indirect immunoenzyme method in three stages and the streptavidin - biotin - peroxidase complex. the antibodies cd68 and hsp27 react with the cells macrophage - like type a and fibroblast - like type b, respectively. the positive controls for the antibodies cd68 and hsp27 were palatine tonsil and breast adenocarcinoma, respectively. the negative controls were the same cases used as positive controls, but a buffer instead of the primary antibody was used for immunohistochemical incubation. the study was approved by the ethics committee of the faculty of medicine of the university complutense of madrid and the federal university of so paulo research ethics committee. all specimens studied during the ninth week showed small spaces or clefts between the primordium of the articular disc and the mandibular condyle that defined the initial formation of the inferior joint cavity. some vascular branches were located in the periphery of the articular disc primordium (figure 1a). all specimens studied during the tenth week showed the organization of the inferior joint cavity complete although crossed by a few tracts of connective tissue. blood vessels in the inferior part of the articular disc primordium were seen during the eleventh week. fusiform - like cells separate the vessels of the inferior articular cavity (figure 1 c, d). the organization of the superior joint cavity began between the squamous part of the temporal bone and the primordium of the articular disc. the superior joint cavity was crossed by few tracts of the connective tissue (figure 1c). numerous vessels were seen in the lateral and medial parts of the tmj (figure 1d). all specimens studied this week showed the joint cavities clearly defined and decrease in the number of septa of connective tissue that crossed them was noted. the vessels in the inferior part of the articular disc did not reach the middle area of the disc (figure 1 e, f). some macrophage - like cells that expressed cd 68 and fibroblast - like type b cells that express hsp27 (figure 2 a, c, e) appeared bottoms of the inferior articular cavity. all specimens studied during the thirteenth week showed the vessels located bottoms of the inferior articular cavity. some vessels surrounded by fusiform cells protruded from the inferior sac bottoms, which corresponds to the primordium of the synovial villi (figure 1 g). panels b and d of figure 2 show the positive control of the antibodies cd68 and hsp27 in palatine tonsil and breast adenocarcinoma samples, respectively. all specimens studied during the ninth week showed small spaces or clefts between the primordium of the articular disc and the mandibular condyle that defined the initial formation of the inferior joint cavity. some vascular branches were located in the periphery of the articular disc primordium (figure 1a). all specimens studied during the tenth week showed the organization of the inferior joint cavity complete although crossed by a few tracts of connective tissue. blood vessels in the inferior part of the articular disc primordium were seen during the eleventh week. fusiform - like cells separate the vessels of the inferior articular cavity (figure 1 c, d). the organization of the superior joint cavity began between the squamous part of the temporal bone and the primordium of the articular disc. the superior joint cavity was crossed by few tracts of the connective tissue (figure 1c). numerous vessels were seen in the lateral and medial parts of the tmj (figure 1d). all specimens studied this week showed the joint cavities clearly defined and decrease in the number of septa of connective tissue that crossed them was noted. the vessels in the inferior part of the articular disc did not reach the middle area of the disc (figure 1 e, f). some macrophage - like cells that expressed cd 68 and fibroblast - like type b cells that express hsp27 (figure 2 a, c, e) appeared bottoms of the inferior articular cavity. all specimens studied during the thirteenth week showed the vessels located bottoms of the inferior articular cavity. some vessels surrounded by fusiform cells protruded from the inferior sac bottoms, which corresponds to the primordium of the synovial villi (figure 1 g). panels b and d of figure 2 show the positive control of the antibodies cd68 and hsp27 in palatine tonsil and breast adenocarcinoma samples, respectively. according to mrida - vela., the tmj joint cavities are not formed synchronously. first, the inferior joint cavity begins to develop in the ninth week as small spaces or fissures appear between the articular disc and the mandibular condyle. subsequently, the superior joint cavity begins to form approximately in the eleventh week of gestation, between the articular disc and the squamous part of the temporal bone. sperber, burdi, and gutcen - toller, who reported that the inferior joint cavity begins to form during the tenth week of gestation, while the superior joint cavity begins to form from the eleventh week of gestation. according to ohnuki, the inferior joint cavity above the mandibular condyle is still being formed during the twelfth week of development, while the superior joint cavity is only visible in the posterior region of the joint. in humans, buccal movements begin during weeks 7 and 8 of development at the level of the incudomalleolar joint. tmj movements help to form the joint cavity and their absence could results in congenital craniofacial anomalies. the presence of blood vessels in the articular disc of the tmj of human fetuses has been described by many authors. van der linden. noted that capillaries were distributed in the periphery of the anterior and posterior portions of the articular disc in human fetuses. however, they did not report any vessels in the central region of the articular disc. wong. observed that in fetuses, 13 to 17.5 weeks of development vessels are mainly located anteriorly and posteriorly, but no evidence of vascularization was found at the center of articular disc. studied 61 specimens of articular disc aged 2 days to 26 years and concluded that vascularization only exists in the first year of life, after which it disappears. interestingly, sab. observed vessels containing red blood cell in the central portion of the tmj articular disc of human fetuses. according to our findings, from the ninth week of development the number of vessels increases around the temporomandibular joint region, sending some vascular branches to the periphery of the articular disc primordium. in specimens at eleven weeks of development, the vessels reach the central portion of the articular disc and fusiform - like cells separate the disc from the inferior articular cavity. during the twelfth week of development, the articular cavities are well defined and the vessels do not reach the central region of the articular disc. these findings can suggest that the vessels are necessary for the vascularization of the articular disc until the synovial membrane is well developed. the synovial membrane of the tmj is areolar and only this type of synovial membrane is characterized by the presence of two cell types in its intima layer : macrophage - like type a cells and fibroblast - like type b cells. their basic function is to engulf, by phagocytosis, the proteins and carbohydrates present in the synovial. fibroblast - like type b cells are ultrastructurally characterized by the presence of a well - developed rough endoplasmic reticulum with numerous secretory granules. macrophage - like type a and fibroblast - like type b cells in the synovial membrane can be identified by immunohistochemistry and transmission electron microscopy in fetuses, adults, and animals before and after birth. ikeda. reported that macrophage - like type a cells in the synovial membrane of the tmj of rats were only visible after birth. found macrophage - like type a and fibroblast - like type b cells only during the gestational period. our findings confirm those of other authors, including who found both macrophage - like type a and fibroblast - like type b cells in the synovial membrane before birth. during the 12 week of development of the peripheral parts of the inferior articular cavity, macrophage - like type a and fibroblast - like type b cells were marked by immunohistochemical reaction with the antibodies cd68 and hsp27, respectively. fibroblast - like type b cells were also marked in the peripheral portions of the superior articular cavity. these findings could suggest that from the twelfth week of development, with the articular cavities well formed, the synovial membrane is functional and the vessels are only seen in the peripheral areas of the articular disc. there is also a third type of cell not yet fully studied called an intermediate lining cell. this study demonstrates that development of the synovial membrane depends on the fibroblast - like type b cells located on the surface of the synovial membrane and macrophage - like type a cells that supply the vessels. our results also suggest that in human specimens at twelve weeks of development, the articular cavities are well formed and the synovial membrane begins to be functional. | the development of the synovial membrane was analyzed in serial sections of 21 temporomandibular joints of human fetuses at 9 to 13 weeks of gestation. sections of two fetuses at 12 weeks of development were used to perform immunohistochemical expression of the markers cd68 and hsp27 on the synovial lining. macrophage - like type a and fibroblast - like type b cells, which express cd68 and hsp27, respectively, were observed at the twelfth week of development. our results suggest that the development of the synovial membrane is related to the vascularization of the joint and the formation of the articular cavities. |
the ideal treatment for asymptomatic bile duct stones found during laparoscopic cholecystectomy has yet to be established. the introduction of laparoscopic cholecystectomy (lc) has not changed the basic rationale for the treatment of cholelithiasis, but it has for common bile duct stones (cbds). the main options are laparoscopic bile duct exploration through the cystic duct or choledochotomy ; conversion to open exploration ; postoperative endoscopic retrograde cholangiopancreatography (ercp) with endoscopic sphincterotomy (es) ; and observation alone. different surgeons deal with this according to their personal preferences and hospital protocols. at our institution, the tendency is to treat bile duct stones in one stage whether the choledocholithiasis is diagnosed preoperatively or found during the procedure. between january 1993 and december 2002, 3118 patients with symptomatic gallstones underwent laparoscopic cholecystectomy. preoperative evidence of choledocholithiasis was defined as a history of jaundice or pancreatitis, elevated total serum bilirubin (> 1.0 mg / dl), alkaline phosphatase (> 147 u / dl), or amylase (> 115 u / dl), or preoperative imaging (ultrasound) demonstrating choledocholithiasis or common bile duct > 7 mm in diameter. ercp was performed preoperatively in 82 of the 437 patients (18.7%) in whom the history, blood tests, and imaging studies strongly suggested bile duct stones. these patients were also compromised with respiratory problems or cardiac disease that increased the operating risk. therefore, 366 patients were operated on laparoscopically : transcystic clearance with transcystic drainage was performed in 96 patients, and transcholedochal clearance with t - tube placement was performed for 270 patients. all patients had intraoperative cholangioscopy utilizing a flexible 7 f fiberscope through the cystic duct or via a choledochotomy to confirm clearance of the biliary system. the mean age of patients was 61 years (range, 31 to 84) ; 138 were men and 228 were women. preoperatively, 44 patients (12%) had clinical, laboratory, or imaging evidence of stones, and operative cholangiography in these patients showed the number of stones to be from 1 to 5 with a diameter of 3 mm to 7 mm. a transcystic catheter was placed in 96 patients (26.2%) and removed after 2 weeks. a t - tube was placed in 270 patients (73.8%) and removed after 3 weeks. in all cases, with transcystic and transcholedochal drainage in situ, no patients developed obstructive jaundice, acute cholangitis, or pancreatitis, and postoperative cholangiography via the transcystic catheter showed residual stones in 2 patients. postoperative hospital stay ranged from 4 days to 12 days, and all patients have been symptom free without evidence of stone recurrence or cholangitis for a mean of 2 years since cholecystectomy. excluding acute cholecystitis, choledocholithiasis is the most common complication of gallstones and occurs in at least 10% to 15% of all patients who undergo cholecystectomy. this prevalence increases dramatically in elderly patients, reaching more than 48% in those who require cholecystectomy patients who are over age 70. not only are biliary tract surgeons of today expected to perform laparoscopic cholecystectomy with the same low morbidity, mortality, and efficacy as in open surgery, but they also have an obligation to manage all stone - related biliary tract disease as efficiently, or more efficiently, than in the past. it is our opinion that the surgeon must manage even these complicated problems in the least invasive manner possible. more than one third of all cases of choledocholithiasis identified during cholecystectomy are unsuspected because preoperative history, clinical signs, and laboratory data are equivocal or normal. in an early series of 500 consecutive cases (open cholecystectomy) with routine ioc, 5% of the patients were found to have unsuspected calculi. a multi - institutional evaluation of the laparoscopic treatment of common bile duct stones also confirmed a high prevalence of unsuspected intraductal calculi. but the greatest value of routine cholangiography is the possibility of recognizing anomalies of the biliary system and detection / documentation of bile duct injuries during surgery. in our series we had only one injury (0.03%) of the hepatic duct that was repaired immediately. intraoperative choledochoscopy is an important part of the treatment of choledocholithiasis, to make sure that all stones are identified and removed. we are convinced that it is important to drain the extrahepatic biliary tree in all patients with choledocholithiasis. in accordance with other authors, we decompress the extrahepatic biliary system after every common bile duct exploration because of the potential for obstruction, secondary to edema at the lower end of the bile duct secondary to surgical manipulation. the management of choledocholithiasis has reached a point in its evolution where more options are available than some institutions can support. the advances over the last 10 years to 15 years in laparoscopic technology have created a seductive atmosphere in which laparoscopy is being considered for managing choledocholithiasis. however, we believe that, when it is possible, it is appropriate to resolve a patient 's pathologic condition in one stage, making use of any method the surgeon has available. | objectives : a minimally invasive approach is considered the treatment of choice for gallbladder stones. we report our experience with the treatment of choledocholithiasis.methods:from january 1993 to december 2002, 3118 patients underwent minimally invasive surgery for symptomatic gallstones, 2681 for gallbladder stones and 437 (14%) for cholecysto-choledocholithiasis.results:we performed endoscopic retrograde cholangiopancreatography and endoscopic sphincterotomy in 71 patients (18.7%) with high operative risks, transcystic clearance and transcystic drainage in 96 cases (26.2%) and transcholedochal clearance with a t - tube in 270 cases (73.8%). in 2 patients, residual stones were removed with endoscopic retrograde cholangiopancreatography and endoscopic sphincterotomy. postoperative stay ranged from 4 days to 12 days. no morbidity or mortality occurred.conclusion:in our experience, one - stage laparoscopic procedure for cholecystocholedocholithiasis is safe and effective in skilled hands. |
the intrapulmonary bronchogenic cyst appears a sharply circumscribed, round or oval nodule or mass, usually in the medial third of the lungs. the lesion do not communicate with the tracheobronchial tree until they become infected, a complication that occurs in about 75% of cases recognized clinically (1, 2). bronchial polyps are rare findings and classified as multiple papillomas, solitary papillomas, and inflammatory polyps (3). bronchial polyp occurs spontaneously or after airway injury such as chronic infection, foreign bodies, asthma, and inhalation injury. we report a case of polypoid intrapulmonary lung cyst, which was the first case among koreans, has been removed by bronchoscopy. a 68-yr - old woman came to our hospital with a month history of discomfort and pain in the right upper chest. she denied a history of fever, upper respiratory infection, or allergies. she had never smoked. on admission her blood pressure was 120/80 mmhg, pulse rate 84/min, respiratory rate 24/min, and body temperature 36.6. cardiac examination showed normal findings. the lungs on auscultation showed a decrease in vesicular sounds in the right upper lung field. laboratory tests revealed normal findings. on spirometry, her forced vital capacity and forced expiratory volume in 1 sec were 2,480 ml (120% of the predicted value) and 2,000 ml (139% of the predicted value), respectively. flexible bronchoscopy revealed a peduncular polyp about 2 cm in length originating from the anterior segment of the right upper lung (fig. after bronchoscopic removal with biopsy forceps of the polyp, cystic lesion of the right upper lung disappeared (fig. 1b) and the discomfort and pain in the right upper chest of patient improved. gross appearance showed polyp with stalk and light - microscopical examination showed a lymphocyte infiltration with epithelial lining cells that was consistent with the diagnosis of a bronchial inflammatory polyp (fig. this is a rare case of polypoid intrapulmonary bronchogenic cyst, which was disappeared after bronchoscopic removal. there were possible diagnoses for the initial radiographic chest findings of the cystic lesion in the right upper lung, including infected cyst, infected large bullae, pulmonary sequestration. bronchogenic cysts are usually solitary, thin walled, unilocular, and roughly spherical in shape. they are filled with either mucoid or serous fluid and do not communicate with the tracheobronchial tree unless they become infected, in which case the cyst fluid may be pus or by pus and air (4). in this case however they may communicate with the tracheobronchial tree and become infected and some enlarge to cause airway obstruction. bronchial polyps are classified as multiple papillomas, solitary papillomas, and inflammatory polyps (3). inflammatory polyps of the airways are now regarded as histopathologically distinct nonneoplastic endobronchial lesions, which in adults are associated with a variety of chronic inflammatory insults such as chronic infection, foreign bodies, asthma, or inhalation injury (5 - 7). generally, benign bronchogenic cysts need not be resected unless they cause symptoms. in treatment of bronchial polyp the developments in bronchoscopic techniques have made it possible to remove superficial tumors by bronchoscopy. removal of the foreign body or inhalation of corticosteroid may resolve bronchial polyp (5). in the present case, bronchial polyp was removed by bronchoscopy and then lung cyst disappeared suggesting that intrapulmonary cyst communicate with bronchus. histologically the bronchogenic cyst wall is lined by a pseudostratified, ciliated epithelium and contains cartilage and strands of smooth muscle important for diagnosis. in the present case, polyp composed of epithelial lining cells without cartilage in their wall may represent intrapulmonary cyst as a result of ingrowth from communicating airway epithelium. | pulmonary bronchogenic cyst in adults is rare and the typical appearance is a sharply circumscribed, round or oval nodule or mass, usually in the medial third of the lungs. bronchial polyps are rare histopathologically distinct nonneoplastic endobronchial lesions and are classified as multiple papillomas, solitary papillomas, and inflammatory polyps. we herein report a patient with polypoid endobronchial lung cyst. a 68-yr - old woman presented with a discomfort and pain in the right upper chest of four weeks??duration. chest radiography revealed a cystic lesion in the right upper lung. computed tomography revealed a 45 cm sized large cyst. neither enlarged mediastinal lymph nodes nor extrabronchial involvements were observed. flexible bronchoscopy revealed a peduncular polyp about 2 cm in length originating from the anterior segment of right upper lung. after bronchoscopic removal of polyp, cystic lesion of the right upper lung disappeared. |
subjects were islet cell antibody negative women who participated in a longitudinal study of the pathogenesis of type 2 diabetes following gdm. briefly, all latino women referred to los angeles county women 's hospital for management of gdm between august 1993 and march 1995 were asked to participate if they of the following criteria : 1) gestational age between 28 and 34 weeks, 2) no current or prior insulin therapy, 3) all fasting serum glucose concentrations 140 mg / dl, or reached the final scheduled study visit 12 years postpartum. women who were pregnant at the time of a scheduled battery of tests were studied at least 4 months after pregnancy and at least 1 month after completion of breastfeeding. all subjects gave written, informed consent for participation in the study, which was approved by the institutional review board of the university of southern california and the los angeles county and the university of southern california medical center. for the present report, which is focused on physiological changes associated with development of diabetes from the first postpartum visit onward, we analyzed data from all subjects who 1) had baseline ogtt, ivgtt, glucose clamp, and body composition studies without diabetes within 30 months postpartum and 2) returned for at least one additional ogtt to determine diabetes status. follow - up data were used up to earlier of either the diagnosis of diabetes by american diabetes association criteria (fasting 126 mg / dl or 2 h 200 mg / dl) or the last visit without diabetes. for the baseline battery of ogtt, ivgtt, glucose clamp, and body composition, subjects came to the general clinical research center on 3 separate days, at least 48 h apart, after 812 h overnight fasts and at least 3 days on an unrestricted diet. on one day, bioelectrical impedance (bia) was measured immediately prior to an ogtt. for bia, subjects lay supine while plastic electrodes were placed on their right hand and foot and a trained technician took dual resistance and reactance readings with a quantom impedance meter (rjl systems, clinton township, mi). for ogtts blood was obtained from an antecubital venous catheter before and 15, 30, 60, 90, 120, and 180 min after the glucose ingestion, placed on ice, and plasma was separated within 20 min and stored at 80c. on a separate day, an ivgtt was performed starting between 0700 and 1000 h. dextrose (300 mg / kg) was injected over 1 min, followed in 20 min by a 5-min infusion of crystalline human insulin (0.03 units / kg). arterialized venous blood was drawn into iced tubes before (n = 2) and for 240 min after (n = 32) the dextrose injection., a glucose clamp was performed starting between 0600 and 0630 h. a primed (0.035 mmol / kg body wt), continuous (2.5 10 mmol / min / kg) infusion of 6,6 h2 d - glucose (tracer) was administered through an antecubital vein for 360 min. a nonprimed infusion of crystalline human insulin (40 mu / min per m body surface area) was administered during the final 180 min of the tracer infusion. dextrose (20% wt / vol in water), containing dideutero - glucose (0.021 mmol / cc) to minimize changes in plasma tracer enrichment, was given to maintain arterialized venous plasma glucose concentrations at 88 mg / dl during the insulin infusion. blood samples for measurement of tracer, hormone/ and metabolite concentrations were drawn into ice - cold tubes at 90, 50, 30, 10, 30, 60, 90, 120, 160, and 180 min relative to the start of the insulin infusion. glucose was measured by glucose oxidase (beckman glucose analyzer ii ; beckman, brea, ca). insulin was measured by a radioimmunoassay (novo pharmaceuticals, danbury, ct) that measured insulin and proinsulin. plasma free fatty acids (ffas) were measured by an enzymatic colorimetric method (wako chemicals, richmond, va). plasma adiponectin and leptin levels were measured using radioimmunoassay kits from linco research. plasma c - reactive protein (crp) and interleukin (il)-6 were measured using crp enzyme - linked immunosorbent assays and ultrasensitive il-6 enzyme - linked immunosorbent assays kits from alpco diagnostics. 6,6, h2-glucose concentrations in infusates and perchloroacetate (pca) supernatants of plasma were measured by gas chromatography and mass spectrometry after conversion of glucose to its aldonitrile penta - acetate derivative. anti bmi was calculated as weight in kilograms divided by the square of height in meters. ivgtt results were analyzed using the minmod program (8) to obtain measures of fractional glucose disappearance due to an increase in insulin above basal (insulin sensitivity ; si). the acute insulin response to intravenous glucose (airg) was calculated by the trapezoid rule as the incremental area under the insulin curve during the first 10 min after the glucose injection. the product of si and airg (the disposition index ; di) was calculated as a measure of acute pancreatic -cell compensation for insulin resistance (8). body fat and fat - free mass were calculated by the formula of kotler. follow - up data were used up to the diagnosis of diabetes or the last visit without diabetes, whichever occurred first. cumulative diabetes incidence rates were estimated using life - table methodology and displayed using kaplan - meier plots. cox proportional hazard regression was used to test for associations between baseline and/or follow - up variables and time to diabetes development. baseline variables were treated as fixed covariates and follow - up variables were treated as time - dependent covariates. follow - up measures included changes from baseline in body weight and fat and disposition index, the presence or absence of additional pregnancies, and use of hormonal contraception. the proportional hazard assumption was evaluated by testing for interaction between the covariate and time in the model ; no significant violation was found for the tested variables. baseline characteristics to convert the glucose in unit of mg / dl to the si unit of mmol / l, multiply the number in the table by 0.0555. basal values are the means of data collected during the last 90 min of the 3-h basal tracer infusion period, and steady - state values are the means of data collected during the final 30 min of the 3-h euglycemic insulin infusion period. all statistical tests were two - sided, and statistical significance was defined as p 0.05. for the present report, which is focused on physiological changes associated with development of diabetes from the first postpartum visit onward, we analyzed data from all subjects who 1) had baseline ogtt, ivgtt, glucose clamp, and body composition studies without diabetes within 30 months postpartum and 2) returned for at least one additional ogtt to determine diabetes status. follow - up data were used up to earlier of either the diagnosis of diabetes by american diabetes association criteria (fasting 126 mg / dl or 2 h 200 mg / dl) or the last visit without diabetes. for the baseline battery of ogtt, ivgtt, glucose clamp, and body composition, subjects came to the general clinical research center on 3 separate days, at least 48 h apart, after 812 h overnight fasts and at least 3 days on an unrestricted diet. on one day, bioelectrical impedance (bia) was measured immediately prior to an ogtt. for bia, subjects lay supine while plastic electrodes were placed on their right hand and foot and a trained technician took dual resistance and reactance readings with a quantom impedance meter (rjl systems, clinton township, mi). for ogtts, subjects drank 75 g of dextrose. blood was obtained from an antecubital venous catheter before and 15, 30, 60, 90, 120, and 180 min after the glucose ingestion, placed on ice, and plasma was separated within 20 min and stored at 80c. on a separate day, an ivgtt was performed starting between 0700 and 1000 h. dextrose (300 mg / kg) was injected over 1 min, followed in 20 min by a 5-min infusion of crystalline human insulin (0.03 units / kg). arterialized venous blood was drawn into iced tubes before (n = 2) and for 240 min after (n = 32) the dextrose injection., a glucose clamp was performed starting between 0600 and 0630 h. a primed (0.035 mmol / kg body wt), continuous (2.5 10 mmol / min / kg) infusion of 6,6 h2 d - glucose (tracer) was administered through an antecubital vein for 360 min. a nonprimed infusion of crystalline human insulin (40 mu / min per m body surface area) was administered during the final 180 min of the tracer infusion. dextrose (20% wt / vol in water), containing dideutero - glucose (0.021 mmol / cc) to minimize changes in plasma tracer enrichment, was given to maintain arterialized venous plasma glucose concentrations at 88 mg / dl during the insulin infusion. blood samples for measurement of tracer, hormone/ and metabolite concentrations were drawn into ice - cold tubes at 90, 50, 30, 10, 30, 60, 90, 120, 160, and 180 min relative to the start of the insulin infusion. glucose was measured by glucose oxidase (beckman glucose analyzer ii ; beckman, brea, ca). insulin was measured by a radioimmunoassay (novo pharmaceuticals, danbury, ct) that measured insulin and proinsulin. plasma free fatty acids (ffas) were measured by an enzymatic colorimetric method (wako chemicals, richmond, va). plasma adiponectin and leptin levels were measured using radioimmunoassay kits from linco research. plasma c - reactive protein (crp) and interleukin (il)-6 were measured using crp enzyme - linked immunosorbent assays and ultrasensitive il-6 enzyme - linked immunosorbent assays kits from alpco diagnostics. 6,6, h2-glucose concentrations in infusates and perchloroacetate (pca) supernatants of plasma were measured by gas chromatography and mass spectrometry after conversion of glucose to its aldonitrile penta - acetate derivative. bmi was calculated as weight in kilograms divided by the square of height in meters. ivgtt results were analyzed using the minmod program (8) to obtain measures of fractional glucose disappearance due to an increase in insulin above basal (insulin sensitivity ; si). the acute insulin response to intravenous glucose (airg) was calculated by the trapezoid rule as the incremental area under the insulin curve during the first 10 min after the glucose injection. the product of si and airg (the disposition index ; di) was calculated as a measure of acute pancreatic -cell compensation for insulin resistance (8). body fat and fat - free mass were calculated by the formula of kotler. follow - up data were used up to the diagnosis of diabetes or the last visit without diabetes, whichever occurred first. cumulative diabetes incidence rates were estimated using life - table methodology and displayed using kaplan - meier plots. cox proportional hazard regression was used to test for associations between baseline and/or follow - up variables and time to diabetes development. baseline variables were treated as fixed covariates and follow - up variables were treated as time - dependent covariates. follow - up measures included changes from baseline in body weight and fat and disposition index, the presence or absence of additional pregnancies, and use of hormonal contraception. the proportional hazard assumption was evaluated by testing for interaction between the covariate and time in the model ; no significant violation was found for the tested variables. baseline characteristics to convert the glucose in unit of mg / dl to the si unit of mmol / l, multiply the number in the table by 0.0555. estimated by bioelectrical impedance. basal values are the means of data collected during the last 90 min of the 3-h basal tracer infusion period, and steady - state values are the means of data collected during the final 30 min of the 3-h euglycemic insulin infusion period. all statistical tests were two - sided, and statistical significance was defined as p 0.05. sixty women had their baseline visits at 15 months postpartum and 12 had their baseline visits at 30 months. at baseline, 26 women had normal glucose levels, 8 had impaired fasting alone, 19 had impaired 2-h glucose alone, and 19 had impaired fasting and 2-h glucose levels using american diabetes association criteria (7). the average annual rate of loss to follow - up was 6.5%. during a median follow - up of 72 months (minimum 12 months ; maximum 142 months), 31 (43%) of the average annual incidence rate of diabetes, calculated by person - years, was 7.2%. the annual diabetes incidence rates were 4.1, 6.9, 7.0, and 14.8% in women whose ogtt values at baseline were, respectively, normal, impaired fasting alone, impaired 2-h alone, and impaired fasting and 2-h glucose together. kaplan - meier plot of diabetes cumulative incidence rate in 72 women without diabetes at entry and with at least one follow - up ogtt. the numbers given by the subjects line included subjects who developed diabetes and who were under follow - up without diabetes by the corresponding follow - up years. weight and glucose increased significantly during follow - up, while acute insulin secretion (airg) and the disposition index (di) fell significantly (table 2). nine women used combination oral contraceptives exclusively for a median of 21 months (range 267). eight women used progestin - only contraception exclusively for a median of 15 months (340). one woman started with a progestin - only contraceptive, was off from any hormonal contraception, and then switched to combination oral contraceptives. rates of change during follow - up to convert the glucose in unit of mg / dl to the si unit of mmol / l, multiply the number in the table by 0.0555. univariate cox regression analysis using each of the baseline variables presented in table 1 revealed that relatively high glucose, relatively low insulin sensitivity, relatively low insulin responses, and relatively low -cell compensation for insulin resistance were associated with development of diabetes (table 3). multivariate cox regression analysis using all baseline variables revealed four that were significantly associated with development of diabetes (table 4). the strongest was -cell compensation for insulin resistance, expressed as the disposition index from ivgtts. the other baseline predictors identified from multivariate analysis were insulin sensitivity, measured as incremental glucose clearance during hyperinsulinemic clamps (low = increased risk), total glucose area during ogtts (high = increased risk), and the 30-min increment in insulin on ogtts (low = increased risk). univariate significant baseline predictors of diabetes variables and units are defined in table 1. multivariate significant baseline predictors of diabetes p 5 years after the index pregnancy (right two bars). b : disposition index during follow - up in women who developed diabetes and in women who remained diabetes free. log scale is depicted to reflect that data were log transformed prior to all data analysis ; geometric means are presented. sixty women had their baseline visits at 15 months postpartum and 12 had their baseline visits at 30 months. at baseline, 26 women had normal glucose levels, 8 had impaired fasting alone, 19 had impaired 2-h glucose alone, and 19 had impaired fasting and 2-h glucose levels using american diabetes association criteria (7). the average annual rate of loss to follow - up was 6.5%. during a median follow - up of 72 months (minimum 12 months ; maximum 142 months), 31 (43%) of the women developed type 2 diabetes. the average annual incidence rate of diabetes, calculated by person - years, was 7.2%. the annual diabetes incidence rates were 4.1, 6.9, 7.0, and 14.8% in women whose ogtt values at baseline were, respectively, normal, impaired fasting alone, impaired 2-h alone, and impaired fasting and 2-h glucose together. kaplan - meier plot of diabetes cumulative incidence rate in 72 women without diabetes at entry and with at least one follow - up ogtt. vertical lines are 95% cis. the numbers given by the subjects line included subjects who developed diabetes and who were under follow - up without diabetes by the corresponding follow - up years. weight and glucose increased significantly during follow - up, while acute insulin secretion (airg) and the disposition index (di) fell significantly (table 2). nine women used combination oral contraceptives exclusively for a median of 21 months (range 267). eight women used progestin - only contraception exclusively for a median of 15 months (340). one woman started with a progestin - only contraceptive, was off from any hormonal contraception, and then switched to combination oral contraceptives. rates of change during follow - up to convert the glucose in unit of mg / dl to the si unit of mmol / l, multiply the number in the table by 0.0555. univariate cox regression analysis using each of the baseline variables presented in table 1 revealed that relatively high glucose, relatively low insulin sensitivity, relatively low insulin responses, and relatively low -cell compensation for insulin resistance were associated with development of diabetes (table 3). multivariate cox regression analysis using all baseline variables revealed four that were significantly associated with development of diabetes (table 4). the strongest was -cell compensation for insulin resistance, expressed as the disposition index from ivgtts. the other baseline predictors identified from multivariate analysis were insulin sensitivity, measured as incremental glucose clearance during hyperinsulinemic clamps (low = increased risk), total glucose area during ogtts (high = increased risk), and the 30-min increment in insulin on ogtts (low = increased risk). univariate significant baseline predictors of diabetes variables and units are defined in table 1. multivariate significant baseline predictors of diabetes p 5 years after the index pregnancy (right two bars). b : disposition index during follow - up in women who developed diabetes and in women who remained diabetes free. log scale is depicted to reflect that data were log transformed prior to all data analysis ; geometric means are presented. this study provides the longest follow - up of which we are aware that used detailed physiological measurements to characterize the natural history of glucose regulation following pregnancies complicated by gdm. two general types of physiological variables were associated with development of type 2 diabetes : the degree of metabolic deterioration at baseline (low insulin sensitivity and -cell function, high glucose levels) and the rate of deterioration thereafter (falling -cell compensation for insulin resistance). to our knowledge, this is the first demonstration that both the degree of deterioration after pregnancy and the rate of deterioration thereafter contribute to the risk of diabetes. our findings are consistent with the concept (4) that gdm most commonly represents detection of a chronic condition (i.e., low and falling -cell compensation for chronic insulin resistance) rather than development of an acute condition (i.e., inability to compensate for acquired insulin resistance) during pregnancy. that concept is strongly supported by serial studies of insulin resistance and -cell compensation in women who develop gdm. those studies reveal that the large majority of the -cell defect observed during the third trimester is present before (2) and after (3,4) pregnancy. moreover, women with gdm increase insulin secretion in parallel to normal women during pregnancy (4), but they do so along a sensitivity - section curve that is characterized by inappropriately low insulin secretion for any degree of insulin resistance. thus, from the physiological standpoint, it appears that gdm is most often a chronic disease characterized by insulin resistance and falling -cell compensation that is simply detected by routine glucose screening during pregnancy. in addition to the physiological variables, three clinical variables provided information about the risk of diabetes after gdm. weight gain was the strongest, consistent with prior clinical observations of shorter duration (10). weight gain is a known risk factor for diabetes ; it can worsen insulin resistance and -cell function as demonstrated previously (1113). indeed, adjustment for weight gain explained part of the association between falling -cell compensation and diabetes risk in this study, suggesting that the impact of falling compensation on diabetes risk may have been mediated at least in part through weight gain. gain in body fat gave similar results to gain in weight, suggesting that increased adiposity is the important component of weight gain accentuating diabetes risk. evidence for association between diabetes and additional pregnancy or progesterone - only contraception was statistically marginal in this study, where only 20 women had one or more additional pregnancies and 8 women used progesterone - only contraception. however, the point estimates for risk were substantial (1.77 for additional pregnancy and 4.28 for progestin - only contraception). moreover, we have previously found both events to be significantly associated with diabetes risk in a much larger clinical cohort of women with prior gdm (10,14,15). the present report adds validity to those prior findings and demonstrates that the risks occur independently of baseline glucose levels, insulin resistance, -cell function, and weight gain. these three variables represent potentially modifiable risk factors for diabetes after gdm. taken together, our findings in hispanic women suggest the following scenario for development of diabetes after gdm. women who have that intolerance are at different stages in progression toward diabetes, in part because they are deteriorating at different rates. women who are the most insulin resistant also have the worst -cell function and highest glucose levels. they are closest to diabetes and develop it soonest, as indicated by the cox regression analysis for baseline predictors of diabetes. faster deterioration is also associated with diabetes, even after adjustment for where women are at baseline. gaining weight, becoming pregnant again, and using progesterone - only contraception can accelerate development of diabetes after gdm. whether there is a common mechanism underlying the effects of these factors on -cell compensation remains to be determined. they are all modifiable and they represent, along with amelioration of insulin resistance (1617), potential clinical approaches to reducing diabetes risk after gdm. in summary, using the longest physiological follow - up of women with prior gdm available to date, we found important differences in both degrees and rates of metabolic deterioration in our exclusively hispanic cohort. the differences, along with the occurrence of weight gain, an additional pregnancy, and use of progestin - only contraception, had important associations with the risk of developing diabetes during more than a decade of follow - up. our findings provide three potentially modifiable clinical risk factors for diabetes in this high - risk group. they also suggest that genetic and environmental determinants of rates of change in -cell compensation for chronic insulin resistance should be an important focus of research to understand the pathophysiology of both gdm and type 2 diabetes that so often follows it. | objectiveto identify physiological and clinical variables associated with development of type 2 diabetes up to 12 years after pregnancies complicated by gestational diabetes.research design and methodsseventy - two islet cell antibody negative nondiabetic hispanic women had oral (ogtt) and intravenous (ivgtt) glucose tolerance tests, glucose clamps, and body composition assessed between 15 and 30 months after pregnancies complicated by gestational diabetes mellitus (gdm). they returned for ogtts at 15-month intervals until they dropped out, developed diabetes, or reached 12 years postpartum. cox regression analysis was used to identify baseline predictors and changes during follow - up that were associated with development of type 2 diabetes.resultsat baseline, relatively low insulin sensitivity, insulin response, and -cell compensation for insulin resistance were independently associated with development of diabetes. during follow - up, weight and fat gain and rates of decline in -cell compensation were significantly associated with diabetes, while additional pregnancy and use of progestin - only contraception were marginally associated with diabetes risk.conclusionsin hispanic women, gdm represents detection of a chronic disease process characterized by falling -cell compensation for chronic insulin resistance. women who are farthest along at diagnosis and/or deteriorating most rapidly are most likely to develop type 2 diabetes within 12 years after the index pregnancy. weight gain, additional pregnancy, and progestin - only contraception are potential modifiable factors that increase diabetes risk. |
the mullerian duct regresses in male due to a glycoprotein secreted by the developing testes called mullerian inhibiting factor (mif). failure to regress may be due to lack of mif or defective mif receptor, resulting in various disorders of regression, and one such disorder is persistent mullerian duct syndrome (pmds). pmds is a rare form of male pseudohermaphroditism characterized by the presence of uterus and fallopian tubes in phenotypically and genotypically normal male (46, xy), with testosterone production and responsiveness. the association between pmds and transverse testicular ectopia (tte) is even more uncommon. a 25-year - old male presented with absent left testis since birth and right sided inguinal swelling for 5 years, and pain in swelling for 2 days. on physical examination, the left scrotum was empty, and right side revealed indirect inguinal hernia with right testis at the root of the scrotum. ultrasonography showed right inguinal hernia with undescended testis in the right inguinal canal and an absent testis on the left side. surgical exploration revealed indirect sac with the right testis. while retrieving the testis, we noticed a complex mass resembling uterus - like structure, and gonad (left) with the fimbria - like structure [figure 1 ]. gonad with fimbria - like structure (above), uterus like structure (middle), and testis (below) both the gonads had vas deferens and vascular supply. the uterus like structure was removed along with left gonad as it was atrophic and sent for histopathology. histopathological study proved it to be uterus [figure 2 ] and left gonad like structure proved to be testis which was atrophic without spermatogenesis [figure 3 ]. testis with absent spermatogenesis chromosomal analysis revealed normal male genotype 46 xy [figure 4 ]. embryologically, between 7 and 8 week of gestation, masculinization occurs in a male fetus. testosterone secreted by the leydig cells in the testis helps in the development of wolffian duct in to vas deferens, epidydimis and seminal vesicle. mif secreted by sertoli cells in the testis, acts locally to suppress the mullerian ducts and causes their regression by 8 and 10 weeks of gestation. pmds results from failure of synthesis of mif, or failure of end organs to respond to mif. the mechanical effect of persistent mullerian duct structures might produce cryptorchidism by preventing normal testicular descent. pmds represents a small fraction of this broad spectrum of male pseudohermaphroditism, characterized by the presence of well - developed or rudimentary uterus, cervix, vagina and fallopian tube in normal 46 xy male. patients with pmds have normal androgen production, male external genitalia and normal penile development. failure of the testis to descend in to the scrotum at birth results in undescended testis. ectopic testis have been reported at various sites including superficial inguinal pouch, suprapubic, femoral, perineal region and the base of penis. migration of the testis to the opposite side where both testis pass through the same inguinal canal is known as tte. partially descended testicles (80 90% of cases) with unilateral cryptorchidism and contralateral inguinal hernia. the term hernia uteri inguinalis is used when uterus is found in the hernia sacsometimes the contralateral testis is found in the hernia sac due to abnormal mobility of the mullerian derivatives, known as ttebilateral cryptorchidism with uterus fixed in pelvis and both testes embedded in round ligaments. partially descended testicles (80 90% of cases) with unilateral cryptorchidism and contralateral inguinal hernia. the term hernia uteri inguinalis is used when uterus is found in the hernia sac sometimes the contralateral testis is found in the hernia sac due to abnormal mobility of the mullerian derivatives, known as tte bilateral cryptorchidism with uterus fixed in pelvis and both testes embedded in round ligaments. a rare form of clear cell adenocarcinoma of the mullerian duct in pmds has been reported. diagnosis is usually made incidentally during surgery for an inguinal hernia or exploration for cryptorchidism. laparoscopy, ultrasonography, and mif detection using bioassay techniques is helpful in diagnosing the syndrome. orchidectomy is indicated when testes can not be mobilized to a palpable location. otherwise orchiopexy is done and patient kept on long - term follow - up. | persistent mullerian duct syndrome (pmds) is a rare form of male pseudohermaphroditism characterized by the presence of mullerian duct structures in a normal male with 46, xy karyotype. transverse testicular ectopia (tte) is rare form of testicular ectopia in which two testes are located on one inguinal side. the opposite scrotum is empty. pmds with tte is rare. we report a case of pmds with tte discovered during surgery for a right inguinal hernia in a 25-year - old male. |
non - typhoid salmonellosis is an infectious disease caused by salmonella species other than salmonella typhi. in most patients, the clinical course of non - typhoid salmonellosis consists of a benign self - limiting gastroenteritis. however, these bacteria are especially problematic in immunocompromised individuals, including patients with malignancy, human immunodeficiency virus, or diabetes, and those treated with corticosteroids or other immunotherapy agents. in addition to enteric symptoms, salmonella species give rise extra - intestinal complications, which are associated with increased mortality rates and graft failure in transplant recipients. salmonella - triggered arthritis may appear 1 to 3 weeks after the onset of infection. this arthritis is frequently asymmetric and migratory and usually involves the large joints. in most patients, this arthritis is self - limiting and lasts a few weeks to several months, but in some patients the disease appears to be chronic in nature. we describe here a patient who experienced non - typhoid salmonella - triggered arthritis in the left hip after undergoing dual living - donor liver transplantation. a 47-year - old man underwent a dual living - donor liver transplantation, consisting of the right lobe from his wife and the left lobe from his brother, due to hepatitis b, liver cirrhosis and hepatocellular carcinoma in february 2010. postoperatively, he was treated with tacrolimus (initially 0.025 mg / kg / day intravenous injection switched into 3.5 mg per oral administration (per os) twice a day) and methyl prednisolone (initially 50 mg intravenous injection, every 6 hours switched into 12 mg per os twice a day). the serum level of tacrolimus was adjusted to 14.6 ng / ml (normal range, 5 - 20 ng / ml) and his graft function was excellent. he discharged in stable condition of day 24 with immunosuppressants (tacrobel 3.5 mg and methylon 12 mg, twice a day), acyclovir (zovirax 200 mg, once a day) and trimethoprim - sulfamethoxazole (400 mg-80 mg, septrin 2 tablets, every other day) to prevent rejection and infection. one week after discharge, he presented at the emergency room with a high fever (39.1), watery diarrhea, severe pain and stiffness in the left hip for 4 days. blood tests showed hemoglobin 11 g / dl, leukocyte count 4,400/mm, platelet count 71,000/mm and normal liver function test but c - reactive protein (crp) concentration was 4.39 mg / dl. blood cultures yielded group d salmonella, which was sensitive to ampicillin, cefixime, and ceftriaxone, and resistant to quinolone. bilateral hip joint magnetic resonance imaging (mri) revealed pyomyositis involving the left iliopsoas and external obturator muscles with abscesses in the iliopsoas muscle as well as diffuse synovial enhancement with increased joint effusion in the left hip joint (fig. he was treated empirically with intravenous ceftriaxone 2 g per day and all immunosuppressive agents were stopped for 3 days. over the next 10 days of intravenous antibiotic treatment, his diarrhea and fever resolved, but pain and limited range of movement of the left hip continued. three weeks later, follow - up mri showed that the abscesses in his muscles had decreased but myositis was still present. diffuse synovial thickening had progressed but the amount of joint effusion had decreased in the left hip (fig. he was maintained on intravenous antibiotics for an additional 3 weeks, following which he was switched from intravenous ceftriaxone to oral cefixime 400 mg per day and sulfamethoxazole - trimethoprim (400 mg/80 mg) 2 tablets per day. after 5 weeks of antibiotic treatment, his joint pain improved progressively and he discharged with a further antibiotic treatment plan for a total of 6 months. an additional mri, performed after 6 months of antibiotic therapy, revealed myositis and arthritis were markedly improved (fig. he had no joint disability and his graft function has remained stable for 22 months after liver transplantation. to our knowledge, non - typhoid or typhoid salmonella - triggered arthritis has not previously been reported in living - donor liver transplant recipients, although it has been described in several renal transplant recipients. salmonella species are gram - negative motile bacilli that are usually transmitted to susceptible hosts by consumption of contaminated foods, including beef, poultry, and eggs. non - typhoid salmonellosis is an infectious disease caused by salmonella species other than salmonella typhi. infection with non - typhoid salmonella usually produces a self - limited gastroenteritis, but may also produce extra - intestinal complications, such as endocarditis, vascular infections, cholecystitis, hepatic and splenic abscesses, urinary tract infections, pneumonia or empyema, meningitis, septic arthritis, and osteomyelitis, all of which are associated with increased mortality rates and graft failure in transplant recipients.1 the severity of illness in individuals with salmonellosis is determined not only by the virulence of the infecting strain but also by host conditions. the most common risk factors include corticosteroid use, malignancy, diabetes, human immunodeficiency virus (hiv) infection, prior antimicrobial therapy and immunosuppressive therapy.2 the rates of bacteremia and extra - intestinal complications are especially high in transplant recipients, approximately 60% and 35%, respectively.3 among the extra - intestinal complications of salmonellosis is salmonella - triggered arthritis, which may be septic or reactive. reactive arthritis is defined as a new onset of swelling of a joint, inflammatory - type back pain, or extra - articular inflammation, occurring within 3 months of the onset of the gastrointestinal symptoms.4 the pathogenesis of reactive arthritis remains obscure, although it is strongly associated with the presence of the major histocompatibility antigen hla - b27, which may predict a more prolonged and severe disease.5,6 the incidence of arthritis following salmonella infections has been reported to range 2 - 15% and incidence of 3.4 % has been reported in renal transplant recipients.1 the median times from onset of gastrointestinal symptoms to the development of arthritis have been found to range from 0 to 90 days, with the usual onset being within 7 to 14 days.4,5,6,7,8,9 the sequence of events that lead to bone and joint infection has not been well characterized and there are no antimicrobial guidelines for the identification of post - salmonella arthritis. ciprofloxacin has good in vitro activity against salmonella owing to its high serum concentrations and intracellular penetration. however, the emergence of ciprofloxacin resistance and cross - resistance to unrelated antibiotics has been described in both immunocompetent and immunocompromised patients as our case. although dual therapies with ciprofloxacin and chloramphenicol or trimethoprim have been used to reduce resistance, salmonella strains resistant to multiple antibiotics have been isolated. third - generation cephalosporins are active in vitro against salmonella species, although the correlation between in vitro sensitivity and clinical response may vary due to differences in tissue and intracellular penetration. ceftazidime has been replaced by ceftriaxone therapy due to its longer half - life, which has enabled once - daily treatment of outpatients.10 moreover, severe prolonged polyarticular reactive arthritis after intestinal salmonellosis is not altered by long - term antibiotic therapy. appropriate physiotherapy, non - steroidal anti - inflammatory agents and local steroid injection have proven useful in the treatment of salmonella - triggered arthritis. however, intra - articular injection of steroids is still considered problematic due to the possibility of secondary infections.9,10 clinical responses are based on the resolution of fever, pain and joint effusion.11 the prognosis of patients with arthritis after salmonella infections has not been well defined. although most patients show resolution of arthritis within 4 months of onset, chronic symptoms can persist for up to 5 years.4 moreover, relapse can occur, even with appropriate therapy. in an israeli series, 2.2% of patients experienced a bacteriologically proven relapse.12 the relapse rate is significantly higher in immunosuppressed patients, with recurrences occurring within a week to 2 years and 19 months after stopping antibiotics.1 it is difficult to adjust the level of immunosuppressants to prevent graft rejection while preserving immunocompetence against pathogens, especially during an active infectious stage. total withdrawal of immunosuppressants has been reported successful in the treatment of refractory septic arthritis caused by salmonella and staphylococcus aureus, together with preservation of graft function, in al long - term renal transplant recipient, although further long - term follow up study is needed to delineate the effect of immunosuppressant withdrawal on renal allograft function.9 in our patient, however, we withdrew immunosuppressive therapy for only 3 days to treat acute bacteremia, as complete withdrawal of such therapy was deemed dangerous for the patient. after resolution of his acute diarrhea and high fever, we resumed immunosuppressants and focused on eradication of ciprofloxacin - resistant salmonella group d. six months of antibiotic treatment have been recommended for hiv patients with salmonella bacteremia susceptible to ciprofloxacin.13 we herein report a dual living - donor liver transplant recipient who had quinolone - resistant, non - typhoidal samonella septic arthritis and that at least 6 months of treatment with cefixime and trimethoprim - sulfamethoxazole would lead to an acceptable outcome, as well as prevention of insidious progress of arthritis and permanent joint damage. | non - typhoid salmonellosis is an infectious disease caused by salmonella species other than salmonella typhi. although the usual clinical course of non - typhoid salmonellosis is a benign self - limiting gastroenteritis, these bacteria are especially problematic in immunocompromised individuals, including patients with malignancies, human immunodeficiency virus, or diabetes, and those receiving corticosteroids or other immunotherapy agents. in addition to enteric symptoms, salmonella species give rise to extra - intestinal complications, including self - limiting arthritis, which appears 1 to 3 weeks after the onset of infection and lasts from a few weeks to several months. in some patients, however, this arthritis spears to be chronic in nature. we describe herein a living - donor liver transplant recipient who experienced non - typhoid salmonella - triggered arthritis in the left hip. the patient recovered uneventfully after 6-month - long antibiotics treatment. clinicians involved in transplantation should be aware of the possibility that transplant recipients, like other immunocompromised individuals, are at risk of salmonellosis and therefore require careful clinical and microbiological evaluation, with the goals of prevention and early recognition of infection. |
having gas and petrochemical industries being constructed and developed in the persian gulf region, many workers have to do jobs with different intensities with regard to the nature of their work, in hot and humid weather. among different workers, those who are working in the construction of buildings, installation of technical equipment, welding, and driving, although the amount of physical activity demand has decreased with regard to technological progression in the construction industry, in many cases, many duties were needed to moderate to severe physical activity however, if there are such conditions in the hot humid weather, this coincidence augments cardiac strain occurrence. performing the tasks in such situations results not only in the increase of blood supply required by large muscles, but also in being exposed to sultry weather and heat excretion through sweating. the prevalence of overweight and obesity is increasing seriously in developing and industrial countries, so much so, esteghamati. have reported that obesity and overweight increased in iran from 13.6 and 32.2 in 1999 to 19.6 and 35.8 in 2005, as well as, 22.3 and 36.3 in 2007. janghorbani. have pointed out the mean overweight and obesity among men to be 42.8 and 11.1%, respectively. furthermore, the prevalence of overweight and obesity among american adults has exceeded the previous 60%. the epidemic studies indicate that overweight and obesity are significant risk factors for some diseases such as diabetes, cardiovascular disease, cancer, and premature death. furthermore, with regard to the fact that fat tissue is considered as a good thermal insulation and has less density than other tissues, so the heat transfer coefficients for muscle and skin tissue are 95 and 85%, respectively, and for fat tissue, 36%. although fat tissue plays a positive role in cold strain, it has a negative effect on heat strain. fat tissue in obese people acts as heat insulation, besides, it increases energy consumption at the time of activity, and generally the level of physical fitness in such people is low. moreover, in many people, a low level of physical fitness causes overweight, so they may have a higher heart rate at the time of physical activity. overweight can be delineated by higher body mass index (bmi) as well as fat tissue percentage. the reduction of performance in hot weather, in obese people, is due to the higher metabolism rate and a slower losing of heat load caused by low area / weight ratio, which has a considerable influence on the capacity and appropriateness of the job. with regard to the high prevalence of obesity and overweight in adults and the workers population, as well the fact that a high percentage of this population are exposed to hot weather and high humidity, simultaneously, in the persian gulf region, particularly in the hot seasons of the year, this question comes to the mind does this coincidence of hot weather and high humidity augment the occurrence of cardiac strain among the workers ? thus, considering the limitation of relevant studies in such climate conditions, during the warm months in the persian gulf region, this study is aimed at determining the relationship between the severity of cardiac strain and overweight in the real field work carried out in the very hot and humid weather of the persian gulf region. this cross - sectional study was conducted on 71 (a total of 350) workers during two months, from june to september 2010, in the persian gulf region. the subjects were engaged in outdoor jobs at two sites of the petrochemical industrial complex that exposed them to heat stress. the subjects were selected by the simple random sampling method. out of the 76 workers in the study ; complete experiments were available from 71 workers. this study was approved by the medical ethics committee of the faculty of medical science at the tarbiat modares university, and all the subjects signed a consent form according to the helsinki declaration. the participants were medically screened for cardiovascular disease, respiratory disease, infectious disease, diabetes, hyperthyroidism, and no medicine use. all subjects were reminded of no drinking coffee and alcohol at the night before the testing day. after resting for 30 minutes in a cool room (wbgt = 22.6 1.9), heart rate at times 20, 25, and 30 minutes (as baseline) then, without separating the measuring devices, the subject was asked to go and begin his work. if the work location was farther than 50 meters from the cool room, the subjects would be transported by car. after starting work, all through, the researcher measured and recorded the heart rate continuously. simultaneous measurements of the heart rate, dry bulb temperature, wet bulb temperature, and globe temperature were carried out. the wbgt index at rest and work was also measured with the wbgt meter (cassella cel). after 60 minutes of heat exposure, the subject was made to stop work and sit on a footstool in the same work station, and the heart rate was measured at the last 30 seconds of each minute of the recovery period, that is, from 30 seconds to one minute (p1) after stop of work, and from 2.5 - 3 minutes (p3) and 4.5 - 5 minutes (p5). assessment of heart rate recovery and reaching the normal rate was done by comparing p1 and p3 ; thus, if p1-p3 10 and p3 90 bpm, it would show that returning to normal heart rate had not happened, and this no - recovery pattern indicated too much strain. to estimate the physical activities, the persian version of the rating perceived exertion of the eston - parfitt was used. in this study, all measurements were performed outdoors, from 9 to 12 pm and 3 to 6 am. at the end of the measurements, the bmi and body surface area (bsa) were calculated, according to the equation, bmi = height (m)/weight (kg) and bsa = 0.20247 weight height, respectively. the maximum heart rate (mhr) was estimated from equation 220 - age. the heart rate reserve (hrr) was calculated as the difference between the maximum heart rate and resting heart rate. the net cardiac cost (ncc) was calculated as the difference between the working heart rate (whr) and resting heart rate (rhr). the relative cardiac cost (rcc) was obtained by expressing the net cardiac cost as the percentage of the heart rate reserve of the precipitants by using the following equation. the relative cardiovascular load (% cvl) was evaluated on the basis of hr as follows : % cvl = 100 [(whr - rhr)/hrmax (8 hours) ], where hrmax (8 hours), was a maximum acceptable hr for a work shift of eight hours, that is, one - third (220 age) + rhr. the cvl evaluates the cardiovascular load or aerobic strain, and can be classified as follows : 100%cvl, intolerable high level, peak loads should be reduced immediately or work must be stopped. with regard to the fact that the heart rate is affected by physical activity, and to neutralize the effect of this ; the heart rate was compared in two groups, with different bmis, when sitting and in low mobility and high mobility standing postures. the physical activity was assessed through direct behavioral observation of the activity, by experienced observers. the obtained data were analyzed with descriptive statistics, pearson correlation, and t - tests, using spss-16. the participants were working in jobs such as welding (n = 17), construction (n = 19), assembly of steel structures and components (n = 10), drivers and operators (n = 15), and supervisors (n = 10). the family of subjects was residing in the fars province (21%), chaharmahal bakhtiari (20%), khuzestan (18%), bushehr (11%), gilan (7%), hamadan (4%), central (4%), and eight other provinces (15%). the body mass index was greater than 25 in 42% of the workers, based on the world health organization (who) criteria. the individual characteristics of workers in three postures, sitting, standing with low mobility, and standing with high mobility, in two groups of body mass index, are shown in table 1. the mean maximum and minimum body mass indices were 25 4, 17.5 and 37 (kg / m), respectively. all of the three levels of working, the means of bmi, weight, and body surface area in the two body mass index groups were different (p > 0.001). the average of age and height in the two body mass index groups was not significantly different (except age in the low mobility group). in the two groups with different bmi, the mean of resting heart rate in the sitting posture (p = 0.021), working heart rate (p = 0.002), and heart rate reserve (p = 0.008) in the low mobility posture were significantly different. physical and physiological characteristics of subjects in different bmi and posture groups the average resting heart rate in the normal weight and overweight groups were 70 11.9 and 75 9.6, respectively. the average maximum heart rate in the normal weight and overweight groups were 190 8.3 and 186 8.4, respectively, the difference between their means was significant (p = 0.023). the average heart rate in the normal weight and overweight groups were 120 14.5 and 110 10.0, respectively, the difference was significant (p = 0.002). the physiological strain indices in the normal weight and overweight groups, at different levels of activity, are presented in table 2. physical workload in two different bmi groups in relation to work posture the mean standard deviation of dry bulb temperature, wet bulb natural temperature, relative humidity, globe temperature, and the wet bulb globe temperature indices were 37.4 3.0, 31.0 2.0, 62 12.8, 30.0 3.9, and 33.3 2.0, respectively. the value of the means at three levels of postures in the normal weight and overweight groups are shown in table 3. the average of all temperatures and wbgt index at three levels of postures in the two bmi groups was not significantly different, and therefore, the heat stress was almost the same in all cases. heat stress in two different bmi groups in relation to work posture the averages of the working heart rate in the normal weight and overweight groups were 101 20.3 and 112 18.9, respectively ; the difference in the mean was significant (p = 0.026). the means of the net cardiac cost in the normal weight and overweight groups were 30.5 17.6 and 36.3 19.0, respectively, the difference in the mean was not significant. the average of relative cardiac cost in the normal weight and overweight groups were 26.6 15.1 and 32.4 16.2, respectively, the difference was not significant (p = 0.073). the averages of the heart rate recovery indicator (p1 - p3) in the normal weight and overweight groups were 6.5 6.9 and 6.6 6.3, respectively ; the difference was not significant. according to the data in table 3, all the three levels of activity, the average of working heart rate, the net cardiac cost, and the relative cardiac cost in the overweight group were higher, when compared with the normal weight group. however, the mean of the working heart rate was different statistically (p = 0.042). the data illustrated in table 3 shows that the average of heart rate recovery after three and five minutes of resting in the overweight group, when compared with the normal weight group, tended to increase, but it was only statistically significant in the low mobility standing posture (p = 0.015). the pearson correlation between the bmi and rhr, ncc, rcc, and whr, adjusted for age, and the wbgt index and activity intensity, was 0.25, 0.27, 0.31 (p 0.001). the average of age and height in the two body mass index groups was not significantly different (except age in the low mobility group). in the two groups with different bmi, the mean of resting heart rate in the sitting posture (p = 0.021), working heart rate (p = 0.002), and heart rate reserve (p = 0.008) in the low mobility posture were significantly different. physical and physiological characteristics of subjects in different bmi and posture groups the average resting heart rate in the normal weight and overweight groups were 70 11.9 and 75 9.6, respectively. the average maximum heart rate in the normal weight and overweight groups were 190 8.3 and 186 8.4, respectively, the difference between their means was significant (p = 0.023). the average heart rate in the normal weight and overweight groups were 120 14.5 and 110 10.0, respectively, the difference was significant (p = 0.002). the physiological strain indices in the normal weight and overweight groups, at different levels of activity, are presented in table 2. the mean standard deviation of dry bulb temperature, wet bulb natural temperature, relative humidity, globe temperature, and the wet bulb globe temperature indices were 37.4 3.0, 31.0 2.0, 62 12.8, 30.0 3.9, and 33.3 2.0, respectively. the value of the means at three levels of postures in the normal weight and overweight groups are shown in table 3. the average of all temperatures and wbgt index at three levels of postures in the two bmi groups was not significantly different, and therefore, the heat stress was almost the same in all cases. the averages of the working heart rate in the normal weight and overweight groups were 101 20.3 and 112 18.9, respectively ; the difference in the mean was significant (p = 0.026). the means of the net cardiac cost in the normal weight and overweight groups were 30.5 17.6 and 36.3 19.0, respectively, the difference in the mean was not significant. the average of relative cardiac cost in the normal weight and overweight groups were 26.6 15.1 and 32.4 16.2, respectively, the difference was not significant (p = 0.073). the averages of the heart rate recovery indicator (p1 - p3) in the normal weight and overweight groups were 6.5 6.9 and 6.6 6.3, respectively ; the difference was not significant. according to the data in table 3, all the three levels of activity, the average of working heart rate, the net cardiac cost, and the relative cardiac cost in the overweight group were higher, when compared with the normal weight group however, the mean of the working heart rate was different statistically (p = 0.042). the data illustrated in table 3 shows that the average of heart rate recovery after three and five minutes of resting in the overweight group, when compared with the normal weight group, tended to increase, but it was only statistically significant in the low mobility standing posture (p = 0.015). the pearson correlation between the bmi and rhr, ncc, rcc, and whr, adjusted for age, and the wbgt index and activity intensity, was 0.25, 0.27, 0.31 (p 27) for the occurrence of thermal disorders was 3.53. under experimental conditions also, it was reported that the body weight, bmi, body fat percent, and a decline in the surface area to mass ratio, were significantly related to cardiac strain increase (deep body temperature and heart rate). in the evaluation of the relation between bmi and thermal fatigue, studied by bate and donoghue, they reported that with an increase in bmi, the risk of thermal fatigue occurrence would be obviously increased, so that the odd ratios for bmi 32, were 1.0, 2.94, and 3.63, respectively. according to the study results, the intensity of cardiac strain among the workers who were overweight or obese, was significantly higher than in those with normal weight. therefore, in order to control the workers cardiac strain, employing people with a bmi of more than 25 for such positions should be withheld, when monitoring people before employment. furthermore, it is proposed that to decline the intensity of cardiac strain in those employed, who are overweight, implementation of some essential interventions, such as, nutrition education and regular physical activity encouragement seems essential. | background : in the hot weather, overweight and obesity are considered as significant risk factors for the incidence of cardiac strain in workers. this study is aimed at comparing the cardiac strain among overweight and normal - weighted workers, in the hot, humid conditions of the south of iran.materials and methods : this cross - sectional study was conducted on 71 workers in the south of iran, in the summer of 2010. the heart rate was measured at rest and at actual work. cardiac strain based on the working heart rate (whr), the relative cardiac cost (rcc), the net cardiac cost (ncc), the load relative cardiovascular (cvl), and heart rate reduction were analyzed in 35 normal weight people (bmi 25), using descriptive statistics and t- tests.results:in 42% of the total workers, the body mass index was more than 25. the average temperature of the two groups was not significantly different. the mean whr in these two groups was 101 20.3 and 112 18.9, respectively (p = 0.026). percentages that exceeded the acceptable limits in parameters of ncc, rcc, whr, cvl, and the brouha index, were significantly higher in overweight people than those in people with normal weight.conclusions:based on the study results, the severity of cardiac strain was higher in overweight workers when compared with normal weight workers. hence, in order to decrease the cardiac strain, selecting overweight individuals for these jobs should be avoided, as also some vital intervention for losing weight, such as, nutrition education and encouraging them to increase their physical activity, should be implemented. |
bilateral pedicle fractures in the spine are uncommon because they have a higher mechanical strength than the pars interarticularis, even in elderly patients.359) furthermore, the pedicle has great intrinsic strength and a short moment arm, and can therefore resist greater cyclic shear forces.1) spondylolysis, or fracture of the pars interarticularis, is the most common injury of the neural arch, and osteoporotic compression fracture is common in adjacent segments after instrumented fusion due to loss of motion at fused segments by altered biomechanics.79) however, bilateral pedicle stress fractures at adjacent cephalad levels after instrumented lumbar fusion are extremely rare because stress concentration on the proximal junction area is associated with severe junctional changes including adjacent compression fractures or stenosis. here, we report a rare case of bilateral pedicle stress fractures, which were not detected before surgery at an adjacent upper level after instrumented lumbar fusion and not associated major trauma. a 77-year - old woman was admitted to our institute with severe back and buttock pain. four years previously, she underwent anterior lumbar interbody fusion (alif) with bone cement augmented screw fixation at the l5-s1 level under a diagnosis of foraminal stenosis. at that time, there were no pedicle fractures at the l4 level (figure 1), and the lowest t - score by dual - energy x - ray absorptiometry (dxa) bone mineral densitometry was -3.8. the patient presented with only mild back discomfort after alif with screw fixation, but the back pain progressively worsened without any history of trauma for 2 months before re - admission. ambulation was limited and the patient was re - admitted to our institute. a physical examination revealed neurologically intact status, but prominent tenderness was present in the low back area and back motion was reduced, especially in backward extension. plain radiographs showed marked irregularity of the l4 pedicle suggesting l4 pedicle fractures at both sides (figure 2a), and magnetic resonance images and computed tomography scans revealed bilateral pedicle fractures through l4 accompanying foraminal stenosis at l4 - 5 level (figure 2b - e). she was treated by posterior lumbar interbody fusion of l4-s1 with rigid screw (figure 3). at her 8-month follow - up assessment, osteoporosis contributes to cephalad compression fractures due to altered biomechanics in adjacent segments caused by junctional failures associated with loss of motion at fused segments.6) fused lumbar segments may increase stress and motion at adjacent unfused segments and accelerate degeneration. however, adjacent fracture of bilateral pedicles after instrumented lumbar fusion is extremely rare and to our knowledge, no such case has been previously reported. furthermore, bilateral pedicle fractures are rare and have only been reported on a few occasions.2458910) in these reported cases, most bilateral pedicle fractures were generally related to previous spine surgery or stress - related activities, and pedicle stress fractures after lumbar fusion surgery occurred at the most proximal or distal level of fusion.6) to our knowledge, three cases of bilateral pedicle stress fracture after lumbar fusion without screw removal have been reported,8910) and two of these cases occurred at the uppermost level of fusion.89) tribus and bradford10) described the case of a patient treated by posterior spinal fusion using segmental instrumentation from t3 to l4 who developed fractures of the lowest l4 pedicle. however, in this case, the adjacent upper level of the l4 pedicle was involved after instrumented l5-s1 fusion. repetitive mechanical stress fractures in the posterior elements are usually located in the pars interarticularis or to a substantially lesser extent, in the pedicle. the pedicle has great intrinsic strength and a short moment arm from the vertebral body, and can therefore resist greater cyclic shear forces.4) as a result, the pedicle is an uncommon location of fracture at the posterior neural arch. rather, fractures are more frequently observed in the isthmus, which corresponds to the weakest zone. nevertheless, bilateral pedicle fractures at adjacent levels can occur due to adjacent pedicles associated with spinal fusion masses may experience abnormal repetitive shear forces and cantilever because of continued motion through intervertebral discs and the pars interarticularis.10) in our patient, the fractures appeared to be fresh, because no sclerotic change was evident at fracture margins in spite of the absence of a trauma history.6) we had performed bone cement augmented screw fixation at l5-s1 level. the overstiffness and enhanced strength may increase the risk of bilateral pedicle fractures of adjacent, non - augmented vertebrae, because of increased biomechanical stress. we fused the l4 - 5 segment to stabilize the pedicle fractures, and the solid stabilization may result in pain relief, and allowed our patient to return to normal activities. we report an uncommon case of bilateral pedicle fractures at the adjacent upper level of previous instrumented fusion without a history of evident trauma. although it is rare, this report gives useful information to spine surgeons regarding the treatment and diagnosis of similar cases. | bilateral pedicle stress fractures are rare even in the elderly. bilateral pedicle fractures are due to post - surgical complications at the level of fusion or stress related activities in most cases. the authors describe a unique case of adjacent l4 bilateral pedicle fractures, which developed 4 years after anterior lumbar interbody fusion with bone cement augmented screw fixation at the l5-s1 level. as far as the authors ' knowledge, no similar case has been previously reported in the literature. the pathophysiological mechanism of this rare entity is discussed with review of relevant literature. |
temporary inferior vena cava (ivc) filters are being used for short - term protection in patients who are at a high risk for pulmonary thromboembolism. the tempofilter ii (b. braun, boulogne, france) is one of the temporary ivc filters, and this is a second - generation long - duration temporary device that is safe, effective and useful in patients who are in a critical thromboembolic situation (1). several episodes of atrial migration or upward displacement have been reported for both the tempofilter i (first - generation device) and the tempofilter ii, but no cases of filter fracture have been reported (1 - 3). here we report on the first case of fracture of one leg of the filter and this leg was embedded in the ivc wall in a patient with deep vein thrombosis. a 62-year - old man presented with a 2-day history of right leg pain and swelling. there was a previous history of stenting for left common iliac vein stenosis five years ago. the axial ct venogram showed a duplicated superficial femoral vein (sfv) and deep venous thrombosis in the right femoral vein, the right popliteal vein and the right calf veins. there was no thrombosis in the infrarenal vena cava and the diameter of the infrarenal vena cava was around 21 mm. catheter - directed thrombolysis and aspiration thrombectomy were planned as treatment for the patient 's dvt. a tempofilter ii was inserted into the inferior vena cava at the infrarenal level under digital subtraction angiography (fig. the following afternoon, catheter - directed thrombolysis using 120,000 iu of urokinase was first performed via the catheter inserted from the right popliteal vein and then aspiration thrombectomy was performed without complication. one week after the procedure, an axial ct venogram demonstrated some residual thrombus in the right sfv and no radiologically evident complications of the filter were noted. three weeks after the procedure, removal of the filter was performed, but the fluoroscopic image of the ivc demonstrated upward displacement of the tempofilter (displacement distance : 3 cm) and fracture of one filter leg (fig. at the time of explantation, we observed that thrombus was present in the filter and the filter was fractured with one missing leg (fig. the patient was asymptomatic and he was discharged without surgical removal of the embedded filter leg. given the long - term complications of permanent filters, the development of a safe and effective temporary filter would be of great benefit for young patients or for patients with a temporary thromboembolic risk that requires short - term protection against pulmonary thromboembolism. however, some temporary, retrievable filters can not be removed and so they must be left as permanent, and this maintains the risk of dvt and caval thrombosis (4). the tempofilter ii is a second generation temporary ivc filter that is introduced and withdrawn by using a tethered catheter with a subcutaneous anchoring device. unlike other temporary filters, the manufacturer validated that retrieval is easy after up to three months without the need for additional equipment (1). other temporary filters often become irretrievable after only two or three weeks as a result of thrombus formation and endothelialization. while the initial experience with the first - generation device (tempofilter i) has been positive (5), several episodes of filter migration to the right atrium due to catheter buckling, one of which was fatal, have suspended further development of this filter (2, 3). the problems related to structural stability were resolved by significant modifications of the filter cone and the tethering catheter. (1) reported a prospective 104-case multicenter study with the tempofilter ii and that study confirms the safety and effectiveness of the tempofilter ii. the filters were successfully removed with no complications in all but one case. while there have been no reported cases of tempofilter fracture in the english medical literature, we found that one leg of the filter had fractured and was embedded in the ivc wall. fortunately, the one fractured filter leg was embedded in the ivc wall without migration and no pulmonary thromboembolism was observed. the patient was asymptomatic and he is currently being examined as an outpatient without surgical removal of the embedded filter leg. filter fracture is very rare and it has been reported in less than 1% of cases (6 - 8). a few cases of a fractured ivc filter migrating to the heart and even causing cardiac tamponade have been reported, suggesting that strenuous physical activity and increased intra - abdominal pressure can lead to the fracture and migration of ivc filters (9, 10). we can postulate that the embedment of one filter leg with constant movement of the tethered catheter can cause fatigue fracture of the embedded filter leg. | tempofilter ii is a device that is used for pulmonary embolism prophylaxis. since the appearance of the tempofilter ii following withdrawal of the tempofilter i, it has been reported that the tempofilter ii is safe, effective and useful. here we report on the first case of a fracture of one leg of the filter and this leg was embedded in the inferior vena cava wall in a 62-year - old man with deep vein thrombosis. |
ureteral obstruction caused by metastasis or invasion of malignancy is generally regarded as a sign of poor prognosis and may be due to compression by the primary or metastatic tumor, retroperitoneal lymphadenopathy, or direct tumor invasion. median survival of patients with a malignant ureteral obstruction is 3.7 to 15.3 months [1 - 3 ]. acute ureteral obstruction associated with renal failure is a urological emergency requiring prompt evaluation and treatment, because renal failure due to a malignant ureteral obstruction is a significant cause of death. current management options include decompression using retrograde ureteral stent (rus) placement or percutaneous nephrostomy (pcn). rus does not usually lead to major complications, is less invasive, better tolerated than pcn, and is considered optimal management for malignant ureteral obstructions. however, a high stent failure rate is a problem encountered by urologists ; therefore, pcn has been commonly used as either a primary or secondary procedure in cases of stent failure. additionally, only a few reports are available that has predicted factors for stent failure - free survival with a malignant ureteral obstruction. therefore, we reviewed our institution 's contemporary experience with treating malignant ureteral obstruction using rus and analyzed the predicting factors for stent failure - free survival. we retrospectively reviewed patients who underwent insertion of a cystoscopic ureteral stent (endo - sof, cook urological, spencer, in, usa) due to a malignant ureteral obstruction between may 2004 and june 2011. direct ureteral obstruction by urolithiasis, bladder cancer, or prostate cancer and extrinsic ureteral compression from lymphadenopathy caused by urological cancer metastasis were excluded. patients who had undergone insertion of a cystoscopic ureteral stent due to a ureteral obstruction from nonurological cancer were included. among 115 patients who underwent insertion of a cystoscopic ureteral stent due to a malignant ureteral obstruction, 71 patients with ureteral obstruction due to nonurological cancer were extracted. stent failure was defined as an increase in serum creatinine of more than twice from nave and worsening hydronephrosis on an imaging study and an inability to replace the ureteral stent during the replacement procedure. performance status (eastern cooperative oncology group criteria), type of cancer, hydronephrosis grade, location of the obstruction, presence of bladder invasion, c - reactive protein (crp), serum albumin, and inflammation - based prognostic score (glasgow prognostic score, gps) were assessed as predictive factors for stent failure. the obstruction level was documented using computed tomography and defined as the upper, middle, or lower ureter as determined by the location above, over, or below the sacroiliac joint. the interval between stent changes was initially planned to be 3 months. in lieu of a formal ethics committee, routine laboratory testing of serum crp and albumin was performed just before insertion of ureteral stents. serum crp was measured by latex turbidimetric immunoassay using a hitachi 7600 analyzer (hitachi, tokyo, japan). the crp limit of detection was 0.03 mg / dl, and 1.0 mg / dl was the upper limit of the normal range. patients with both elevated crp (> 1.0 mg / dl) and low albumin (1.0 mg / dl) and low albumin (1.2 mg / dl) just before insertion of the ureteral stent was elevated in 33 patients (46.5%) and most cases were associated with bilateral obstruction (17, 65.44%, p=0.015, data not shown). sixteen (22.5%) and 11 patients (15.5%) had gross hematuria and flank pain, respectively. in the 71 patients who underwent rus, the stent failed to indwell in 15 patients (21.1%) at the first visit, and stent failure occurred in 15 patients (21.1%) later. the median periods for stent failure - free survival and overall survival were 5 and 7.7 months, respectively. a univariate analysis indicated that hypoalbuminemia (1.2 mg / dl were not associated with stent failure - free survival (table 2). a multivariate analysis revealed that the presence of a mid or lower ureteral obstruction (hr, 3.27 ; 95% ci, 1.19 to 8.95 ; p=0.007), elevated serum creatinine before ureteral stent placement (> 1.2 mg / dl ; hr, 2.16 ; 95% ci, 1.02 to 4.57 ; p=0.044), and gps1 (hr, 7.22 ; 95% ci, 2.89 to 18.0 ; p=0.001) were associated with stent failure - free survival (table 3). the median age of the 71 patients was 55 years (range, 19 to 85 years). forty - six patients (64.8%) were female, and 25 patients (35.2%) were male. twenty - six patients (36.6%) had a bilateral ureteral obstruction, 29 (40.8%) had a left - sided unilateral ureteral obstruction, and 16 (22.5%) were right sided. gastric cancer (30, 42.3%) was the most common cause of malignant ureteral obstruction. other types of cancer that caused ureteral obstruction were colon cancer (13, 18.3%), rectal cancer (10, 14.1%), and cervical cancer (11, 15.5%). seventeen patients (23.9%) had an upper level ureteral obstruction, 22 (31.0%) had a mid ureteral obstruction, and 32 (45.1%) had a lower ureteral obstruction. serum creatinine (> 1.2 mg / dl) just before insertion of the ureteral stent was elevated in 33 patients (46.5%) and most cases were associated with bilateral obstruction (17, 65.44%, p=0.015, data not shown). sixteen (22.5%) and 11 patients (15.5%) had gross hematuria and flank pain, respectively. in the 71 patients who underwent rus, the stent failed to indwell in 15 patients (21.1%) at the first visit, and stent failure occurred in 15 patients (21.1%) later. the median periods for stent failure - free survival and overall survival were 5 and 7.7 months, respectively. a univariate analysis indicated that hypoalbuminemia (1.2 mg / dl were not associated with stent failure - free survival (table 2). a multivariate analysis revealed that the presence of a mid or lower ureteral obstruction (hr, 3.27 ; 95% ci, 1.19 to 8.95 ; p=0.007), elevated serum creatinine before ureteral stent placement (> 1.2 mg / dl ; hr, 2.16 ; 95% ci, 1.02 to 4.57 ; p=0.044), and gps1 (hr, 7.22 ; 95% ci, 2.89 to 18.0 ; p=0.001) were associated with stent failure - free survival (table 3). our results suggest that the presence of a mid or lower ureteral obstruction, gps 1, and elevated serum creatinine before ureteral stent placement were associated with poor stent failure - free survival in patients with a malignant ureteral obstruction. the management of extrinsic malignant ureteral obstruction is a difficult situation in which the urologist balances patient quality of life, renal preservation and risk of complication in the setting of a poor prognosis. obstruction of upper urinary tract lesions occurs in both benign and malignant disease, and the focus of treatment is the smooth elimination of urine. obstruction due to benign diseases was treated by removing the lesions, whereas obstruction due to malignant diseases was treated by relieving the obstruction rather than removing the causative lesions. ureteral obstruction from malignancy may be due to compression by the primary or metastatic tumor, retroperitoneal lymphadenopathy, or direct tumor invasion ; therefore, renal function can be improved in either case and maintained by early detection and appropriate urinary diversion. experience with ureteral stents finney and hepperlen. described the technique in 1978, and stents were subsequently used for patients with malignancies that were causing external compression of the ureter. the development of endoscopy tools and techniques occurred in the late 1980s, and the previous treatment concept that surgical treatment for patients with malignant ureteral obstruction had priority was changed in the early 1990s [12 - 15 ]. several studies have reported that predictors of stent failure are baseline serum creatinine, no treatment after rus, gross tumor invasion noted at cystoscopy, degree of hydronephrosis, type of cancer, and male gender. izumi. reported that male gender and type of cancer are predictors of stent failure and jeong., also showed that baseline serum creatinine and no treatment after rus are predictors of stent failure. in addition, other studies have revealed that gross tumor invasion noted at cystoscopy and the degree of hydronephrosis are predictors of stent failure [16 - 18 ]. therefore, currently, there is a lack of consensus on the precise predictors of stent failure in malignant obstruction. in the present study, the presence of a mid or lower ureteral obstruction, gps 1, and elevated serum creatinine (> 1.2 mg / dl) before ureteral stent placement were independently associated with stent failure - free survival, whereas type of cancer, male gender, and degree of hydronephrosis were not associated with stent failure - free survival. patients with malignancy and weight loss have many reasons for poor baseline renal function and after insidious obstruction, the renal reserve may be insulted and less likely to recover. this mechanism may be associated with poor stent failure - free survival. in the present study, serum creatinine in cases of bilateral obstruction more elevated than unilateral obstruction, therefore, this is may be associated with poor stent failure - free survival. in the previous studies, jeong. suggested that this poor survival time might be due to the extensive metastasis to the retroperitoneum, which results in periureteric metastasis. also, chung. demonstrated that decreased flow was found to a greater degree in ureters with simulated proximal rather than distal obstruction. resistance to flow is associated with diameter and length, and if the obstruction is closer its origin resistance to flow is increased. however, in the present study, the presence of a mid or lower ureteral obstruction was associated with stent failure - free survival. therefore, further studies are needed to identify difference in stent failure - free survival according to the level of ureteral obstruction. in addition, the systemic inflammatory state characteristic of advanced cancer and reflected in crp elevation may have bearing on prognosis. furthermore, serum albumin is frequently low in patients with cancer, fueled by malnutrition and inflammation, so hypoalbuminemia may influence prognosis as well. by combining crp and albumin determinants as the gps, the independent association of these indices with poor prognosis has been demonstrated in various types of cancer. in the present study, cpr, hypoalbuminemia and gps were associated with early ureteral stent function failure on univariate analysis, and only gps was independently associated with short sffs on multivariate analysis. this was probably due to worsening of ureteral obstruction resulting from rapid progression of malignancy that had systemic inflammation, in line with prior findings. to our knowledge this is the first report that gps is an independent predictor of sffs. however, the results should be interpreted carefully because the patient characteristics differed among studies. although an intrinsic ureteral obstruction due to benign disease such as stone disease, ureteral stricture, or an ureteropelvic junction obstruction is usually successfully managed in the long term by rus, the incidence of stent failure is significantly higher in cases of malignant ureteral obstruction. in the present study, the stent success rate was 42%, which was lower than the 72% reported by donat and russo, and the 92% reported by rosenberg.. this discrepancy may be explained as a result of biocompatibility and differences in patient characteristics. complications caused by rus placement may include storage symptoms such as increased frequency and nocturia, hematuria, low abdominal pain, and encrustation. in addition, ascending pyelonephritis can occur due to vesicoureteral reflux. currently, the limitations associated with conventional treatments for ureteral obstructions highlight the need for a novel treatment that can maintain ureteral patency while minimizing the deterioration of patient quality of life. several types of metallic stents have been used in the palliative treatment of malignant ureteral obstructions. their study identified a 35% rate of failure in patients with malignant ureteral onbstruction treated with a metallic ureteral stent. this outcome is comparable to the failure rates historically observed with traditional polyurethane based stents for malignant ureteral obstruction. in addition, kim. reported that they assessed the efficacy and safety of insertion of a polytetrafluoroethylene membrane - covered self - expandable metallic stent (uventa stent) for palliation of malignant ureteral obstruction. in their study, the uventa stents were not obstructed during follow - up, so that the overall patency rate was 100%, but de novo ureteral obstruction developed in 4 ureters. further studies and long term follow - up would be necessary to assess the role of these stents in the treatment of malignant ureteral obstruction. first, the patients that initially underwent rus but failed due to severe obstruction were categorized into the stent failure group. as a result furthermore, the patient population was relatively irregular, because of the various types of cancer. second, we did not evaluate poststent systemic therapy because most of enrolled patients were systemic therapy - off state. finally, cases of removing a ureteral stent due to severe complications were not included. further studies are needed to identify the predictive factors for stent failure - free survival in a large cohort with malignant ureteral obstructions. ureteral obstruction due to extrinsic malignant compression is a poor prognostic sign and should prompt an open discussion with the patient and family. in the present study, the presence of a mid or lower ureteral obstruction, gps 1, and serum creatinine before ureteral stent insertion > 1.2 mg / dl were helpful for predicting poor stent failure - free survival. in addition, pcn and metallic stents are also considered for patients who have these factors. | purposeto determine predictive factors for stent failure - free survival in patients treated with a retrograde ureteral stent for a malignant ureteral obstruction.materials and methodswe retrospectively reviewed 71 patients who underwent insertion of a cystoscopic ureteral stent due to a malignant ureteral obstruction between may 2004 and june 2011. performance status, type of cancer, hydronephrosis grade, location of the obstruction, presence of bladder invasion, c - reactive protein (crp), serum albumin, and inflammation - based prognostic score (glasgow prognostic score, gps) were assessed using a cox proportional regression hazard model as predicting factors for stent failure.resultsa univariate analysis indicted that hypoalbuminemia (1.2 mg / dl ; hr, 2.16 ; 95% ci, 1.02 to 4.57 ; p=0.044) were associated with stent failure - free survival.conclusionsa mid or lower ureteral obstruction, gps 1, and serum creatinine before ureteral stent insertion > 1.2 mg / dl were unfavorable predictors of stent failure - free survival. these factors may help urologists predict survival time. |
acute variceal hemorrhage (avh) from esophageal varices (ev) or gastric varices (gv) is a devastating complication of portal hypertension. it is a leading cause of death in cirrhotic patients, particularly in those with hepatic decompensation. early cohort studies observing the natural course of patients with avh revealed that the short - term mortality rate was as high as 50%, with uncontrolled active hemorrhage and recurrent bleeding as the major causes of death [13 ]. as a witness of progress in modern medicine, the prognosis of avh has remarkably improved for the last 3 decades, although the short - term mortality (conventionally defined as within 6 weeks of each episode) in recent series remained approximately 1520%. the improved outcome of cirrhotic patients with avh probably results from advancement in the multidisciplinary approaches that include pharmacological therapy (vasoactive agents, antibiotic prophylaxis), endoscopic intervention (band ligation for ev, variceal obliteration for gv), transjugular intrahepatic portosystemic shunt (tips), and surgery. being an essential part in the management of acute upper gastrointestinal (ugi) bleeding, endoscopy plays important roles in the confirmation of bleeders, stratification of risks, control of active hemorrhage, and prevention of the first and recurrent bleeding in cirrhotic patients with avh. the purpose of this paper is to provide a concise and updated review on the use of endoscopy in managing patients with avh. patients with avh frequently present with unstable hemodynamics because bleeding characteristically occurs not only massively but also rapidly. therefore, restoration of circulatory volume by intravenous fluid resuscitation should be carried out immediately at patients ' arrival. blood component therapy usually is needed to correct anemia and bleeding tendency (coagulopathy as well as thrombocytopenia). a quick assessment for the indications of airway protection by endotracheal intubation is mandatory, in that the concern of suffocating aspiration is substantial in patients with massive hematemesis, impaired consciousness, and delirious status. ideally, risks of circulatory collapse and airway compromise should be minimized before patients are transported to endoscopy rooms. intravenous administration of erythromycin prior to endoscopy may be considered in cirrhotic patients presenting with hematemesis, because brisk bleeding and large quantity of residual blood in the ugi tract often obscure endoscopic views, add difficulty of therapeutic intervention, and increase chance of aspiration. as a motilin receptor agonist the efficacy of erythromycin in cleansing stomach and thereby improving quality of endoscopy has been demonstrated in randomized controlled trials [68 ]. recently, altraif and colleagues reported in a double - blind randomized trial that erythromycin of 125 mg intravenously administered 30 minutes before endoscopy as compared with placebo significantly increased the proportion of a clear stomach (48.9% versus 23.3%, p 15 hours after presentation to the hospital) was an independent risk factor associated with mortality (odds ratio 3.67 ; 95% confidence interval, 1.27~10.39). the sooner the better concept, in that the association between risk of death and endoscopy timing was nonlinear and mortality did not decrease with every hour earlier of endoscopy. they found whether endoscopy was performed within 4, 8, or 12 hours within initial assessment at hospital did not influence recurrent bleeding, blood transfusion, need for rescue therapy, length of hospitalization, or mortality. of note, only half of all patients with avh were enrolled into analysis in their study, because the results of those with initial unstable hemodynamics were not reported. in view of the understandable difficulty to perform a randomized trial to compare different endoscopy timings in this setting based on currently available data, we believe the rule is to perform endoscopy within 15 hours of presentation, but meanwhile we also acknowledge there is no evidence to support rushing endoscopy in avh patients, particularly in those with stable hemodynamics. therefore, while delaying endoscopy for more than 15 hours should be avoided, endoscopists may wait in the first few hours to allow emergency resuscitation, optimal medication, and perhaps preparation for a cleaner stomach to be carried out. determination of bleeding source by upper gi endoscopy has important prognostic value in cirrhotic patients with acute ugi bleeding, since patients with variceal bleeding (definite or probable) fared significantly worse than those who bleed from other sources [23, 24 ]. in addition, active bleeding on endoscopy was shown to predict 5-day treatment failure and 6-week mortality [24, 25 ]. with regard to the risk prediction for patients with endoscopically confirmed avh, measurement of hepatic venous pressure gradient (hvpg) is arguably the best method to stratify risk of untoward outcomes. it has been demonstrated that an initial hvpg > 20 mmhg most reliably identified those patients whose clinical course would evolve poorly. furthermore, a large body of evidence supports reduction of greater than 20% of the initial hvpg value convincingly indicates risk reduction in recurrent bleeding and mortality. however, application of hvpg measurement is regrettably not widespread around the world, and in reality is not incorporated into daily practice in the vast majority of institutions. fortunately, there is evidence suggesting that easily obtainable clinical variables, as compared with hvpg, may have similar accuracy in predicting failure of treatment during the acute phase of a bleeding episode (5 days). among the various clinical parameters that have been investigated, indicators of hepatic reserve (child - turcotte - pugh classification, model for end - stage liver disease (meld) score), markers of bleeding severity (active bleeding on endoscopy, presentation with hematemesis, amount of blood transfusion, hemoglobin level), underlying liver disease or comorbidity (etiology, hepatocellular carcinoma, portal vein thrombosis), complications during bleeding episodes (encephalopathy, bacterial infection, renal dysfunction), and failure of initial treatment (uncontrolled active hemorrhage, recurrent bleeding) have been shown to predict clinical outcomes [20, 24, 2932 ]. endoscopic identification of avh patients at risk of unfavorable outcomes may be crucial in guiding subsequent management. garcia - pagan and colleagues reported in a randomized trial that ev patients with hepatic decompensation (child - turcotte - pugh scores between 7 and 13) and persistent bleeding at endoscopy would benefit from early tips performed within 72 hours. the one - year survival rate was 86% versus 61% (p <.001) in patients randomized to early tips as compared with those who were assigned to receive optimal pharmacotherapy plus endoscopic band ligation. therefore, it stands to reason that patients with actively bleeding and compromised liver function may require aggressive therapy to be implemented as early as a continuation to endoscopic therapy rather than as a rescue measure for treatment failure. although the prognostic factors for avh have been extensively studied, those for cirrhotic patients with all sources of acute ugi bleeding remain sparsely explored. undoubtedly, endoscopy is urgently indicated in cirrhotic patients presenting with ugi bleeding, but it takes time to resuscitate the patients, transfuse blood components, and administer intravenous medications. therefore, risk stratification explicitly for variceal bleeding may not be applicable for clinicians managing patients in the emergency department, since not all cirrhotic patients bleed from varices. previous studies that investigated prognostic indices independent of the source of bleeding not only incorporated endoscopic data but also allowed subjective criteria [23, 24 ]. in our opinion, criteria based on subjective judgment may not be reliable, particularly in the busy emergency setting. for example, uncovering and staging ascites and encephalopathy relies on expertise and is not free of interobserver variation. we believe a useful stratification system in the setting of emergency room should ideally be built on simple, objective, and readily available parameters. to this end, we have retrospectively studied 542 consecutive episodes of acute ugi bleeding from 389 cirrhotic patients in order to develop a prognostic model consisting of pre - endoscopic clinical factors that were routinely available in the first hour at hospital. we revealed that 6-week mortality was independently associated with male gender, hypoxemia on arrival, hepatocellular carcinoma and another malignancy, serum bilirubin, and prothrombin time (table 1). the performance of a model built on these 6 variables was superior to the meld score in predicting 6-week mortality, with c statistic of 0.84 and 0.71 respectively (p =.002). presumably, earlier risk stratification may guide earlier modification of therapeutic approaches to improve the outcomes of those at risk. further research is now warranted to elucidate how pre - endoscopic risk stratification will influence the early management for cirrhotic patients presenting with acute ugi bleeding. screening endoscopy is mandatory to confirm the presence, to determine the size, and to uncover the stigmata of varices in cirrhotic patients, particularly in those with decompensated status [3638 ]. historically endoscopic sclerotherapy had been used in preventing the first bleeding from esophageal varices prior to the era of band ligation, but it is no longer recommended in this indication because the risk of complications may outweigh the potential benefits [40, 41 ]. evl is technically infeasible for small esophageal varices defined as size < 5 mm or f1 according to the classification proposed by beppu., whereas nonselective beta - blocker (nsbb) may slow the growth of small ev and thereby prevent the first variceal hemorrhage. in patients with medium to large (or f2-f3) ev, risk of future bleeding band ligation is as at least effective as nsbb for primary prophylaxis of ev bleeding [4346 ]. the decision to use evl or nsbb should be individualized according to the local resources and expertise, patients ' preference and characteristics, tolerability of side effects, and contra - indications to either therapy. in fact, more than half of patients preferred evl over nsbb use for fear of side effects from beta - blockade, such as light - headedness, shortness of breath, fatigue, and poor memory. because poor tolerability to nsbb is not uncommon and the response of hvpg to pharmacological therapy can not be reliably assessed by clinical parameters, we usually perform evl for primary prophylaxis in our institutes. there is no doubt that band ligation and nsbb are effective, respectively, to prevent first bleeding in the ev with medium to large size, but it remains unknown whether combination therapy with both treatment modalities is more effective than either therapy alone. sarin. reported in a randomized controlled trial that propranolol plus evl and evl alone were not different in bleeding related death, although there was less recurrence of varices in the combination group (5.6% versus 15.3%, p =.03)., propranolol plus evl as compared with propranolol alone resulted in lower rate of first bleeding from the high risk ev (6% versus 31%, p =.03), and higher bleeding - free survival rate (96% versus 69%, p =.04) during the 18-month followup in cirrhotic patients awaiting liver transplantation. nevertheless, lo and colleagues demonstrated that evl plus nadolol was not only not more effective that nadolol alone for primary prophylaxis of ev bleeding but also associated with more adverse events (68% versus 40%, p =.06). as the controversy goes on, currently combination therapy with evl plus nsbb can not be recommended in patients whose ev has not bled. evl is the recommended endoscopic therapy whenever feasible to control active ev bleeding, because it is unambiguously safer and more effective than sclerotherapy [5153 ]. occasionally, sclerotherapy may be substituted if evl is technically difficult, for example, in a repeatedly ligated esophagus with scarred mucosa that is difficult to be sucked into the cap. it is important to carefully scrutinize the bleeding stigmata (e.g., hematocystic spot, white nipple) in those without ongoing bleeding at endoscopy. localization of the origin of bleeding is essential for successful endoscopic therapy, inasmuch as evl should be initiated at or just below the bleeding point. if the bleeder can not be clearly localized, ligation may start at the gastroesophageal junction and then advance upward spirally. while active bleeding at endoscopy mandates immediate hemostasis, absence of ongoing hemorrhage during endoscopy should not be erroneously regarded as reassuring to reserve endoscopic therapy. in a randomized trial, lo and colleagues compared evl plus terlipressin versus terlipressin alone in cirrhotic patients presenting with acute inactive ev bleeding and demonstrated that evl was effective in reducing 5-day rebleeding rate (0% versus 15%, p =.006), treatment failure rate (2% versus 24%, therefore, evl can not be spared in cirrhotic patients with inactive bleeding ev at endoscopy if another bleeding source is unlikely. injection therapy with tissue glue (e.g., n - butyl-2-cyanoacrylate and 2-octyl - cyanoacrylate) to obliterate varices has become the endoscopic treatment of choice for isolated gastric varices (igv) and gastroesophageal varices extending beyond cardia (gov2). regrettably, there is considerably less data regarding the endoscopic therapy in controlling active gv hemorrhage, in contrast to the overwhelming evidence supporting the role of evl in ev bleeding. glue injection using cyanoacrylate for acute gv bleeding achieves high rates of immediate hemostatsis, eventual eradication, and low treatment failure - related mortality rate. consistent results from randomized trials provide convincing evidence to support the superiority of obliteration therapy over either sclerotherapy [5759 ], or band ligation [60, 61 ]. while the techniques to achieve variceal obliteration vary in different institutes, it has been adopted in our daily practice to inject a mixture of n - butyl-2-cyanoacrylate and lipiodol (1:1) without contrast agent. despite the efficacy and generally acceptable safety profile of injection therapy with tissue adhesives, thromboembolism infrequently occurs and represents the most fearful complication of cyanoacrylate injection that may potentially lead to infarction of multiple organs [62, 63 ]. use of thrombin or fibrin has been explored in the management of acute gv bleeding with promising preliminary results [6466 ]. theoretical advantages of thrombin injection include biocompatibility and minimal mucosal damage, whereas possibility of transmissible infectious disease and excessive cost are major concerns. before data from controlled trials comparing it with cyanoacrylate is available, thrombin injection should better be viewed as experimental and ideally be confined in the setting of clinical studies. as long as the portal hypertension persists, it is simply the natural course of varices to rebleed, with 1-year rebleeding rate approximating 60%. since gastroesophageal varices result from portal hypertension and occurrence of variceal hemorrhage depends directly on hydrostatic pressure of portal system (as reflected by hvpg), presumably the best treatment to prevent recurrent bleeding is to reduce the severity of portal hypertension, and that is the pathophysiological basis for the efficacy of nsbb. in view of the high recurrence rate, preventive measures for recurrent bleeding should be instituted right after acute bleeding episode is controlled. it is recommended that patients receive secondary prophylaxis before they are discharged from hospital for an bleeding episode, especially for those with large varices, red color signs, and decompensated cirrhosis. consistent with its superior role in primary prophylaxis and controlling active hemorrhage, evl remains the preferred endoscopic treatment for secondary prevention of ev bleeding. evl, again, outperforms sclerotherapy in this indication in terms of lower complication rate and higher efficacy [6870 ]. singh. reported in a meta - analysis that combination of evl and sclerotherapy as compared with evl alone was not more effective in preventing recurrent ev bleeding, but was associated with higher complication events such as esophageal stricture. in our opinion, endoscopic sclerotherapy has no role in the secondary prophylaxis of ev bleeding. with regard to variceal obliteration by tissue adhesives, there was a randomized trial demonstrating similar rebleeding rates between histoacryl injection and nsbb administration, but the former treatment was associated with a higher complication rate (47.6% versus 10%, p <.03). moreover, we are unaware of any trial comparing efficacy and safety of glue injection with that of evl in the secondary prophylaxis of ev bleeding. in contrast to the scenario of primary prophylaxis, in which combination therapy with evl and nsbb does not fare better than either therapy alone, combining endoscopic therapy plus pharmacological therapy is recommended in the setting of secondary prophylaxis. a meta - analysis including 23 studies showed that rates of rebleeding (both from all sources and specifically from varices) are lower with combination of endoscopic therapy (either sclerotherapy or evl) plus drug therapy than with either therapy alone. therefore, cirrhotic patients recovering from acute ev bleeding should receive nsbb and have their varices eradicated by band ligation. in those who are unable or unwilling to undergo evl although some studies proposed an interval of 1 to 2 weeks [69, 70, 74 ], others advocated an interval of 1 - 2 months of band ligation for obliteration of ev [75, 76 ]. yoshida. found a short interval between sessions of evl might even be detrimental by showing that the overall rates of variceal recurrence and additional treatment were both higher in patients with evl at a biweekly interval than those with a bimonthly protocol. generally, we do not repeat sessions of evl within 2 weeks because prior ligation - related mucosal ulceration may not have healed by that time and thereby may influence the following deployment of ligating bands. as far as efficacy is concerned, tips may be a more effective modality than endoscopic therapy to prevent recurrent bleeding. according to a meta - analysis, patients undergoing tips had a lower rebleeding rate than those receiving endoscopic treatment (19% versus 47%, p <.001). the risk of hepatic encephalopathy, development of shunt stenosis, and the cost of a covered stent make tips traditionally considered as rescue therapy in patients with repeated avh. however, as aforementioned in the section of risk prediction, early tips strategy (< 72 hours) in high - risk patients improves survival significantly and may lead to paradigm shift in the future. despite the relative paucity of data in the efficacy and safety of using endoscopy to prevent recurrent hemorrhage from gv, tissue adhesives injection using n - butyl - cyanoacrylate is a reasonable choice for patients bleeding from igv1 or gov2, similar to control of acute bleeding, [55, 78 ]. for those who have bled from gov1, either tissue adhesives injection or band ligation may be used, depending on the location of varices, technical feasibility, and expertise of the endoscopist. unless it is technically infeasible, we recommend band ligation for ev and gov1 at the same time. endoscopy should not be delayed for more than 15 hours as it is associated with increased risk of in - hospital mortality, although otherwise the data is insufficient for embracing the sooner the better active bleeding at endoscopy in decompensated cirrhotic patients predicts poor outcomes and may warrant more aggressive treatment, such as early tips right after endoscopic therapy. band ligation is the endoscopic modality of choice in primary prophylaxis, hemostasis of active bleeding, and secondary prophylaxis of ev bleeding. although occasionally sclerotherapy may still be performed for hemostatic control of acute ev bleeding, it should no longer be used in the prophylactic setting. tissue glue injection to attain variceal obliteration is now the preferred endoscopic therapy to control and prevent bleeding from fundic and isolated gv. the paucity of data in the management of gv warrants more research, particularly large controlled trials, to define the evidence - based standard of care. even though substantial improvement has been achieved for the last several decades in the management of cirrhotic patients with avh, there is undoubtedly plenty room for continuing improvement in this still highly lethal medical emergency. | playing a central role in the modern multidisciplinary management of acute gastroesophageal variceal hemorrhage, endoscopy is essential to stratify patient at risk, control active hemorrhage, and prevent first as well as recurrent bleeding. before endoscopic procedure, antibiotic prophylaxis along with vasoactive medication is now routine practice. intravenous erythromycin effectively cleanses stomach and may improve the quality of endoscopy. the timing of endoscopy should be on an urgent basis as delay for more than 15 hours after presentation is associated with mortality. active variceal bleeding on endoscopy in a patient with hepatic decompensation heralds poor prognosis and mandates consideration of aggressive strategy with early portosystemic shunting. band ligation has become the preferred modality to control and prevent bleeding from esophageal varices, although occasionally sclerotherapy may still be used to achieve hemostasis. addition of pharmacotherapy with nonselective beta blockade to endoscopic ligation has become the current standard of care in the setting of secondary prophylaxis but remains controversial with inconsistent data for the purpose of primary prophylaxis. gastric varices extending from esophagus may be treated like esophageal varices, whereas variceal obliteration by tissue glue is the endoscopic therapy of choice to control and prevent bleeding from fundic and isolated gastric varices. |
decision - making capacity is fundamental to an individual 's independence and invariably deteriorates at some point along the dementia continuum, compromising autonomy in financial matters, medical care [2, 3 ], and informed consent. even at the earliest stages of alzheimer 's disease (ad), decision making can be affected, and certain features of the disease may have direct implications for decision making capacity in everyday life. previous work has demonstrated an important role for metacognitive factors including memory awareness and disease awareness in determining the likelihood that an individual would demonstrate capacity to make a decision about a treatment for ad. while global cognition also influenced capacity in this study, there was not a direct relationship between these variables such that individuals at any given level of cognition could have been judged as competent or not competent. consideration of the elements that constitute capacity reveals why memory awareness may have clear relevance for decisions about a memory treatment. decision - making capacity has been conceptualized in terms of four core components including (1) understanding = the ability to comprehend information relevant to the decision (e.g., risks and benefits), (2) appreciation = the ability to apply the information to one 's own situation, (3) reasoning = the ability to consider and compare potential consequences of various decisions, and (4) expression of choice = the ability to communicate a stable choice [6, 7 ]. if an individual is unaware that his or her memory is impaired, then he or she may be unlikely to appreciate the personal benefits of a proposed memory treatment, or to accurately anticipate and reason through the personal consequences of a decision. in fact, reasoning and appreciation of benefit were the decision - making indices most related to disordered awareness in the above study. the integral role for self awareness in treatment decisions raises the question of whether decisions regarding everyday functions are also compromised by reduced memory awareness. indeed, individuals with mci and dementia are often unaware not only of cognitive deficits but of functional limitations as well [1, 811 ]. such unawareness may explain why a patient who needs assistance with daily tasks might make a less than optimal decision as to how to best carry out the task. for example, reduced awareness of cognitive and functional changes in patients with dementia can result in unsafe behaviors such as continued driving. the current study sought to examine how awareness influences everyday decision making capacity related to medication management, a critical daily responsibility for many older adults. medication management and adherence can be challenged by cognitive and metacognitive limitations in the context of dementia, and lack of illness awareness has been raised as a particular threat that should alert clinicians to potentially poor adherence. we cross - sectionally examined the effects of memory awareness on decision making capacity related to medication management in early ad. we proposed that individuals with reduced awareness would make suboptimal decisions about medication management (e.g., failure to use assistive devices or enlist help from family members). specifically, we hypothesized that reduced awareness would be associated with lower capacity scores secondary to impaired scores on the appreciation and reasoning indices. what is especially novel about this study is that we investigated the influence of awareness on everyday decision making, using both subjective and objective metrics to comprehensively characterize memory awareness. we have shown that objective metamemory tools capture the clinical phenomenon of disordered awareness in ad and have the potential to advance our understanding of its etiology, nature, and consequences. while related, however, clinical ratings of awareness and metamemory scores also have the potential to capture different aspects of self - assessment. for example, while a subjective clinical rating of awareness characterizes an individual 's broad perception of their memory, the objective metamemory task measures individuals ' ability to systematically evaluate themselves on an item - by - item basis in the context of a specific task. investigation of both awareness variables in relation to decision making may further elucidate the manner in which self awareness may affect capacity. moreover, examining the precise factors which contribute to decision making will better equip clinicians and researchers to identify individuals with impairments in their capacity for navigating important decisions on a daily basis. given the cognitive demands of the metamemory task and our interest in studying capacity in early ad, only patients with mild - to - moderate ad, defined as a score of 19 or greater on the mini - mental state examination (mmse) were recruited at two separate centers, columbia university medical center and the university of pennsylvania. excluded were individuals with ongoing moderate - to - severe psychiatric conditions, and individuals with history of head injury, stroke, and other neurologic illnesses that might impact cognition and/or the presentation of ad. columbia university medical center (cumc)17 individuals with mild ad were recruited through the cumc alzheimer 's disease research center and received comprehensive neurologic and neuropsychological evaluations that were reviewed in a diagnostic consensus conference attended by neurologists and neuropsychologists. diagnoses of alzheimer 's disease were made according to the neurologic disorders and stroke - alzheimer 's disease and related disorders association (ninds - adrda) criteria. all participants provided informed consent and were reimbursed $ 30.00 for participation.50 healthy elders were recruited from three sources : the healthy control database available through the alzheimer 's disease research center at cumc, local senior centers, and market mailing procedures that target a diverse group of elders in new york city with a range of ethnic and educational backgrounds. controls were thoroughly screened by interview to exclude individuals with neurologic, psychiatric, or severe medical disorders. participants were considered eligible for the study if they were age 55 or above, and scored at least 24 on the mmse. 17 individuals with mild ad were recruited through the cumc alzheimer 's disease research center and received comprehensive neurologic and neuropsychological evaluations that were reviewed in a diagnostic consensus conference attended by neurologists and neuropsychologists. diagnoses of alzheimer 's disease were made according to the neurologic disorders and stroke - alzheimer 's disease and related disorders association (ninds - adrda) criteria. 50 healthy elders were recruited from three sources : the healthy control database available through the alzheimer 's disease research center at cumc, local senior centers, and market mailing procedures that target a diverse group of elders in new york city with a range of ethnic and educational backgrounds. controls were thoroughly screened by interview to exclude individuals with neurologic, psychiatric, or severe medical disorders. participants were considered eligible for the study if they were age 55 or above, and scored at least 24 on the mmse. university of pennsylvania25 individuals with mild ad were recruited through the university of pennsylvania penn memory center. eligible patients and their family members enrolled at the center, who agreed to be contacted for research studies, were sent a letter describing the study. a research assistant then called the contact person, and explained the study in more detail. 25 individuals with mild ad were recruited through the university of pennsylvania penn memory center. eligible patients and their family members enrolled at the center, who agreed to be contacted for research studies, were sent a letter describing the study. a research assistant then called the contact person, and explained the study in more detail. participants were seen for a two - hour test session including a clinical rating of awareness, capacity interview, metamemory testing, depression questionnaire, and a test of global cognition. clinical ratings of awareness were not obtained on healthy elders as clinical diagnosis of ad was the criteria against which self - report was measured. for the purposes of the capacity interview, informants were contacted prior to the test session to obtain the relevant information. this study was approved by the institutional review board at both medical centers and all individuals provided informed consent prior to participation. mini - mental state examination this commonly used 30-item measure of global cognition assesses orientation, attention, language, visuospatial functioning, and memory. this commonly used 30-item measure of global cognition assesses orientation, attention, language, visuospatial functioning, and memory. philadelphia repeatable verbal learning test (pvlt) the pvlt is a list - learning task modeled after the 9-word california verbal learning test [18, 19 ] in which participants are required to learn 9 words (comprising three different categories : fruit, tools, and furniture) over the course of five trials. the primary dependent variable was the recognition discriminability index, a variable that has been shown to reflect hippocampal integrity and to be differentially affected in ad versus other dementias. the pvlt is a list - learning task modeled after the 9-word california verbal learning test [18, 19 ] in which participants are required to learn 9 words (comprising three different categories : fruit, tools, and furniture) over the course of five trials. the primary dependent variable was the recognition discriminability index, a variable that has been shown to reflect hippocampal integrity and to be differentially affected in ad versus other dementias. assessment of capacity for everyday decision making (aced) this semistructured interview consists of 30 items spanning the four abilities that constitute decision making capacity : understanding, appreciation, reasoning, and expression of choice. this assessment tool was designed to evaluate a person 's capacity to solve problems with each of three daily decisions related to medication management, meal preparation, or finances. in the current study, analyses of decision - making capacity were restricted to those individuals who completed the medication management interview, as only 5 individuals completed the meal preparation and finances interviews.the interview takes approximately 20 minutes to administer and is based on a discussion with a knowledgeable informant who confirms whether the participant has difficulty managing medications and what the current procedure is for managing the patient 's medications. briefly, the understanding subscale (010) assesses the individual 's ability to comprehend information about options for medication management for individuals who have memory problems (e.g., use of a pillbox or assistance from another person), the advantages of these options, and the disadvantages. to reduce demands on memory, this information is presented orally to the subject and also printed on a sheet ; the interviewer reminds the subjects that this information is available if they have forgotten what was presented orally by the examiner. in order to receive credit for answers, subjects must respond in their own words and not read verbatim from the sheet. the appreciation subscale (08) assesses individuals ' ability to apply the information to themselves by asking them if they have any problems with remembering to take their medication and whether they think one of the suggested approaches to managing medications would benefit them or perhaps make things harder for them. the reasoning subscale (010) requires subjects to evaluate options for medication management in comparison to one another and to imagine what the effects of each option would be on their daily life. finally, expression of choice (02) requires subjects to state a preference for managing their medications that is consistent with their reasoning throughout the interview.every interview was audio recorded for scoring and reliability purposes. all interviews with participants with ad were scored by group consensus, and one out of every three interviews with healthy elders was consensus scored. at cumc, the consensus group included at least two research assistants and the pi (sc). a random sample of interviews obtained at cumc were reviewed and scored by the team at penn to ensure reliability across sites. this semistructured interview consists of 30 items spanning the four abilities that constitute decision making capacity : understanding, appreciation, reasoning, and expression of choice. this assessment tool was designed to evaluate a person 's capacity to solve problems with each of three daily decisions related to medication management, meal preparation, or finances. in the current study, analyses of decision - making capacity were restricted to those individuals who completed the medication management interview, as only 5 individuals completed the meal preparation and finances interviews. the interview takes approximately 20 minutes to administer and is based on a discussion with a knowledgeable informant who confirms whether the participant has difficulty managing medications and what the current procedure is for managing the patient 's medications. briefly, the understanding subscale (010) assesses the individual 's ability to comprehend information about options for medication management for individuals who have memory problems (e.g., use of a pillbox or assistance from another person), the advantages of these options, and the disadvantages. to reduce demands on memory, this information is presented orally to the subject and also printed on a sheet ; the interviewer reminds the subjects that this information is available if they have forgotten what was presented orally by the examiner. in order to receive credit for answers, subjects must respond in their own words and not read verbatim from the sheet. the appreciation subscale (08) assesses individuals ' ability to apply the information to themselves by asking them if they have any problems with remembering to take their medication and whether they think one of the suggested approaches to managing medications would benefit them or perhaps make things harder for them. the reasoning subscale (010) requires subjects to evaluate options for medication management in comparison to one another and to imagine what the effects of each option would be on their daily life. finally, expression of choice (02) requires subjects to state a preference for managing their medications that is consistent with their reasoning throughout the interview. all interviews with participants with ad were scored by group consensus, and one out of every three interviews with healthy elders was consensus scored. at cumc, the consensus group included at least two research assistants and the pi (sc). a random sample of interviews obtained at cumc were reviewed and scored by the team at penn to ensure reliability across sites. clinical ratings of awarenesstesting sessions began with a brief interview to allow the examiner to make a clinical judgment regarding participant awareness of memory loss in participants with ad. examiners asked participants to discuss their opinions of their memory abilities at the current time and assigned a score ranging from 1 to 4 on a modified version of the anosognosia rating scale. participants were scored according to the following four point ordinal rating system : 1 = full awareness (spontaneous complaint or ready admission of memory loss along with the recognition that the loss is consequential and abnormal. loss related to dementia or neurologic disease) ; 2 = moderate awareness (spontaneous admission of memory loss ; however, loss is discussed in the context of normal age related changes. no discussion of diagnosis) ; 3 = shallow awareness (inconsistent or transient recognition of memory loss, or uncertainty regarding memory loss. patients may acknowledge inconsequential memory loss) ; 4 = no awareness (matter - of - fact denial of impairment in response to direct questions regarding memory loss). if spontaneous responses did not clearly fit into a specific rating category (e.g., my memory is bad), the examiner queried as appropriate (e.g., do you have a sense of why your memory is bad ?) to extract sufficient information for assigning a score of 14. responses were recorded verbatim and scored before moving onto the remainder of the battery. testing sessions began with a brief interview to allow the examiner to make a clinical judgment regarding participant awareness of memory loss in participants with ad. examiners asked participants to discuss their opinions of their memory abilities at the current time and assigned a score ranging from 1 to 4 on a modified version of the anosognosia rating scale. participants were scored according to the following four point ordinal rating system : 1 = full awareness (spontaneous complaint or ready admission of memory loss along with the recognition that the loss is consequential and abnormal. loss related to dementia or neurologic disease) ; 2 = moderate awareness (spontaneous admission of memory loss ; however, loss is discussed in the context of normal age related changes. no discussion of diagnosis) ; 3 = shallow awareness (inconsistent or transient recognition of memory loss, or uncertainty regarding memory loss. patients may acknowledge inconsequential memory loss) ; 4 = no awareness (matter - of - fact denial of impairment in response to direct questions regarding memory loss). if spontaneous responses did not clearly fit into a specific rating category (e.g., my memory is bad), the examiner queried as appropriate (e.g., do you have a sense of why your memory is bad ?) to extract sufficient information for assigning a score of 14. responses were recorded verbatim and scored before moving onto the remainder of the battery. task developmentthe current metamemory test, a modified episodic feeling of knowing task, was designed as part of a larger ongoing study on metamemory in ad and thus has two characteristics that require attention. first, all healthy elders were randomly assigned to one of three task conditions (described below). therefore, in the current study, analyses were conducted to ensure comparable metamemory performance across all conditions before collapsing scores across healthy elders. second, metamemory stimuli were slightly modified several times over the course of the study to improve the psychometric properties of the task. therefore, analyses were conducted to ensure comparable metamemory performance across stimuli sets in both the healthy elder and ad groups. the current metamemory test, a modified episodic feeling of knowing task, was designed as part of a larger ongoing study on metamemory in ad and thus has two characteristics that require attention. first, all healthy elders were randomly assigned to one of three task conditions (described below). therefore, in the current study, analyses were conducted to ensure comparable metamemory performance across all conditions before collapsing scores across healthy elders. second, metamemory stimuli were slightly modified several times over the course of the study to improve the psychometric properties of the task. therefore, analyses were conducted to ensure comparable metamemory performance across stimuli sets in both the healthy elder and ad groups. task instructions and formatthe examiner read the following instructions, during this task, i am going to tell you about five people. i will tell you their name and something about their background. your task is to try to remember this information as best you can. immediately after the first learning trial (e.g., haxby wrote a nonfiction book about space travel ; corbett was a former mayor in nevada, etc.), predictions for memory performance were acquired one at a time for each item by providing written questions on 8.5 11 paper (e.g., who was a former mayor of nevada ?) and the following prompt read aloud by the examiner : there are eight possible answers on the next page. once predictions were recorded, participants were provided with eight answer choices and asked to select the correct answer. the answer choices included the correct response, the correct answers for the remaining 4 stimuli (to control for basic familiarity effects), and 3 new distractors. in the standard condition, the tester then moved onto the next item. all participants with ad and approximately one third of healthy elders completed the standard condition ; the remaining two thirds of the healthy elders completed the query and feedback conditions. in the query condition, participants were asked whether they thought their answer was correct (postdiction) prior to moving onto the next item. in the feedback condition, the examiner told the participant if their answer was correct or incorrect prior to moving onto the next item. this process was repeated for learning trials 24 resulting in a total of 20 metamemory judgments. stimuli were presented in the same order across each of the four learning trials ; questions and answer choices were presented in a pseudorandom order. the examiner read the following instructions, during this task, i am going to tell you about five people. i will tell you their name and something about their background. your task is to try to remember this information as best you can. immediately after the first learning trial (e.g., haxby wrote a nonfiction book about space travel ; corbett was a former mayor in nevada, etc.), predictions for memory performance were acquired one at a time for each item by providing written questions on 8.5 11 paper (e.g., who was a former mayor of nevada ?) and the following prompt read aloud by the examiner : there are eight possible answers on the next page. once predictions were recorded, participants were provided with eight answer choices and asked to select the correct answer. the answer choices included the correct response, the correct answers for the remaining 4 stimuli (to control for basic familiarity effects), and 3 new distractors. in the standard condition, the tester all participants with ad and approximately one third of healthy elders completed the standard condition ; the remaining two thirds of the healthy elders completed the query and feedback conditions. in the query condition, participants were asked whether they thought their answer was correct (postdiction) prior to moving onto the next item. in the feedback condition, the examiner told the participant if their answer was correct or incorrect prior to moving onto the next item. this process was repeated for learning trials 24 resulting in a total of 20 metamemory judgments. stimuli were presented in the same order across each of the four learning trials ; questions and answer choices were presented in a pseudorandom order. dependent variableresolution, or the relative accuracy of self - judgments, reflects the extent to which accuracy is high when predictions for performance are high, and accuracy is low when predictions are low. the nonparametric goodman - kruskal gamma statistic, a rank order correlation, was used to measure resolution. gamma compares the relative number of concordant and discordant prediction / accuracy pairs, discarding ties, or instances in which either the rating or accuracy in one pair is equal to that in another pair. limitations of gamma include a tendency to be pulled to an extreme value on the basis of only one concordance or discordance and a possibility that no score can be calculated in the event of all ties. although there are many potential metamemory metrics, gamma was used in the current study based on its selective association with clinical ratings of memory awareness in ad in a previous study from our lab, and its robustness as a measure of self - specific processes in nondemented elders. resolution, or the relative accuracy of self - judgments, reflects the extent to which accuracy is high when predictions for performance are high, and accuracy is low when predictions are low. the nonparametric goodman - kruskal gamma statistic, a rank order correlation, was used to measure resolution. gamma compares the relative number of concordant and discordant prediction / accuracy pairs, discarding ties, or instances in which either the rating or accuracy in one pair is equal to that in another pair. limitations of gamma include a tendency to be pulled to an extreme value on the basis of only one concordance or discordance and a possibility that no score can be calculated in the event of all ties. although there are many potential metamemory metrics, gamma was used in the current study based on its selective association with clinical ratings of memory awareness in ad in a previous study from our lab, and its robustness as a measure of self - specific processes in nondemented elders. 15-item geriatric depression scale (gds)the gds measures a variety of depressive symptoms (e.g., sadness, hopelessness, and worthlessness) and has demonstrated adequate validity against the 30-item questionnaire as well as concurrent validity with other measures of depression. the gds measures a variety of depressive symptoms (e.g., sadness, hopelessness, and worthlessness) and has demonstrated adequate validity against the 30-item questionnaire as well as concurrent validity with other measures of depression. the following results were considered significant at p <.05. clinical ratings of awareness19% (n = 8) of ad participants were rated as fully aware of their memory loss, 26% (n = 11) were rated as moderately aware, 38% (n = 16) were rated as having shallow awareness, and 17% (n = 7) were judged to have no awareness of memory loss. 45% of the sample (full moderate awareness) was collapsed into the aware ad (aad) group, and 55% of participants (shallow no awareness) were compiled into the unaware ad (uad) group. consistent with our previous report, the awareness groups were indistinguishable on the basis of age, gender, global cognition, or memory. see table 1. however, awareness groups in the current study differed slightly in years of education, with the aware group having approximately two more years of formal schooling (p =.04). there was not a statistically significant difference in the proportion of aware (29%) versus unaware (10%) individuals who managed their medication independently (x = 1.954, p =.16). 19% (n = 8) of ad participants were rated as fully aware of their memory loss, 26% (n = 11) were rated as moderately aware, 38% (n = 16) were rated as having shallow awareness, and 17% (n = 7) were judged to have no awareness of memory loss. 45% of the sample (full moderate awareness) was collapsed into the aware ad (aad) group, and 55% of participants (shallow no awareness) were compiled into the unaware ad (uad) group. consistent with our previous report, the awareness groups were indistinguishable on the basis of age, gender, global cognition, or memory. see table 1. however, awareness groups in the current study differed slightly in years of education, with the aware group having approximately two more years of formal schooling (p =.04). there was not a statistically significant difference in the proportion of aware (29%) versus unaware (10%) individuals who managed their medication independently (x = 1.954, p =.16). metamemory task condition and stimuli setas part of a larger study, healthy elders received one of three conditions of the metamemory test. of those who had a calculable metamemory score in the current study (35 of 50 healthy elders), 31% received the standard task condition, 40% the query, and 29% the feedback. there was no difference in metamemory score as a function of task condition, f(2, 32) = 1.63, p =.213. there was also no difference in metamemory as a function of stimuli set in the healthy elders f(2, 32) =.10, p =.905, or ad group f(1, 33) =.68, p =.415. therefore, all metamemory scores were collapsed across task condition and stimuli set for the current analyses. as part of a larger study, healthy elders received one of three conditions of the metamemory test. of those who had a calculable metamemory score in the current study (35 of 50 healthy elders), 31% received the standard task condition, 40% the query, and 29% the feedback. there was no difference in metamemory score as a function of task condition, f(2, 32) = 1.63, p =.213. there was also no difference in metamemory as a function of stimuli set in the healthy elders f(2, 32) =.10, p =.905, or ad group f(1, 33) =.68, p =.415. therefore, all metamemory scores were collapsed across task condition and stimuli set for the current analyses. metamemory scoresgamma scores were calculable for 35 of 42 participants in the ad group (83%) and 35 of 50 healthy elders (70%). the remaining participants demonstrated no variability in their predictions (yes, maybe, and no) or in their accuracy. in a replication of our previous findings, gamma scores in the ad group were significantly lower in the unaware group than in the aware group (p =.015). gamma scores were calculable for 35 of 42 participants in the ad group (83%) and 35 of 50 healthy elders (70%). the remaining participants demonstrated no variability in their predictions (yes, maybe, and no) or in their accuracy. in a replication of our previous findings, gamma scores in the ad group were significantly lower in the unaware group than in the aware group (p =.015). decision making capacityin the ad group, analyses examining capacity were restricted to the 34 individuals who completed the medication management version of the aced. mean scores for the aced total score and subscores by awareness group are presented in table 2. a multivariate general linear model was used to compare aced scores across the two ad groups and healthy elders. analyses revealed that aced scores were significantly lower in each of the ad groups than in the healthy elders (f(2, 67) = 17.63, uad, p <.01 ; aad, p =.01). performance on the aced did not vary as a function of whether or not participants managed their medications independently at home f(1, 32) =.011, p =.92. in the ad group, analyses examining capacity were restricted to the 34 individuals who completed the medication management version of the aced. mean scores for the aced total score and subscores by awareness group are presented in table 2. a multivariate general linear model was used to compare aced scores across the two ad groups and healthy elders. analyses revealed that aced scores were significantly lower in each of the ad groups than in the healthy elders (f(2, 67) = 17.63, uad, p <.01 ; aad, p =.01). performance on the aced did not vary as a function of whether or not participants managed their medications independently at home f(1, 32) =.011, p =.92. memory awareness and capacitya univariate analysis of variance was used to examine capacity scores as a function of clinically determined awareness level in the ad group. total aced score was not significantly different across groups (p =.065), but performance on the appreciation index was lower in the uad group as compared to the aad group (p =.007). this difference was driven both by problems appreciating one 's own difficulty with managing medications f(1, 32) = 5.596, p =.024, as well as the overall ability to appreciate the potential benefits and harms of receiving assistance with medication management, f(1, 32) = 4.34, p =.045. when examined individually, neither appreciation of potential benefits nor appreciation of harms were significantly different as a function of awareness. bivariate correlations were then used to examine the correlates of decision making in ad. clinical rating of awareness was the only characteristic that correlated with everyday decision making in ad (r =.41, p =.007). a univariate analysis of variance was used to examine capacity scores as a function of clinically determined awareness level in the ad group. total aced score was not significantly different across groups (p =.065), but performance on the appreciation index was lower in the uad group as compared to the aad group (p =.007). this difference was driven both by problems appreciating one 's own difficulty with managing medications f(1, 32) = 5.596, p =.024, as well as the overall ability to appreciate the potential benefits and harms of receiving assistance with medication management, f(1, 32) = 4.34, p =.045. when examined individually, neither appreciation of potential benefits nor appreciation of harms were significantly different as a function of awareness. bivariate correlations were then used to examine the correlates of decision making in ad. clinical rating of awareness was the only characteristic that correlated with everyday decision making in ad (r =.41, p =.007). the current study investigated the selective influence of memory awareness in alzheimer 's disease on everyday decision - making capacity related to medication management. as predicted, and consistent with karlawish and colleagues ' findings in a study of decisions regarding memory treatment, awareness was particularly important for decision making capacity in the current study. that is, in comparison to demographic variables, global cognition, memory, and mood, only memory awareness was associated with everyday decision making. in particular, memory awareness was clearly associated with performance on the ability to appreciate personal difficulty with managing medications, as well as the advantages and disadvantages of receiving assistance with medication management. these results suggest that reduced memory awareness in ad may pose a particularly important threat to decision making, not only for medical treatment and research, but for everyday issues as well. one of the advantages of the current study was the multiple metrics used to characterize memory awareness. in addition to rating participants from an overall, global, and clinical perspective, objective metamemory testing was used to quantify memory awareness rigorously and at a more local level. previous work by our lab has established that scores on this metamemory test track with clinical ratings of awareness, and application of both methodological approaches increases our confidence that we are capturing the phenomenon of disordered awareness in a reliable fashion. moreover, it enables consideration of the specific awareness components which may or may not influence the outcome of interest, in this case, everyday decision - making capacity. findings from the current study reinforce the idea that in the context of early ad, awareness varies significantly, and this variation is not simply a reflection of global cognition, demographic variables, mood, or memory. instead, it is a selective deficit. its clinical consequences are important and relate to caregiver burden [27, 28 ], the success of behavioral interventions, decisions to continue driving, and medical decision making. it is thus reasonable to expect that memory awareness may also influence everyday decisions that should be based in part on the fact that one 's memory is impaired. examination of medication management decisions allowed us to answer this question, given the idea that optimal decisions about medication management would require appreciation of one 's memory limitations and the need to accommodate one 's approach to this cognitively demanding daily routine in light of these limitations. examination of capacity scores across awareness groups in ad indeed revealed that decision making - capacity related to medication management was diminished in the unaware group, specifically on the appreciation subscale. the appreciation subscale queries the subjects to declare if they (1) have any problems with remembering to take medications, (2) think that using an assistive device (e.g., pillbox) to help them remember to take their medications could benefit them, (3) think that having someone administer their medications to them could benefit them, and (4) think that either of these options might actually make things worse for them. these answers are always interpreted and scored in the context of the information provided by the informant. that is, if a subject denied a problem with remembering to take medications and this was contradicted by the informant, then a score of zero would be assigned for the first appreciation item. the remaining appreciation items required subjects to consider whether or not receiving assistance with medication management (in the form of a device such as a pillbox or help from another individual) could possibly benefit them or perhaps make things worse for them, and to justify their responses with a logical reason. points were deducted if the response reflected false beliefs (e.g., i would not get any benefit from a pillbox because i do not have any trouble managing my medications) or illogical reasoning. in contrast, the understanding subscale does not require the individual to consider the decision from a personal standpoint, but simply to reiterate the options for someone who might need assistance with medication management, to consider the benefits in general, and the disadvantages in general. it is thus apparent why appreciation scores would be most affected by awareness : subjects must recognize their own difficulty with managing their medications. interestingly, results indicate that unaware subjects not only had difficulty recognizing their problems with medication management (which might be expected), but they also had difficulty appreciating the potential advantages and disadvantages of receiving assistance. the current findings nicely demonstrate that awareness selectively affects a component of capacity that requires application of information to oneself and not those indices which at face value have little to no metacognitive demands. we should emphasize that we are not arguing that primary cognitive functions are not important for capacity ; of course, they contribute to performance across the various capacity subscales. indeed, in the current study, both the aware and unaware ad groups had reduced capacity scores in relation to healthy elders, and studies consistently demonstrate associations between capacity and aspects of neuropsychological functioning in different clinical populations (see for a review). what is interesting about the findings in the current study is that in the context of very - mild - to - mild ad, the primary factor driving capacity was self - awareness rather than global cognition. moreover, this was not simply a reflection of the task 's hypothetical component (which could be cognitively demanding) as actual medication management status, that is, whether or not the participant received assistance with medication management at home, was comparable across awareness groups and unrelated to aced scores. we can not exclude the possibility that a specific cognitive deficit such as executive dysfunction mediated the association between capacity and awareness, as both variables have been associated with this cognitive deficit [3133 ], and future work should address this possibility. however, a compelling study by koren and colleagues demonstrating a selective role for metacognitive rather than executive abilities in predicting capacity to consent to treatment in individuals with schizophrenia speaks to this issue. specifically, on a modified version of the wisconsin card sorting test (wcst) in which participants were asked to indicate whether or not they wanted each sort counted toward their overall score, multiple aspects of metacognition were related to capacity, whereas conventional wcst moreover, recent work from our lab examining nondemented elders supports the idea that self - referential processing is independent from more general executive abilities. in sum, it appears that aspects of metacognition are dissociable from more general executive abilities and may hold a particularly critical role for decision - making capacity. this brings us to a second issue addressed in the current study, which was whether or not specific measures of memory awareness are differentially associated with capacity. interestingly, despite the association between the two memory awareness measures in the current study (clinical ratings of awareness and metamemory scores), there was no association between metamemory scores and capacity (table 3). this discrepancy touches on the differences between the two awareness metrics and urges us to consider what forms of memory awareness are captured uniquely by each measure despite their shared variance. we have argued in the past that objective metrics of memory awareness are critically needed for furthering our understanding of disordered awareness in ad, particularly its nature and etiology, and have shown that such metrics in fact capture clinical differences in awareness. thus, use of objective metrics would be ideal when attempting to shed light on the specific metacognitive errors that give rise to the clinical phenomenon of disordered awareness, for example. however, it may be that under certain circumstances, broad clinical impressions capture an element of self awareness that is practically relevant and lost when measured at the item level for a number of different reasons. specifically, the metamemory task requires individuals to make item - by - item predictions about performance (i.e., local level) whereas the clinical rating of awareness queries for a much broader assessment of oneself (i.e., global level). each type of self - assessment might affect the other such that general impressions about oneself might bias answers at the local level, and impairment at the local level might lead to impairment at the global level. however, global impressions of one 's functioning may hold more weight for decision making than local assessments on a specified task, because the former represent beliefs about oneself in general rather than within the confines of a specific task.. moreover, an overall self - assessment that is outside the context of a task does not provide the opportunity for an individual to evaluate themselves online and incorporate any feedback about task performance ; in that sense, it better approximates an individual 's general sense of themselves and their abilities. again, this type of self - awareness is what appears to be critical in the context of a decision - making paradigm in which individuals are required to consider a particular issue and to come to a decision about the issue in a context - free (task - free) environment. for example, the current capacity assessment was not conducted by requiring individuals to engage in a task (e.g., fill prescriptions, organize pills in a pillbox and prospectively remember to take these pills) before making a decision about how best to approach this task in daily life. perhaps this more specific form of capacity assessment would be more highly related to awareness as measured through metacognitive testing. this raises an interesting question about the manner in which decision - making capacity is assessed and might be assessed in the future. for example, some capacity instruments like the one used in this study intentionally reduce demands on episodic memory when evaluating capacity in patients with ad. thus, patients are not penalized for this particular cognitive limitation, and other elements of decision making can be examined more directly. similarly, perhaps heightening subjects ' awareness in the context of a capacity evaluation would allow examination of decision making when an individual is provided with all information relevant to the decision. this might be achieved in a number of ways including (1) making explicit statements (e.g., you have been diagnosed with alzheimer 's disease and this disease causes your memory functioning to decline over time), (2) providing a context in which subjects might recognize memory failures (e.g., cognitive testing), or (3) conducting metacognitive testing to inform the patient about his or her memory awareness. while there may be ethical issues to consider for each of these approaches, the advantage would be that decision making could be evaluated once subjects possess accurate information about their abilities and would thus not be biased in subjects who have metacognitive deficits. alternatively, it may be that assessments of capacity are best when they approximate the real world. if so, then an effort to facilitate awareness in the artificial context of a capacity evaluation would provide an inaccurate depiction of an individual 's decision - making capacity in everyday life. for example, marson and colleagues do not provide materials that reduce memory demands in their capacity assessment tools, and in fact, memory becomes a highly predictive factor for performance in individuals with dementia [35, 36 ]. this may be a better representation of certain decision - making situations encountered by individuals in daily life, and thus an important part of the capacity evaluation. there is not likely to be one correct approach, and the specific circumstances of each capacity question and evaluation may have to drive the manner in which capacity is assessed, with the goal of balancing the patient 's autonomy and safety as best as possible. future work should further consider these issues and explore the feasibility and utility of different forms of capacity assessment in individuals with dementia. in sum, results from the current study identify memory awareness as an important component of everyday decision making in ad and shed light on aspects of capacity assessments that may warrant additional consideration depending on the particular issue at hand. first, it should be emphasized that overall judgments of capacity were not made in a dichotomous fashion by expert raters as has been done in other studies. as such, the current results do not comment on whether awareness directly affects the rating of an individual as capable or not capable to make daily decisions about medication management. rather, current results allow the examination of the factors which contribute to decision making capacity, and which might be expected to lead to the judgment of an individual as not capable of making a decision. second, memory data were unavailable for the individuals seen at the university of pennsylvania ; however, the correlation coefficient (r =.10) reported based on the individuals at cumc is not suggestive of even a borderline statistical association between memory and capacity. moreover, the manner in which capacity was measured in the current study was specifically selected to eliminate the demands on episodic memory, so an association was not anticipated. third, the cognitive battery used in the current study was extremely limited as this was not the focus of the current investigation. however, future work should examine the extent to which specific aspects of cognition covary with awareness and capacity and whether or not deficits in any area of cognition might mediate the association between capacity and awareness. finally, the current study did not include an objective measure of medication management [37, 38 ], a component which would have allowed us to directly examine the association between capacity, awareness, and the skills necessary for medication management. despite these limitations, the current study provides important information on the factors which influence everyday decision making in ad, highlighting the critical role for memory awareness and raising considerations for the manner in which capacity is currently assessed. | memory awareness in early alzheimer 's disease (ad) influences capacity to provide informed consent for a memory treatment. this study investigated the extent to which aspects of memory awareness influence everyday decision - making capacity about medication management in ad. 42 participants with mild ad and 50 healthy elders underwent clinical ratings of memory awareness, metamemory testing, and an interview of everyday decision - making capacity regarding medication management. 45% of ad subjects were classified as aware (aad) and 55% as unaware (uad) based on clinical ratings and supported by metamemory testing (p =.015). capacity was impaired in each of the ad groups as compared to the healthy elders f(2, 67) = 17.63, uad, p <.01 ; aad, p =.01). within the ad group, capacity correlated selectively with awareness as measured with clinical ratings (r =.41, p =.007) but not objective metamemory testing (r =.10, p =.60). appreciation scores were lower in uad as compared with aad f(1,35) = 8.36, p =.007. unawareness of memory loss should heighten clinicians ' concern about everyday decision - making capacity in ad. |
this is a case report of a male patient affected by autism spectrum disorder (asd) with an 11 mb mosaic trisomy of the pericentromeric region of chromosome 8 and maternal uniparental disomy (hupd / iupd) of the same chromosome. uniparental disomy (upd) is defined by the presence or inheritance of a pair of homologous chromosomes from one parent in a diploid genome 1. upd is classified as (i) heterodisomic (hupd), denoted by the presence of both uniparental homologues, (ii) isodisomic (iupd), denoted by the presence of two copies of one homologue, or (iii) a mixture (hupd / iupd) of both 2. a rescued meiosis nondisjunction error associated with meiotic recombination and possible additional somatic exchange between chromatids can result in the mixed (hupd / iupd) forms 3. autism spectrum disorder is a lifelong neurodevelopmental disorder characterized by deficits in social communication and interaction, repetitive and restrictive behavior, extensive clinical and etiologic heterogeneity, as well as, a remarkably rising global prevalence rate. a number of studies have provided evidence supporting the particular role of upd in asd, epilepsy and intellectual disability (i d) 4. furthermore, these neurodevelopmental conditions are also commonly associated with de novo constitutional copy number variations (cnvs) and/or complex chromosomal rearrangements affecting single or multiple chromosomes 5. the proband is currently sixyearold born to nonconsanguineous parents of greek descent with a birthweight of 3.8 kg. the pregnancy was complicated by polyhydramnios, and dilatation of the pelvicalyceal system identified by ultrasound and the latter resolved spontaneously at birth. he had a normal growth pattern but had gastroesophageal (ge) reflux for the first few months of life. there was difficulty in breastfeeding during the first 2 months but was exclusively breastfed till the age of 15 months. at 18 months, he developed an allergy to cow milk (ige = 334 iu / ml). he developed chronic asthma with virally induced exacerbations but grew out of it at 4 years of age. his hearing was tested by brain stem evoked potential at the age of 2 years due to delayed speech and was found to be normal. he has hypermetropia in the left eye, and patching the right eye improved visual acuity. an evaluation at the age of 3 years showed that he can not use a tricycle and can throw a ball but can not kick it. another evaluation at the age of 5 years showed that he could follow the orders of three words, and his fine motor and gross motor skills were at 4 years. he could copy 3d objects in 3d, has no concept of money, and does not dress himself but became toilet trained. he has hyperphagia, and at the age of 6 years, his weight is 28 kg (75th % ile), height is 123 cm (90th % ile), and the head circumference is 55 cm (more than two sd above the mean for age and gender). the proband had two assessments for his neurodevelopment and was diagnosed with mild mental retardation without designation of an iq figure, attention deficit hyperactivity disorder (adhd), and pervasive developmental disordernot otherwise specified (pddnos), those based on griffith scale. the diagnosis of pddnos is due to lack of communication and abstract thinking, temper tantrums, poor interaction with other children, and obsession with buttons, as well as based on the autism diagnostic observation schedule (ados). gbanded chromosomal analysis showed a mosaic (60%) marker chromosome (47,xy,+mar) (fig. cytogenetic analysis of gbanded chromosomes showed an unidentified marker present in 60% of the metaphases examined. the marker observed is composed of the pericentromeric region of chromosome 8 and is likely the remnant of a trisomy rescue. dna was extracted from peripheral blood of the proband and his parents after obtaining an informed consent. the consents and the research project were approved by the qatar biomedical research institute institutional review board (qbriirb), which operates according to the accords of the declaration of helsinki. genomewide snp genotyping was performed, utilizing the omniexpress array (700k) on an illumina (san diego, california) platform. genotyping quality assessment, snp calling, and copy number variation (cnv) analysis the analysis showed the absence of paternal contribution to the child 's chromosome 8, except for an 11.29 mb duplicated region, thus indicating both a partial pericentromeric trisomy of chromosome 8 and a maternal uniparental disomy. the pericentromeric trisomy (38,989,81350,283,147 ; p11.22q11.21) is present in the proband but not in the parents [de novo ]. the region is demarcated by the markers (rs4733980, rs7001086) and contains 38 proteincoding genes. a complex pattern of mixed heterodisomy and isodisomy (hupd / iupd) indicated by the intermittent regions of loss of heterozygosity (loh) (172,93917,483,118 and 77,032,017119,348,323) and heterozygosity (17,483,11838,989,813 and 50,283,14777,032,017) on the child 's chromosome 8 was also observed. this chromosomal pattern changed with further molecular evaluation using microsatellite genotyping analysis. to validate the identified trisomy, quantitative realtime pcr (qrtpcr) was performed using the sybr green pcr master mix on a 7900 fast realtime pcr system (applied biosystems, foster city, california). primer sets were designed to amplify two exons in genes (adam18, exon 12, 393 bp and efcab1, exon 2, 403 bp) and three intronic regions (chr8:28.2 mbp, 172 bp ; 38.98 mbp, 402 bp ; 50.28 mbp, 391 bp) located on the p and q arm, using the webbased tool primer3 (http://bioinfo.ut.ee/primer3/) (table s1). the exonic dna fragments within the trisomic region showed two copies in the parents and three copies in the child. the three intronic dna fragments peripheral to the trisomy showed two copies in the parents and the child, thus confirming the trisomy (fig. (a) pedigree schematic showing clinically unaffected (unshaded) parents and affected (shaded) proband. circle and squares symbolize female and male family members, respectively. (b) genotypes corresponding to the 21 analyzed microsatellite markers are shown underneath every individual. alternating regions indicated by (i) in the proband denote heterodisomy of maternal origin, (ii) denotes a single, maternally inherited, isodisomic region, and () denotes the trisomic region (38.950.3 mbp) where maternal and paternal genetic contribution is noted. (c) qrtpcr analysis of three regions (i) 28. 2 mbp, (ii) 38.9 mbp and (iii) 50.3 mbp, peripheral to the trisomy reveals two copies in the proband and parents. qrtpcr of () marked regions at positions (adam18 exon 12, 39.4 mbp) and (efcab1 exon 2, 49.6 mbp). microsatellite marker analysis was performed using 48 polymorphic markers spanning chromosome 8 to determine the parental origin of the identified regions of complex disomy (hupd / iupd). the genotypes of the child and both parents for 21 informative microsatellite markers (table s2) showed homozygosity in the child but not in the mother in one loh region (75,958,125137,687,554), thus confirming maternal isodisomy for this region. conversely, identical heterozygous fragment sizes in the mother and affected child confirmed the observation of maternal heterodisomy at the rest of the chromosome including region (172,93917,483,118), which was suggested by the snp array to be isodisomic. maternal and paternal inheritance was noted for markers within the pericentromeric region of trisomy (fig. the identified cnv was systematically compared to cnvs present in the database of genomic variants (dgv) (http://dgv.tcag.ca/dgv/app/home) to assess its frequency in control populations. the cnv was considered novel if it did not coincide with any cnvs reported in the database. no duplications matching or overlapping with the trisomy identified here were found in control population studies upon searching the dgv database. furthermore, the identified cnv was compared to cnvs reported to cause known chromosomal imbalance syndromes that are documented in the decipher grch37 database (https://decipher.sanger.ac.uk/index). multiple reports of overlapping duplications on chromosome 8 in patients with comparable phenotypes, including seizures, autistic features, and intellectual disability were identified (table 1). details of primers utilized in qrtpct analysis including pcr product position and amplification conditions. fragment size, gc content and annealing temperature are shown the clinical phenotype, number of cases, and chromosomal makeup for each reported duplication are shown. using the sfari database for asd candidate genes 6, a number of chromosome 8 genes that have been implicated in various forms of de novo or inherited asd and/or intellectual disability were identified (table s3). simultaneously occurring, multiple chromosomal abnormalities are usually rare in a single patient 2. mixed (hupd / iupd) is most often observed in unidentified small supernumerary marker chromosome (ssmc) cases 7. for a significant number of upd cases, undetected mosaicism, recessive genetic disease, and pathogenic structural rearrangement the collective effect of these events possibly explains the observed clinical phenotypes in this patient, which directly depends on the gene content of the chromosome involved. for the majority of chromosomes, upd is without phenotypic effects. however, in certain chromosomal segments, it can result in clinically recognizable phenotypes due to imprinting effects the result of inherited differences in gene expression 9. a role for this mechanism is not suspected in this case, as evidence supporting the presence of imprinted genes on chromosome 8 has not been reported 10. another diseasecausing mechanism in upd is reduction to homozygosity of a disease variant in the iupd region, for which the parent of origin is heterozygous. evidence of this mechanism was noted in a male patient with the autosomal recessive chromosomal instability disorder, nijmegen breakage syndrome (nbs), due to maternal isodisomy of chromosome 8 11. there are four genes (fabp5, sdc2, vps13b, and ext1) within the iupd region, which have been previously implicated in autism 12, 13, 14, 15, but we did not explore this mechanism in this report. based on literature evidence, the role of ssmcs in asd is well documented and should be examined here. asd, developmental delay and mental retardation were noted, respectively, in 22, 83, and 39% of the 18 ssmc(8) cases without upd(8) reported in the literature 16. although 70% of ssmc carriers are clinically asymptomatic, duplications extending past the pericentromere to 8p11.22 or to 8q11.22 have been particularly identified in association with developmental delay and asd 17, 18. table s4, summarizes the nine pericentromeric ssmc(8) cases reported specifically with asd among other neurologic and physical presentations. two of these cases were found to display small supernumerary ring chromosome 8 19, 20. six of the nine cases displayed varying degrees of cognitive impairment and developmental delay with no physical malformations. however, cases 3, 4, and 9 were reported with both cognitive delay and physical findings including polydactyly (case 3), dysmorphic facial features (case 4), overgrowth syndrome, scoliosis, single palmar crease, umbilical hernia, and deafness (case 9) 19, 21. only case 7 was found to display the same phenotypic features noted in our case including developmental delay, asd, and hypotonia 22. case 7 describes a 6yearold male with a de novo, mosaic, chromosome 8derived ssmc (38.9447.85 mb ; p11.23q11.1). the pericentromeric derived ssmc shares 8.9 mb of the 11 mb trisomic region described in our patient, which explains the similarity in phenotypic presentation among the two cases. several genes contained within the trisomic region of our patient have been previously linked to neuronal function. in particular, chrnb3 (cholinergic receptor, nicotinic, beta 3), which encodes the subunits of neuronal cholinergic receptors, has been reported within trisomies identified in several patients with rare, de novo asdspecific cnvs 23. approximately, 1.3% of ssmc cases present with upd 24, 25. there are several reported cases of smc(8) coincident with complex, maternally, or paternally derived (hupd or iupd) 7. to our knowledge, none have been described in association with asd ; however, various degrees of developmental delay, intellectual disability, and dysmorphism were reported in these cases, summarized in table s5, 11, 26, 27, 28. since to date no known phenotypes are associated with upd(8), it is plausible that upd(8) does not contribute to the clinical phenotype in our patient. in this present case, the observation of three haplotypes at the centromere is suggestive of a trisomy rescue from a meiosisi nondisjunction, therefore resulting in maternal hupd 24. the region of isodisomy possibly reflects a meiotic, postzygotic recombination event ; however, further investigation is required to validate these speculations. furthermore, upd resulting from trisomy rescue is often associated with placental or fetal mosaicism. however, it is challenging to isolate the specific clinical effects of the upd from those caused by the mosaic trisomy. as such, this is the first report of a patient with asd and i d presenting with a complex chromosomal, maternal upd and ssmc(8). despite reports of their contribution to the observed phenotype, the exact mechanisms underlying these chromosomal aberrations are not entirely clear. based on our reported case and on review of literature findings, we recommend testing for ssmc(8) in patients presenting with asd, developmental delay, and hypotonia. in the presence of an identifiable ssmc(8), exploration of upd is also recommended to achieve better understanding of phenotype genotype correlation pattern. genes reported in the safari gene database to be implicated in asd on chromosome 8. summary of literaturedescribed cases presenting with autism among other clinical presentations due to smc derived from chromosome 8. details of the age, gender and clinical presentation noted in each case is included. | key clinical messagevarious chromosomal anomalies including small supernumerary marker chromosome (ssmc) and uniparental disomy (upd) have been described in association with intellectual disability and autism spectrum disorder. based on our reported findings, we recommend that patients with ssmc(8) be evaluated for autism spectrum disorder (asd) for early institution of therapy. in the presence of an identifiable ssmc, exploration of upd is also recommended to further investigate the role of chromosome 8 upd in asd. |
regional anesthetic techniques with adjuvants are commonly used to improve block characteristics and extend analgesic effect into the postoperative period. for majority of ophthalmic procedures regional anesthesia in the form of peribulbar and there is less hemodynamic instability, less respiratory depression, better postoperative analgesia, and less nausea and vomiting with regional anesthesia and hence safer and more effective than general anesthesia. retrobulbar block provides adequate rapid anesthesia, akinesia and control of intraocular pressure as well as postoperative analgesia. but sometimes, it may cause serious complications such as globe perforation, brainstem anesthesia, and retrobulbar hemorrhage. peribulbar block is a rapid, simple and safe technique but requires larger volume of local anesthetic solution to produce the desired akinesia. the duration of the block depends on the type and composition of the local anesthetic mixture injected. hence, the present study was carried out to determine effect of adding adjuvants to local anesthetic solution in peribulbar blocks for superior analgesia. the addition of adjuvant drugs such as opioids, clonidine, ketamine, and prostigmine to the local anesthetic mixture might prolong the duration of the block. these drugs have been used along with local anesthetics for extradural, intrathecal and peripheral nerve plexuses blocks to produce more intense and prolonged analgesia. opioids are commonly added to local anesthetic solutions to increase intensity and duration of anesthesia by acting on opioid receptors present on the central nervous system. clonidine, an alpha 2-agonist, is found to prolong anesthesia and analgesia of local anesthetics and duration of retrobulbar block. hyaluronidase as an adjuvant to anesthetic mixture helps in spreading injected mixtures thus accelerating the onset time and improving the quality of block. the combination of fentanyl and clonidine added to local anesthetics has an advantage of decreasing the side effects of both the drugs due to smaller dose used. after approval from institutional ethics committee p. d. u. medical college, rajkot, a controlled prospective clinical study was carried out in the year 20142015 on 105 patients of age > 15 years, of either sex including adult and geriatric age group undergoing ophthalmic surgeries under peribulbar block of the american society of anaesthesiology grade i and ii and informed written consent was obtained from all the patients. exclusion criteria included - coagulation abnormalities, known allergic reaction to drug, local infection at the puncture site, patients with impaired orbital and periorbital sensation, patients with complicated vitreous hemorrhage as retinal detachment, extensive epiretinal membrane, dropped nucleus or intraocular lens, patients with posterior staphyloma, patients with axial length more than 28 mm or patient 's refusal. all the patients were made familiar with standard 10 cm visual analog scale (vas) preoperatively. for elimination of bias in the study blinding of investigators, participants, and assessors of both the study groups was done. three groups were made : control group s : 5 ml lignocaine 2% + 3 ml bupivacaine 0.5% + 1 ml hyaluronidase (250 iu) + 1 ml normal salinefentanyl group f : 5 ml lignocaine 2% + 3 ml bupivacaine 0.5% + 1 ml hyaluronidase (250 iu) + 1 ml fentanyl (25 g diluted to normal saline 1 ml)clonidine group c : 5 ml lignocaine 2% + 3 ml bupivacaine + 1 ml hyaluronidase (250 iu) + 1 ml clonidine (25 g diluted to normal saline 1 ml). control group s : 5 ml lignocaine 2% + 3 ml bupivacaine 0.5% + 1 ml hyaluronidase (250 iu) + 1 ml normal saline fentanyl group f : 5 ml lignocaine 2% + 3 ml bupivacaine 0.5% + 1 ml hyaluronidase (250 iu) + 1 ml fentanyl (25 g diluted to normal saline 1 ml) clonidine group c : 5 ml lignocaine 2% + 3 ml bupivacaine + 1 ml hyaluronidase (250 iu) + 1 ml clonidine (25 g diluted to normal saline 1 ml). all patients underwent a preanesthetic check - up before surgery, and all the routine and specific investigations were documented. standard monitors such as electrocardiogram, noninvasive blood pressure, and pulse oximeter were applied, and patients vitals were recorded, intravenous (iv) line was secured and patients were premedicated with injection glycopyrolate (0.2 mg iv, injection ondansetron (4 mg) iv and injection ranitidine (50 mg) iv. in peribulbar block, short beveled fine 25 g needle was inserted in inferotemporal part of lower orbital rim at the junction of medial 2/3 and lateral 1/3 at infraorbital notch. it was proceeded 12 mm vertically and tilted 15 superiorly, injecting local anesthetic mixture around the globe, total 10 ml of solution was injected, around 68 ml in peribulbar region and 2 ml in lower eyelid. compression was given over the globe for 10 min with pinky ball (ball was released after first 5 min and then was compressed again to prevent central retinal artery occlusion). evaluation of the success of the block was done by scoring the mobility of eyeball at 1, 3, 5, 10, and 15 min after the end of injection. movements were scored by three - point scoring system in the four quadrants as : 0 = akinesia (ocular movement 1 mm but 4 mm). 0 = akinesia (ocular movement 1 mm but 4 mm). giving a maximum aggregate score of 8 for the four muscles, a score of 0 in all four directions was taken to indicate a successful block. if not achieved within 15 min after injection, the case was excluded from the study. the onset of globe akinesia was recorded from the time of injection of local anesthetic until complete globe akinesia (score 0). lid akinesia was assessed by asking the patient to open both the eyes widely followed by squeezing them maximally. onset of lid akinesia was defined as time elapsing from injection of local anesthetic solution until complete lid paralysis. duration of globe akinesia was recorded from time of injection of local anesthetic mixture till recurrence of muscle movements (score 8). the duration of the lid akinesia was recorded from the time of injection of local anesthetic mixture till full recurrence of lid movements. globe anesthesia was assessed by a gentle touch on the conjunctiva with a cotton swab. duration of globe anesthesia was recorded from the time of injection of local anesthetic till the complete disappearance of sensation. agitated, anxious or restlessco - operative, oriented and tranquilresponsive to commands onlybrisk response to light glabellar tap or loud auditory stimulussluggish response to light glabellar tap or loud auditory stimulusno response to light glabellar tap or loud auditory stimulus. agitated, anxious or restless co - operative, oriented and tranquil responsive to commands only brisk response to light glabellar tap or loud auditory stimulus sluggish response to light glabellar tap or loud auditory stimulus no response to light glabellar tap or loud auditory stimulus. duration of lid and globe akinesiatime of rescue analgesic given when vas 4 and total number of rescue analgesic given. duration of lid and globe akinesia time of rescue analgesic given when vas 4 and total number of rescue analgesic given. postoperatively, patients were monitored for pulse, blood pressure, spo2, sedation and complications (if any). the patients were asked to make a vertical mark on the line to indicate the intensity of their pain and vas was scored by measuring from the left side, how far the patient marked towards the maximum pain end. when vas of 4 postoperatively, rescue analgesia was given in the form of injection diclofenac sodium 1.5 mg / kg i.m. all the patients were observed for incidence of any side effects and complications such as nausea, vomiting, hypotension, bradycardia, globe perforation, oculocardiac reflex, and chemosis during intra- and post - operative period. in peribulbar block, short beveled fine 25 g needle was inserted in inferotemporal part of lower orbital rim at the junction of medial 2/3 and lateral 1/3 at infraorbital notch. it was proceeded 12 mm vertically and tilted 15 superiorly, injecting local anesthetic mixture around the globe, total 10 ml of solution was injected, around 68 ml in peribulbar region and 2 ml in lower eyelid. compression was given over the globe for 10 min with pinky ball (ball was released after first 5 min and then was compressed again to prevent central retinal artery occlusion). evaluation of the success of the block was done by scoring the mobility of eyeball at 1, 3, 5, 10, and 15 min after the end of injection. movements were scored by three - point scoring system in the four quadrants as : 0 = akinesia (ocular movement 1 mm but 4 mm). 0 = akinesia (ocular movement 1 mm but 4 mm). giving a maximum aggregate score of 8 for the four muscles, a score of 0 in all four directions was taken to indicate a successful block. if not achieved within 15 min after injection, the case was excluded from the study. the onset of globe akinesia was recorded from the time of injection of local anesthetic until complete globe akinesia (score 0). lid akinesia was assessed by asking the patient to open both the eyes widely followed by squeezing them maximally. onset of lid akinesia was defined as time elapsing from injection of local anesthetic solution until complete lid paralysis. duration of globe akinesia was recorded from time of injection of local anesthetic mixture till recurrence of muscle movements (score 8). the duration of the lid akinesia was recorded from the time of injection of local anesthetic mixture till full recurrence of lid movements. globe anesthesia was assessed by a gentle touch on the conjunctiva with a cotton swab. duration of globe anesthesia was recorded from the time of injection of local anesthetic till the complete disappearance of sensation. agitated, anxious or restlessco - operative, oriented and tranquilresponsive to commands onlybrisk response to light glabellar tap or loud auditory stimulussluggish response to light glabellar tap or loud auditory stimulusno response to light glabellar tap or loud auditory stimulus. agitated, anxious or restless co - operative, oriented and tranquil responsive to commands only brisk response to light glabellar tap or loud auditory stimulus sluggish response to light glabellar tap or loud auditory stimulus no response to light glabellar tap or loud auditory stimulus. duration of lid and globe akinesiatime of rescue analgesic given when vas 4 and total number of rescue analgesic given. duration of lid and globe akinesia time of rescue analgesic given when vas 4 and total number of rescue analgesic given. postoperatively, patients were monitored for pulse, blood pressure, spo2, sedation and complications (if any). the patients were asked to make a vertical mark on the line to indicate the intensity of their pain and vas was scored by measuring from the left side, how far the patient marked towards the maximum pain end. vas was recorded hourly. when vas of 4 postoperatively, rescue analgesia was given in the form of injection diclofenac sodium 1.5 mg / kg i.m. all the patients were observed for incidence of any side effects and complications such as nausea, vomiting, hypotension, bradycardia, globe perforation, oculocardiac reflex, and chemosis during intra- and post - operative period. table 1 shows demographic characteristics of the three groups. there was no statistically significant difference among three groups in terms of age, weight, sex, and duration of surgery. the mean time of onset of globe akinesia was significantly faster in group f (6.94 3.55 min) and c (6.70 3.3 min) as compared to group s (10.91 3.74 min). similarly, the mean time of onset of lid akinesia was significantly faster in group f (4.08 1.73 min) and c (4.03 1.72 min) as compared to group s (6.97 3.61 min). characteristics of the block mean duration of globe akinesia was 117.78 10.42 min in group s, 207.71 13.54 min in group f and 213.52 14.52 min in group c signifying longer duration of globe akinesia in group f and c as compared to group s. furthermore, the mean duration of lid akinesia was significantly longer in group f (143.14 7.86 min) and c (162.06 17.1 min) as compared to group s (87.64 9.76 min). group f (217.7143 12.67 min) and group c (258.82 14.50 min) had a longer duration of analgesia in comparison to group s (258.82 14.50 min). the mean duration of sensory blockage was longer in group f (79.71 7.64 min) and c (80.88 7.81 min) compared to group s (67.63 7.925 min). s, mean blood pressure ranges from 90.9 6.12 to 88.6 15.5, in group f from 90.1 4.92 to 88.3 3.49, and in group c, it ranges from 92.2 2.48 to 90.2 4.34. there is no statistically significant difference in mean blood pressure of the patients between the three groups (p 0.05). mean arterial pressure changes figure 2 shows the mean pulse rate changes during surgery. in group s, mean pulse rate ranges from 75.22 8.94 to 76.82 6.52, in group b from 78.62 6.25 to 80.34 6.34 and group c from 78.85 3.37 to 81.33 1.10. the result shows that there is no statistically significant difference in pulse rate of the patients between the three groups. of 35 patients in group s, 11 patients required first analgesic dose in 2 h and 24 patients required it in 3 h. in group f 34 patients required the first analgesic in 4 h and only one required it in 3 h. in group c, 33 patients required the first analgesic in 4 h and 2 required it in 5 h. these results show there is a longer duration of analgesia in group f and c as compared to group s. first analgesic requirement injection of local anesthetic mixtures in neural blocks may sometimes lead to unsatisfactory surgical conditions for relatively lengthy operations. local anesthetics act through blockage of selective sodium channels, on the other hand, opioids, act on opioid receptors and cause an increase in potassium conductance. hence, the combination of local anesthetic and opioid provides superior anesthesia by inhibiting multiple areas of neuronal excitability. its mechanism of action involves direct action on peripheral alpha 2 receptors and blockage of conduction of type c fibers. our study compares the effect of the addition of fentanyl and clonidine to local anesthetic solution in peribulbar block. results obtained in our study are comparable to study carried out by maha mi oussef.. they studied the effect of fentanyl versus that of clonidine when used as adjuvants to bupivacaine in peribulbar block and showed that the addition of either clonidine or fentanyl to the local anesthetic during peribulbar block results in a rapid onset and longer duration of the block with a longer period of postoperative analgesia (group f 262 min 9.71), group c 298 11.16 min). the addition of clonidine was found to prolong the duration of the block more than that of fentanyl. fahmy ng. studied the effect of adding fentanyl and/or clonidine to local anesthetic on prolongation of the peribulbar block in cataract surgery and concluded that the addition of clonidine alone to local anesthetic solution in peribulbar block prolonged the duration of block for up to 2 h only. the addition of fentanyl alone to local anesthetic solution could not prolong the duration of the block, while the addition of both clonidine and fentanyl to local anesthetic solution prolonged the duration of the block for up to 3 h. prolongation of analgesia by clonidine is also supported by the study carried out by bharti. in which he added 1 g / kg clonidine to local anesthetic mixture and found a significant increase in the duration of anesthesia and analgesia after peribulbar block. gupta and gurunath also showed that adding clonidine to local anesthetic mixture prolongs the duration of sensory blockage. our results are also consistent with the study done by abo el enin. in which they studied the effect of fentanyl addition to local anesthetic in peribulbar block and observed that addition of fentanyl to local anesthetic mixtures fastens the onset and prolongs the duration of akinesia and postoperative analgesia in peribulbar block. have also reported that addition of clonidine to 1% ropivacaine has no clinically significant benefit. apart from nausea and vomiting, chemosis was also reported in our study. a study by bharti further researches may be carried out for different doses of the adjuvants like clonidine and fentanyl to confirm the results of our study. compared the three doses of clonidine in peribulbar block and concluded that addition of 1 g / kg clonidine with anesthetic mixture significantly prolonged duration of analgesia and anesthesia after peribulbar blocks with limited side effects. the addition of fentanyl or clonidine as an adjuvant to local anesthetic solution in peribulbar block provides faster onset and prolonged duration of globe and lid akinesia and postoperative analgesia with stable intraoperative hemodynamics. | objective : to compare the effect of addition of fentanyl and clonidine as adjuvants to bupivacaine and lignocaine in peribulbar block.methods:the study was conducted on 105 adult patients of either sex, of asa grade i and ii undergoing ophthalmic surgeries. patients were randomly divided into 3 groups of 35 each. all the patients were given peribulbuar block with 5ml lignocaine 2% + 3 ml bupivacaine 0.5% + 1 ml hyaluronidase (250 iu). in addition to this 1 ml normal saline was added to group s, 25 g fentanyl to group f and 25 g clonidine to group c. onset and duration of globe and lid akinesia, duration of sensory blockage and analgesia, hemodynamic parameters, number of rescue analgesic and visual analogue score were recorded.results:the mean time of onset of globe and lid akinesia was significantly faster in group f and group c compared to group s, mean duration of globe and lid akinesia was longer in group f (207.71 + 13.54 and 143.14 + 7.86 min) and group c (213.52 + 14.52 and 162.06 + 17.1 min) compared to group s (117.78 + 10.42 and 87.64 + 9.76 min). the mean duration of analgesia was significantly longer in group f (217.71 + 12.67) and c (258.82 + 14.50 min) as compared to group s (131.39 + 9.63 min).conclusion : addition of fentanyl or clonidine as adjuvant to local anaesthetic in peribulbar block provides faster onset and prolonged analgesia compared to local anaesthetic alone. |
wernicke 's encephalopathy is an acute or subacute neurological disorder characterised by ocular abnormalities leading to ophthalmoplegia, ataxia and altered mentation due to thiamine (vitamin b1) deficiency. the patients he described suffered from mental confusion, eye movement disorders, and ataxia. in 1887, the russian psychiatrist sergei korsakoff published reports describing a syndrome of anterograde amnesia and confabulation. other conditions associated with undernutrition such as recurrent dialysis, hyperemesis, starvation, gastric plication and cancer may also cause wernicke 's encephalopathy. a 56-year - old woman with colon cancer was admitted to the hospital with fecaloid discharge from a laparotomy incision during the previous 3 weeks. the patient had been in excellent health until about 2 months earlier when she had been diagnosed with a left - sided colon cancer. she had been categorised as having stage iii adenocarcinoma of the colon, and adjuvant calcium folinate and 5-fluorouracil (5-fu) had been planned for 6 months. two weeks after the first cycle she was admitted to the hospital due to a discharge from an abdominal incision scar. on the day of admission her temperature was 37c, the pulse was 92 bpm, and respirations were 16 per minute. abdominal examination revealed a left lower quadrant fecaloid discharge located on the laparotomy incision of a previous surgery. she was ordered nil by mouth and parenteral fluids including dextrose in water were started. eight hours after starting parenteral nutrition, she suddenly developed epilepsy and loss of consciousness. upon neurological examination, complete blood count revealed wbc 9,760 per microliter, hb 9.8 g / dl, hct 31.1%, plt 280,000 per microliter. clinical chemistry showed a glucose level of 123 mg / dl, urea 27 mg / dl, creatinin 0.5 mg / dl, ast 38 iu, alt 41 iu, alp 128 iu, total bilirubin 0.41 mg / dl, na 138 meq / l, k 4.1 meq / l, ca 9.6 mg / dl and albumin 3.5 g / dl. bilateral, increased thalamic and periaquaeductal signal intensities suggested wernicke 's encephalopathy in light of the relevant history and physical findings of the patient (fig. 1). serum thiamine level was measured and reported as 19.8 pg / l (normal : 2575). this constellation of findings was consistent with wernicke 's encephalopathy precipitated by 5-fu infusion for metastatic colon cancer. rapid institution of thiamine treatment 100 mg per day reversed her symptoms gradually in about 10 days. her level of consciousness and cooperation improved and orientation in time and location was restored. her cycloplegia and nystagmus were no longer detectable after 10 days of treatment although ataxic gate persisted somewhat. subsequent mri scan of the brain showed complete resolution of the abnormalities detected one month previously (fig. it possesses a critical role in energy metabolism serving as a cofactor for key enzymes such as alpha - ketoglutarate dehydrogenase, pyruvate dehydrogenase, and transketolase in krebs cycle and pentose phosphate pathway of the cells. since thiamine is used in the final metabolism of carbohydrates and many amino acids, deficiency of this vitamin causes many disorders related to cardiovascular, neuromuscular, and gastrointestinal systems. diet deficient in thiamine for 23 weeks results in symptoms. since thiamine - dependent enzymes play a major role in cerebral energy utilization, thiamine deficiency results in necrosis in the medial thalamus and periaquaeductal gray matter and irreversible brain damage due to derangements in the blood - brain barrier. common causes include alcoholism, eating disorders, malnutrition, gastric bypass surgery, magnesium deficiency, hyperemesis gravidarum, disorders associated with prolonged vomiting, prolonged parenteral nutrition, hiv infection, chronic dialysis, and finally cancer. infections, electrolyte imbalances, cerebral metastasis, chemotherapeutics, and opioid overdose are common causes. as the incidence of wernicke 's encephalopathy in cancer patients is not known, a high index of suspicion is needed for diagnosis to prevent the morbidity and mortality associated with this condition. chronic malnutrition contributes to thiamine deficiency in cancer patients especially after prolonged nausea and vomiting associated with chemotherapy. tumors with a high proliferation index such as leukemias and sarcomas deplete thiamine rapidly especially after parenteral hyperalimentation or glucose load. in the present case, the presence of an enterocutaneous fistula may have exacerbated nutritional loss including thiamine, although there has been no report in the literature which associates wernicke 's encephalopathy with enteral fistulae. another interesting association is that the patient 's symptoms and signs were precipitated by 5-fu infusion, although we can not differentiate it confidently from wernicke 's encephalopathy precipitated by glucose infusion. however, wernicke 's encephalopathy associated with glucose infusion usually occurs after an average of 14 days after the start of glucose infusion. a literature search suggests that high - dose doxifluridine, ifosfamide, and 5-fu can cause encephalopathy resembling a wernicke - korsakoff syndrome. basu and dickerson concluded that cancer patients were unable to convert thiamine pyrophosphate, the active form of the vitamin, since the excretory level of thiamine was high, making a deficient state unlikely. we also detected a near - normal thiamine level in the blood of our patient. however, the gold standard test to detect thiamine deficiency is to measure red cell transketolase activity. this test is expensive and not widely available. basu. also showed in rats that treatment with 5-fu resulted in decreased liver and spleen concentrations of thiamine without affecting the urinary excretory levels supplemented with large doses of the vitamin. in conclusion, this case illustrates how thiamine can rapidly reverse neurologic signs and symptoms if recognized early even though the association with 5-fu is a rather rare entity in a patient with colon cancer. | wernicke 's syndrome, caused by thiamine deficiency, is most commonly associated with alcoholism but can also occur in patients who are malnourished or have malabsorption of nutrients for other reasons. since the classic triad of encephalopathy, nystagmus and ataxia occurs simultaneously in only 1033% of cases, a high index of suspicion is needed in any patient with confusion and memory loss. in this case report, we present a 56-year - old female patient with metastatic colon cancer complicated with enterocutaneous fistula. she developed wernicke 's encephalopathy precipitated by 5-fluorouracil infusion. replacement with thiamine rapidly reversed her neurologic symptoms and signs. |
the study was a collaborative effort of the disease control department of the burkina faso ministry of health and the world health organization. district - level aggregated surveillance data (weeks 117, 2012) and official population data were used. at the subdistrict level, population and surveillance data (weeks 116, 2012) the weekly alert and epidemic status for kombissiri and its subdistricts were determined by using the 2012 meningitis incidence thresholds and the 2014 revised guidelines, which have a lower alert threshold (4,9). in 2005, subdistrict zones were created in burkina faso to improve disease surveillance accuracy and timeliness ; districts were subdivided into zones of 30,000 persons (table). within these zones, population numbers are similar, unlike numbers in health facility (hf) catchment areas, which in kombissiri range from 440 to 25,000 persons. in burkina faso, zone data are infrequently analyzed, except in kombissiri (figure 1). defined as time between weekly attack rate crossed at least the alert threshold (5 cases / week/100,000 population) and descended below the alert threshold, (i.e., from alert to alert). defined as time between weekly attack rate crossed the alert threshold and reached the epidemic threshold (10 cases / week/100,000 population) (i.e., from alert to epidemic). defined as time between weekly attack rate crossed the epidemic threshold and descended below the epidemic threshold again (i.e., from epidemic to epidemic). defined as time between weekly attack rate crossed the epidemic threshold and reached maximum incidence (i.e., from epidemic to peak). # # a reactive immunization campaign with acwy polysaccharide vaccine was conducted during week 18, 2012. zones within kombissiri district, burkina faso. on a national level, 13 (21%) of the 63 districts in burkina faso n. meningitidis serogroup w caused 82.6% (384/465) of confirmed cases in these districts, in which no reactive vaccination against this serogroup had been recently conducted. kombissiri had the highest cumulative attack rate (car) and was affected longer than other districts. of 136 suspected meningitis cases in kombissiri, 44 (32%) were confirmed : 36 (82%) of those were caused by n. meningitidis serogroup w, 1 (2%) was caused by n. meningitidis serogroup x, and 7 (16%) were caused by streptococcus pneumoniae. the outbreak in kombissiri reached alert and epidemic thresholds during weeks 7 and 11, respectively ; a total of 9 weeks were spent in these phases (table). at the subdistrict level, alert and epidemic thresholds were reached at weeks 1 and 3, respectively, 6 and 8 weeks, respectively, earlier than at the district level. alert and epidemic thresholds were first crossed in zone 2, and contiguous zones were gradually affected from week 6 onward (figure 2). zones 1 and 2 (outbreak epicenters) were the only zones in the epidemic phase for > 2 continuous weeks. in these zones, the average alert and epidemic phases were longer than those at the district level (12.5 vs. 9.0 weeks), the preepidemic phase was shorter (2.0 vs. 4.0 weeks), the time to peak was longer (6.5 vs. 1.0 weeks), and the epidemic phase started earlier (week 3 [zone 2 ] and week 8 [zone 1 ] vs. week 11) and was longer (10 vs. 3 weeks) (table). the average car and peak incidence in zones 1 and 2 were higher than those for kombissiri (car 223.3 vs. 105.2 cases/100,000 population ; peak incidence 42.6 vs. 14.4 cases / week/100,000 population) and other districts (table). the levels were also higher than those in 2012 serogroup w epidemics with district - level documentation in the gambia (car 111 cases/100,000 population) and benin (car 123.7 cases/100,000 population ; peak incidence 16.7 cases / week/100,000 population) (10,11). if the 2014 recommendations for epidemic detection had been used in kombissiri, the district - level preepidemic phase would have been 4 weeks longer, reaching the alert threshold in week 3 rather than 7 (13 weeks total in alert and epidemic phases). at the zone level, in the outbreak epicenter, the alert phase would have begun 1 week earlier in zone 1 ; no change would have been seen for zone 2, which was in the alert phase since week 1. weekly meningitis alert status and epidemic status at the district (a) and subdistrict (zone) (b) level in kombissiri district, burkina faso, during epidemiologic weeks 116, 2012. during the 2012 serogroup w meningitis epidemic in burkina faso, localized subdistrict epidemics occurred before those identified at the district level. subdistrict epidemics were also of longer duration and greater intensity. at the subdistrict level, the epidemic spread from the 2 epicenter zones to other contiguous zones ; if a subdistrict - level epidemic risk assessment had triggered district - level interventions in kombissiri, meningitis surveillance and microbiologic testing could have been intensified (alert phase) and epidemic control measures could have been implemented (epidemic phase) up to 6 and 8 weeks earlier, respectively, by using epidemic response guidelines in use at the time and up to 2 weeks earlier by using 2014 guidelines. at the district level, the short time between crossing the epidemic threshold and attaining the epidemic peak (average < 1 week) leaves a limited window for effective intervention because reactive immunization has a moderate effect on natural epidemic evolution after an epidemic has peaked (12). in this epidemic, the time between crossing the epidemic threshold and reaching peak was much longer when characterized at the zone level (3 and 10 weeks, respectively, in the 2 outbreak epicenters). characterizing the epidemic risk at the subdistrict level with the 2014 alert threshold for a population of 30,000 yielded critical time gains, particularly during the epidemic preparedness phase. early interventions with longer implementation windows improve response efficiency and might have halted this epidemic before it spread throughout kombissiri. timelier, targeted interventions could potentially be mounted at district or even subdistrict levels if the epidemic risk is localized. too little evidence is available now to consider modifying the epidemic threshold ; lowering the threshold might trigger unnecessary resource - intensive interventions, using limited vaccine supplies. however, modifications should be reconsidered once dynamics of non serogroup a meningitis epidemics are better understood at the subdistrict level (4). the advantages of reducing the spatial scale of epidemic risk assessment were recognized when n. meningitidis a was driving the epidemiology of meningitis in africa and phenomena detected at hf level developed into large epidemics (13,14). however, outbreaks caused by non - a meningococcal meningitis serogroups have less resonant patterns, and analysis of the epidemic risk at a level within the subdistrict (e.g., hf level) may lack sensitivity (4). nevertheless, it is essential that good quality data be readily available at that level for finer analysis when needed. the infrequent use of subdistrict - level data is partly due to their limited routine availability. collection of subdistrict - level data was difficult when enhanced surveillance was the predominant approach for surveillance in the meningitis belt, but the transition toward case - based strategies (according to which meningitis cases are individually described at the hf level) should help fill this gap (9,15). | in 2012, neisseria meningitidis serogroup w caused a widespread meningitis epidemic in burkina faso. we describe the dynamic of the epidemic at the subdistrict level. disease detection at this scale allows for a timelier response, which is critical in the new epidemiologic landscape created in africa by the n. meningitidis a conjugate vaccine. |
addressing pulmonary function disorders is the most important measure in maintaining quality of life in stroke patients, especially in those who experience asymmetrical breathing due to impairment of respiratory muscles (such as the diaphragm) on the affected side1 and abnormal thoracic extension2. impaired pulmonary function limits physical activities3, and compromises pulmonary hygiene due to the inappropriate removal of mucus inside the airway, inducing complications such as pneumonia4. abnormal respiratory function reduces forced vital capacity (fvc), forced expiratory volume in the first second (fev1), and peak cough flow (pcf)4. methods used to increase respiratory ability include stretching the pectoralis major5, pursed - lip breathing6, and inspiratory muscular training7. among stroke patients undergoing rehabilitation, consequently, thoracic mobilization may increase trunk mobility9.thoracic movements are related to cervical movements10. therefore, thoracic and cervical mobilization may increase trunk mobility, improving respiratory function. however, few studies have examined the relationship between thoracic and cervical mobilization and respiratory function in stroke patients. this study aimed to conduct thoracic and cervical mobilization in stroke patients and to determine its effect on respiratory function. the subjects were 21 stroke patients admitted to m rehabilitation hospital in daegu metropolitan city, south korea. the purpose and scope of the study was explained, and informed consent was obtained according to the ethical standards of the declaration of helsinki. subject selection was based on the following : no previous respiratory disease, no diagnosis of lung disease based on radiography and physical examination, and no cognitive dysfunction (i.e., mmse - k scores exceeding 24) that could lead to impaired cooperation (i.e., due to severe aphasia or dementia). a physiotherapist blinded to the assessment results entered all demographic data into a computer program designed to perform stratified randomization. subjects were randomly divided into a control group (cg, n = 11), whose members did not undergo thoracic and cervical joint mobilization, and an experimental group (tcmg, n = 10), whose members underwent thoracic and cervical joint mobilization. both groups were simultaneously given physiotherapy treatments consisting of 30 min of exercise, 15 min of rehabilitation ergometer training, and 15 min of functional electrical stimulation. additionally, members of the tcmg had 30 min of thoracic and cervical joint mobilization. during thoracic mobilization, subjects were seated on a height - adjustable therapeutic table and asked to cross their arms. the therapist conducted flexion, extension, lateral flexion, and thoracic rotation while wrapping the patient s crossed arms with the therapist s left hand, and the therapist s right hand on the patient s thoracic vertebrae. the therapist wrapped the right arm around the patient s face, up to the back of the patient s neck, and put the hand on the segment to be mobilized. the segmental spinous process just below the segment to be mobilized was fixed with the left hand using the index finger and thumb. respiratory function was measured using a pulmonary function calculator in a micro spirometer (micro medical ltd., uk). we defined fvc as the amount of air that can be forcibly exhaled from the lungs after taking the deepest breath possible ; additionally, we defined fev1 as the volume of air that can be forced out in one second after taking the deepest breath possible. pcf was measured using a peak flow meter (cardinal health 232 ltd., uk) to measure cough capability. the subjects assumed the same posture as previously described, taking breaths as deep as possible followed by forcibly coughing as strongly as possible. a paired t - test was conducted to compare changes in the values of respiratory and cough function according to the length of time the groups had undergone mobilization. to test statistical significance, the significance level was set to 0.05. the results, which were statistically processed using pasw 18.0 for windows, are expressed as mean and standard deviations. the general characteristics of the subjects are listed in table 1table 1.general characteristics of the subjects (number or meansd)variablescontrol group (n=11)experimental group (n=10)gender (male / female)3/83/7paretic side (left / right)7/44/6age (years)76.58.776.510.2time since stroke (months)29.617.830.912.9height (cm)162.17.6160.06.5weight (kg)51.59.456.78.0. a paired t - test was conducted to compare respiratory function of the subjects before and after the exercise. these results are shown in table 2table 2.comparison of pre and post pulmonary function (meansd)groupvariablespreposttcmgfvc (ml)1,589.0323.21,692.0336.1fev1 (ml)1,275.0340.71,365.0324.7cgfvc (ml)1,531.8674.91,557.3608.6fev1 (ml)1,240.9545.21,280.0541.3tcmg : thoracic and cervical mobilization group, cg : control group, fvc : forced vital capacity, fev1 : forced expiratory volume at 1 second, p<0.05. however, the tcmg had a statistically significant increase in fvc, from 1,589.0323.2 ml to 1,692.0336.1 ml. however, the tcmg saw a statistically significant increase in fev1, from 1,275.0340.7 ml to 1,365.0324.7 ml. changes in pcf between the groups are shown in table 3table 3.comparison of pre and post coughing function (meansd)groupvariablespreposttcmgpcf (ml)149.065.7157.569.8cgpcf (ml)137.794.7141.494.8tcmg : thoracic and cervical mobilization group, cg : control group, pcf : peak cough flow, p<0.05. however, the tcmg showed a statistically significant increase in pcf, from 149.0 65.7 tcmg : thoracic and cervical mobilization group, cg : control group, fvc : forced vital capacity, fev1 : forced expiratory volume at 1 second, p<0.05 tcmg : thoracic and cervical mobilization group, cg : control group, pcf : peak cough flow, p<0.05 stroke patients generally have reduced respiratory function due to reduced thoracic space caused by reduced trunk mobility. this study sought to determine the effects of thoracic and cervical joint mobilization on respiratory capabilities of stroke patients. fvc and fev1, are well - known indicators of respiratory capabilities ; stroke patients are known to have low fvc and fev1 values due to damage sustained by the central nervous system s respiratory system regulators4, 12. in a previous study, the fvc values of healthy male and female subjects were 3,600 ml and 2,500 ml, respectively, while their fev1 values were 2,500 ml and 1,800 ml, respectively13. comparatively, our study subjects had lower fvc and fev1 values. the tcmg showed improved fvc and fev1following the exercise, indicating that thoracic and cervical mobilization can improve the respiratory function of stroke patients. thoracic mobilization increases facet joint sliding in the thoracic vertebrae and normalizes the joint capsule, thereby improving thoracic flexibility while expanding the thorax9. mobilization exercises help facilitate thoracic movement during inhalation, increasing thoracic expansion and improving pulmonary function. abnormal thoracic movements can induce cervical dysfunction15, and thoracic mobilization can increase cervical range of motion and reduce cervical pain16. therefore, not only has the cervical or thoracic spine been treated to correct deformation or relieve pain, but concurrent mobilization of the cervical and thoracic spine has also been attempted in recent years11. a recent report states that abnormal cervical and thoracic posture such as a forward - leaning head and kyphosis are related in stroke patients10. this study treated the cervical and thoracic spine concurrently, inducing thoracic expansion and increased pulmonary capability through an effective increase in movement. impaired cough capability following a stroke can trigger a higher incidence of aspiration and reduced cleaning ability in the cilia of the mucosa, increasing chest infection susceptibility4. a pcf of 300 l / min or higher is typical among healthy persons. at 160270 l / min, patients are vulnerable to viral infections. at less than 160 l / min, the self - cleaning action of the muco - ciliary pathways may be significantly impeded17. the subjects had pcf lower than 160 l / min., thus indicating low cough capability. thus, appropriate intervention is required to improve their cough capabilities. to improve cough capability, patients need to strengthen expiratory muscles, have an appropriate inhalation ability prior to cough, and have the ability to maintain a small airway to increase intrathoracic pressure18. the exercises used in this study can increase thoracic movement, helping patients inhale appropriately prior to coughing. these exercises can also facilitate expiratory muscle (e.g., the abdominal muscles) lengthening prior to contraction, increasing cough capability. the finding sindicate that these exercises improve thoracic movement, resulting in improved pulmonary and cough function.. future research on improvements in the pulmonary function of stroke patients is needed. a multifaceted study on trunk movements, posture, and respiratory functions should be undertaken. | [purpose ] this study aimed to conduct thoracic and cervical mobilization in stroke patients and determine its effects on respiratory function. [subjects and methods ] twenty - one stroke patients were studied. subjects were divided into a control group (control group, n=11) who did not undergo thoracic and cervical joint mobilization, and an experimental group (thoracic and cervical mobilization group, n=10) who underwent thoracic and cervical joint mobilization. forced vital capacity and forced expiratory volume in the first second, well - known indicators of respiratory capabilities, were measured. peak cough flow was measured as an indicator of cough capability. [results ] after the exercise, respiratory function in the thoracic and cervical mobilization group showed statistically significant improvements demonstrated by increases in forced vital capacity, forced expiratory volume in the first second, and peak cough flow. [conclusion ] the findings indicate that thoracic and cervical mobilization can improve the thoracic movements of stroke patients resulting in improved pulmonary function. |
a recent paper by martinez. in this journal reported the genome sequencing and analysis of five additional dermatophyte species, bringing the total number to seven (1). in this commentary, we will situate this report in the context of current dermatophyte genomics and speculate on the future of the field based on the advances made in aspergillus genomics after the first three aspergillus genomes were sequenced in 2005 (24). dermatophytes are a uniquely pathogenic group of fungi that cause most common fungal infections globally (5). dermatophytic fungi are contained within three genera, trichophyton, epidermophyton, and microsporum. in the united states alone, millions of individuals seek treatment for dermatophyte infections annually, translating into an economic burden estimated at $ 400 million per year (6). moreover, large - scale epidemics have been reported in american troops in conflicts in europe and an urban childcare center outbreak (7, 8). the knowledge surrounding the mode by which these pathogens cause disease is insufficient, perhaps due to lack of research utilizing modern molecular tools. due to this deficiency genetic tools have been underutilized in the characterization of these fungi, resulting in a lack of sequenced dermatophyte genomes and their pathogenicity (9). as noted, seven whole - genome sequences of dermatophyte species have now been generated (see the broad institute s dermatophyte comparative database at http://www.broadinstitute.org/annotation/genome/dermatophyte-comparative/multihome.html) : the nuclear genome and mitochondrial sequences of microsporum canis, microsporum gypseum, trichophyton equinum, trichophyton rubrum, and trichophyton tonsurans (1), as well as the availability of arthroderma benhamiae and trichophyton verrucosum genome sequences (10). in their comparative study, martinez. (1) report that the sequenced dermatophytes are enriched relative to other human - associated fungi with four gene families that contribute to their ability to cause disease, an observation that mirrors the original analysis of the first two dermatophyte genomes (10). these include (i) proteases, secreted to degrade skin, that reportedly act as virulence factors ; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to the skin niche ; (iii) secondary metabolites, compounds that act as toxins, immune system modulators, or signals in the interactions between fungus and host ; and (iv) a class of proteins (lysm) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host s immune response to the fungi. overall, these genome sequence identifications are important for revealing genome components that have the potential to further our understanding of the pathogenicity of dermatophytes. the availability of these sequence and analysis data will provide researchers large amounts of useful information that will provide power to studies aimed to decipher and interpret the molecular basis of host colonization, invasion, and specialization. the observations about the dermatophyte genomes are reminiscent of the observations made on the first three aspergillus genomes that were sequenced and analyzed. this is not surprising given that all dermatophytes and aspergilli belong to the same phylum, ascomycota. characterization and analysis of many virulence - associated traits in aspergillus species (1) may be useful in the search for such traits in dermatophyte genomes. additionally, aspergillus pathogens have been the subject of medically important research, targeting genes associated with replication cycles and secreted enzymes involved in secondary metabolite production. the genome sequences of aspergillus fumigatus, aspergillus nidulans, and aspergillus oryzae were reported in back - to - back nature papers in 2005 (24). shortly after that publication event, the sequence of aspergillus flavus was completed (11). a. fumigatus and a. flavus cause invasive aspergillosis in immunocompromised patients, an ability that positions them as the more important fungal pathogens in this group. all of these fungi but a. oryzae are environmental saprophytes whose niche is decaying plant material. a. oryzae, whose genome sequence revealed it to be essentially a derivative of ancestral a. flavus, has experienced centuries of human cultivation as a key ingredient in the production of sake, miso, soy sauce, and other japanese foods. at the time of the genome sequence publications, all but a. nidulans were presumed to be asexual. as was the case for dermatophytes, the genome analysis of these aspergilli revealed several striking features, including a surprising abundance of secondary metabolite biosynthetic gene clusters, a full set of sexual cycle genes even in the presumed asexual strains, and an abundance of secreted degradative enzymes. the aspergillus genomes inspired a burst of studies that leveraged these genome sequences, as overviewed in fig. 1 (1220). subsequent post - genome sequence studies have revealed the identity of numerous products of the secondary - metabolite biosynthetic clusters and the roles of some of them in a. fumigatus and a. flavus virulence ; have identified conditions for in vitro sexual cycles in a. fumigatus and a. flavus, which has led to genetic analysis studies of these organisms that are now under way ; and have supported studies of the roles of many of the secreted proteases and other degradative enzymes in virulence. in addition, multiple strains of a. fumigatus and a. flavus have been sequenced, with the total of a. fumigatus sequences completed or under way approaching 100 (http://gsc.jcvi.org/projects/gsc/a_fumigatus/index.php). the timeline highlights some of the numerous critical studies since the publication of the first three aspergillus genome sequences in 2005. the cited papers should be taken as representative, as no rigorous prioritization was imposed in selecting papers to highlight in the timeline. sexual reproduction has been suggested as a means to revamp the virulence of fungi via meiotic recombination, which increases the population diversity, and via mating on the human host. these may be associated with antifungal resistance or the rate of pathogenicity of dermatophytes (21). like the story of sex in a. fumigatus, some dermatophytes, which were once assumed to be asexual, based on these studies, prediction of unexposed sexual cycles can be assumed from the dermatophytes containing functional sex genes. recently, identification of the mating type locus (mat) of five dermatophytes (m. canis, m. gypseum, t. equinum, t. rubrum, and t. tonsurans) with comparable virulence were reported using bioinformatic tools (23). furthermore, successful mating of t. rubrum with arthroderma simii suggests that these species have the benefits of sex, including cross - species sexual recombination and adaptation, that may outweigh the efficiencies of an asexual clonal expansion (24). in the aspergillus species, the apn2 and sla2 genes, encoding a dna lyase and cytoskeleton protein, flank the mat loci (25). however, those mat genes for dermatophytes are essentially identical and linked on one side of the mat locus (23). the discovery and characterization of the mat locus of dermatophytes allows further studies in the pathogenesis to be explored. the importance of these proteins in avoiding detection by the host immune system is supported by the observation that during dermatophyte infection, defective or absent cell - mediated immunity predisposes the host to chronic or recurrent dermatophyte infection (26). previously, expression of hydrophobin has been demonstrated to inhibit immune recognition in a. fumigatus (27). dermatophytes a. benhamiae and t. verrucosum, both shown to activate human inflammatory infections, also display moderate expression of a surface hydrophobin gene, suggesting a possible role in immune response functions (10). discovery of the abundance of secondary - metabolite biosynthetic clusters in the aspergillus genomes has led to the identification of the products of many of these clusters and the roles of some of them in virulence. a similar abundance of these clusters has now been noted in the reported dermatophyte genome sequences. for example, melanin, which is an important virulence determinant in aspergillus (28), was also isolated from dermatophytes (m. canis, m. gypseum, t. equinum, t. rubrum, and t. tonsurans) in vitro and during infection, suggesting a similar role in aspergillus and dermatophyte pathogenesis (29). moreover, t. rubrum produces xanthomegnin, a toxin produced by aspergillus in culture and in the human host (30). transcriptome analysis revealed differential expression of secondary - metabolite genes during dermatophyte and aspergillus infections, underscoring their importance in the colonization of tissues and potentially in the manipulation of the host inflammatory response (30). future studies will undoubtedly leverage the genome sequences of these clusters in dermatophytes to identify their secondary - metabolite products and their potential specific roles in virulence. given the recent major progress in the development of broad - scale transcriptional and genome sequence - dependent analyses of dermatophytes (10, 3032) and a selection of functionally characterized genes (33, 34), full genome sequences will fulfill a critical urgency in the need to develop molecular genetic techniques to study these pathogens. molecular studies of dermatophyte genomics and pathogenicity have been undertaken in spite of the limited number of sequenced genomes. for example, vermout and colleagues used rna silencing as a potential functional genomics tool in m. canis to identify two proteases, sub3 and dppiv, coding for subtilisin and dipeptidyl peptidase, respectively (35). previous studies have also demonstrated the association of increased keratinase with increased disease symptoms in m. canis (36). several studies have used proteomics to characterize secreted and conidial proteins in t. rubrum, a. benhamiae, and m. canis (10, 37, 38), but these have been limited in number and applicability by the lack of genome sequence. now that they can be coupled to genome sequences, these and other omics methods, such as metabolomics, glycomics, and lipidomics, will be more powerful, and accordingly, will strengthen the understanding and characterization of dermatophyte pathogenesis. it is clear that the availability of additional dermatophyte genomes will accelerate and enhance molecular studies of these pathogenic fungi. it is therefore most appropriate to celebrate the publication of these new dermatophyte genomes and to note this event as a consequential milestone in the efforts to manage the terrible diseases caused by this group of fungi. the aspergilli and the dermatophytes are closely related, and the new dermatophyte genome sequences reveal features similar to those in the aspergilli. this observation suggests commonality in how these fungi survive and thrive in a mammalian host. specific features of the dermatophytes and aspergilli diseases such as invasiveness, fatality, and organ involvement have resulted in research communities with disproportionate funding support that favored more rapid advancement in the aspergilli than in dermatophytes. another important factor that favored the aspergilli was the strength of a. nidulans as a model organism and the mature community that had developed around this model prior to the genome sequence publications. however, studies of these two groups of fungi have been, and will continue to be, synergistic, with each community taking lessons from the other. we project that the rate of progress in dermatophyte genomic research will accelerate now in much the same way aspergillus research accelerated following the publication of the aspergillus genomes in 2005 and 2006. | abstractdermatophytes are a uniquely pathogenic group of fungi that cause most common fungal infections globally. the major cause of athlete s foot is trichophyton rubrum, a pathogen of human skin. a recent paper in this journal reported the sequencing and analysis of five additional genome sequences, including that of trichophyton rubrum. these five join the existing two additional genome sequences to bring the total to seven dermatophyte genome sequences, a notable milestone in the study of these fungi. these additional genomes set the stage for future genome - supported studies on the biology, pathogenicity, and host specificity of this important group of pathogens. to predict how this future might play out, we review the history of aspergillus genomics since the initial publication of the first three aspergillus genome sequences in 2005, an event that stimulated important studies of the pathogenic aspergillus species. from these 7 years of aspergillus history, we offer some speculation on the future of dermatophyte studies supported by the genome sequences given the similarities, differences, and relative levels of support for studies in these two groups of fungi and the diseases they cause. |
prior studies have confirmed the existence of extensive functional connectivity disruptions in schizophrenia patients, resulting in disorders of information processing in the brain and corresponding behavioral and psychological symptoms. for example, patients with schizophrenia have shown decreased interhemispheric coherence compared to normal controls. studies have also shown that coordination defects in the bilateral visual pathway were associated with a reduction of the schizophrenia patients abilities to process vocabulary. it has been demonstrated that antipsychotic agents can improve the functional connectivity of the brain in schizophrenia, and changes in the functional connectivity are related to improvements in psychotic symptoms, resulting in certain however, the relationship between the disease state of schizophrenia and the interhemispheric functional connectivity of the brain is largely unknown. in address this issue, we compared the interhemispheric functional connectivity among schizophrenia patients with complete remission, patients with incomplete remission, and healthy controls using a newly developed method of voxel - mirrored homotopic connectivity (vmhc). previous studies have shown that the corpus callosum, which connects the two hemispheres, is damaged in patients with schizophrenia, and this damage is relatively stable with little change across different disease states. accordingly, we hypothesized that vmhc would be reduced in schizophrenia patients and that the degree of this reduction may not be associated with the disease state of the schizophrenia, i.e., the reduction in vmhc may serve as one trait marker of schizophrenia. this study was conducted between january 2013 and october 2014, which was approved by the ethics committee of the tianjin anning hospital (no. 2012-h-007). all participants provided signed, informed consent. a total of 94 patients with schizophrenia and 55 healthy controls were included in this study. the diagnosis of schizophrenia was determined by two experienced psychiatrists according to the structured clinical interview for dsm - iv axis i disorders diagnostic criteria. the inclusion criteria were age between 18 and 60 years, and right - handedness. the exclusion criteria included magnetic resonance imaging (mri) contraindications, poor image quality, any neurological disorder, a history of traumatic brain injury, and a history of drug abuse. additional exclusion criteria for healthy controls included a history of any mental disorder and a first - degree relative with any mental disorder. the severity of psychiatric symptoms in the patients with schizophrenia was quantitatively assessed using the positive and negative syndrome scale (panss). the standard definition of clinically complete remission in this study was a panss score 60 and the absence of psychotic symptoms for at least 3 months before scanning, as judged by a clinician 's diagnosis. this study included 36 schizophrenia patients with complete remission and 58 patients with incomplete remission. one patient in the complete remission group and 8 patients in the incomplete remission group had never received any medication, and the rest of the patients were receiving atypical antipsychotic medications during the mri examinations. the antipsychotic dosages are reported in table 1 as the chlorpromazine equivalents were calculated based on clinically equivalent dosing estimates. for each schizophrenia patient, the chlorpromazine equivalent was estimated according to the antipsychotic drugs and dosages used in the latest week before the mri scan. demographic and clinical information of schizophrenia patients with complete or incomplete remission, and healthy controls f value ; value ; t value. with regard to the severity criterion, a score of mild (3) or less was required for all eight core symptoms of panss, including p1 delusions, p2 conceptual disorganization, p3 hallucinatory behavior, n1 blunted affect, n4 social withdrawal, n6 lack of spontaneity, g5 mannerisms / posturing, and g9 unusual thought concept. with regard to the time criterion, the symptom severity mentioned above must have been maintained for at least 6 months. incomplete remission : these criteria comprise a severity criterion and a time criterion. with regard to the severity criterion, a score of (4) or more was required for all eight core symptoms of panss. mri data were acquired using a 3.0-tesla mr scanner (discovery mr750, general electric, milwaukee, wi, usa). all participants underwent three - dimensional (3d) high - resolution t1-weighted structural imaging and resting - state functional magnetic resonance imaging (fmri) scanning. the brain volume sequence was applied in the 3d high - resolution structural imaging with the following parameters : repetition time (tr)/echo time (te)/inversion time (ti) = 8.2/3.2/450 ms ; flip angle (fa) = 12 ; field of view (fov) = 256 mm 256 mm ; matrix = 256 256 ; slice thickness = 1 mm ; and no gap in 188 sagittal slices. the resting - state fmri data were obtained using the gradient - echo single - shot echo planar imaging sequence, and the scan parameters were as follows : tr / te = 2000/45 ms ; fov = 220 mm 220 mm ; matrix = 64 64 ; fa = 90 ; slice thickness = 4 mm ; gap = 0.5 mm ; 32 interleaved axial slices ; and 180 volumes. all participants were asked to close their eyes, relax, move as little as possible, think of nothing in particular, and not fall asleep during the resting - state fmri scans. the resting - state fmri data were preprocessed using the statistical parametric mapping software (spm8 ; http://www.fil.ion.ucl.ac.uk/spm). the preprocessing procedures were as follows : (1) exclusion of the first 10 volumes, reflecting the time needed for the bold signal to reach the steady state and for the participants to adapt to the environment ; (2) time correction ; (3) realignment ; (4) nuisance covariates regression (including six head movement parameters, their first time derivations, and the signals of white matter, cerebrospinal fluid, and the whole brain) ; (5) the band - pass filter (frequency range of 0.010.08 hz) ; (6) spatial normalization of the resting - state fmri data to the standard montreal neurological institute space by combination of the segmentation registration of the individual high - resolution structural images ; and (7) spatial smoothing using a 6 mm 6 mm 6 mm gaussian kernel. the vmhc was calculated using the data processing assistant for resting - state functional mr imaging toolkit (dparsf, state key laboratory of cognitive neuroscience and learning, beijing normal university, china). after preprocessing, the fmri images were divided symmetrically into right and left brain hemispheres, with the center level as the plane of symmetry. for each case, pearson 's correlation coefficient was calculated for the time series of each voxel and its corresponding voxel in the mirrored hemisphere. to increase the normality of the distribution, fisher 's z - conversion was conducted for the correlation coefficients. the statistical analysis of the demographic and clinical data was carried out using spss version 19.0 software (spss, chicago, il, usa). one - way analysis of variance (anova) and chi - square test were used to test differences in age and gender, respectively, among the three groups. two - sample t - test was used to test differences in dosage of antipsychotic agent and disease duration between two patient groups. multiple comparisons were corrected using a false discovery rate (fdr) method with the corrected p < 0.05. the brain region with a significant vmhc difference was defined as the region of interest (roi), and the mean vmhc value in the roi was extracted for post hoc analysis, i.e., pair - wise comparisons across the three groups. in addition, the roi - based correlation analyses with panss scores were performed for the two patient groups using a partial correlation analysis while controlling for age and sex. this study was conducted between january 2013 and october 2014, which was approved by the ethics committee of the tianjin anning hospital (no. 2012-h-007). all participants provided signed, informed consent. a total of 94 patients with schizophrenia and 55 healthy controls were included in this study. the diagnosis of schizophrenia was determined by two experienced psychiatrists according to the structured clinical interview for dsm - iv axis i disorders diagnostic criteria. the inclusion criteria were age between 18 and 60 years, and right - handedness. the exclusion criteria included magnetic resonance imaging (mri) contraindications, poor image quality, any neurological disorder, a history of traumatic brain injury, and a history of drug abuse. additional exclusion criteria for healthy controls included a history of any mental disorder and a first - degree relative with any mental disorder. the severity of psychiatric symptoms in the patients with schizophrenia was quantitatively assessed using the positive and negative syndrome scale (panss). the standard definition of clinically complete remission in this study was a panss score 60 and the absence of psychotic symptoms for at least 3 months before scanning, as judged by a clinician 's diagnosis. this study included 36 schizophrenia patients with complete remission and 58 patients with incomplete remission. one patient in the complete remission group and 8 patients in the incomplete remission group had never received any medication, and the rest of the patients were receiving atypical antipsychotic medications during the mri examinations. the antipsychotic dosages are reported in table 1 as the chlorpromazine equivalents were calculated based on clinically equivalent dosing estimates. for each schizophrenia patient, the chlorpromazine equivalent was estimated according to the antipsychotic drugs and dosages used in the latest week before the mri scan. demographic and clinical information of schizophrenia patients with complete or incomplete remission, and healthy controls f value ; value ; t value. with regard to the severity criterion, a score of mild (3) or less was required for all eight core symptoms of panss, including p1 delusions, p2 conceptual disorganization, p3 hallucinatory behavior, n1 blunted affect, n4 social withdrawal, n6 lack of spontaneity, g5 mannerisms / posturing, and g9 unusual thought concept. with regard to the time criterion, the symptom severity mentioned above must have been maintained for at least 6 months. incomplete remission : these criteria comprise a severity criterion and a time criterion. with regard to the severity criterion, a score of (4) or more was required for all eight core symptoms of panss. mri data were acquired using a 3.0-tesla mr scanner (discovery mr750, general electric, milwaukee, wi, usa). all participants underwent three - dimensional (3d) high - resolution t1-weighted structural imaging and resting - state functional magnetic resonance imaging (fmri) scanning. the brain volume sequence was applied in the 3d high - resolution structural imaging with the following parameters : repetition time (tr)/echo time (te)/inversion time (ti) = 8.2/3.2/450 ms ; flip angle (fa) = 12 ; field of view (fov) = 256 mm 256 mm ; matrix = 256 256 ; slice thickness = 1 mm ; and no gap in 188 sagittal slices. the resting - state fmri data were obtained using the gradient - echo single - shot echo planar imaging sequence, and the scan parameters were as follows : tr / te = 2000/45 ms ; fov = 220 mm 220 mm ; matrix = 64 64 ; fa = 90 ; slice thickness = 4 mm ; gap = 0.5 mm ; 32 interleaved axial slices ; and 180 volumes. all participants were asked to close their eyes, relax, move as little as possible, think of nothing in particular, and not fall asleep during the resting - state fmri scans. the resting - state fmri data were preprocessed using the statistical parametric mapping software (spm8 ; http://www.fil.ion.ucl.ac.uk/spm). the preprocessing procedures were as follows : (1) exclusion of the first 10 volumes, reflecting the time needed for the bold signal to reach the steady state and for the participants to adapt to the environment ; (2) time correction ; (3) realignment ; (4) nuisance covariates regression (including six head movement parameters, their first time derivations, and the signals of white matter, cerebrospinal fluid, and the whole brain) ; (5) the band - pass filter (frequency range of 0.010.08 hz) ; (6) spatial normalization of the resting - state fmri data to the standard montreal neurological institute space by combination of the segmentation registration of the individual high - resolution structural images ; and (7) spatial smoothing using a 6 mm 6 mm 6 mm gaussian kernel. the vmhc was calculated using the data processing assistant for resting - state functional mr imaging toolkit (dparsf, state key laboratory of cognitive neuroscience and learning, beijing normal university, china). after preprocessing, the fmri images were divided symmetrically into right and left brain hemispheres, with the center level as the plane of symmetry. for each case, pearson 's correlation coefficient was calculated for the time series of each voxel and its corresponding voxel in the mirrored hemisphere. to increase the normality of the distribution, fisher 's z - conversion was conducted for the correlation coefficients. the statistical analysis of the demographic and clinical data was carried out using spss version 19.0 software (spss, chicago, il, usa). one - way analysis of variance (anova) and chi - square test were used to test differences in age and gender, respectively, among the three groups. two - sample t - test was used to test differences in dosage of antipsychotic agent and disease duration between two patient groups. multiple comparisons were corrected using a false discovery rate (fdr) method with the corrected p < 0.05. the brain region with a significant vmhc difference was defined as the region of interest (roi), and the mean vmhc value in the roi was extracted for post hoc analysis, i.e., pair - wise comparisons across the three groups. in addition, the roi - based correlation analyses with panss scores were performed for the two patient groups using a partial correlation analysis while controlling for age and sex. the three groups showed no significant differences in age (one - way anova, f = 0.249, p = 0.780) or gender (chi - square test, = 0.040, p = 0.980). the complete remission group and the incomplete remission group exhibited no significant differences in the dosage of antipsychotic agent (two - sample t - test, t = 0.051, p = 0.960) or the disease duration (two - sample t - test, t = 0.443, p = 0.659). the differences in vmhc among the three groups are shown in figure 1 and table 2. the vmhc was significantly different in the visual region (inferior occipital and fusiform gyri) and the sensorimotor region (paracentral lobule) across the three groups (p < 0.05, fdr corrected). pair - wise comparisons in the post hoc roi - based analysis showed that the vmhc of the visual and sensorimotor regions in schizophrenia patients with complete remission and incomplete remission was lower than that in healthy controls ; however, there was no significant difference between the two patient subgroups (p < 0.05, bonferroni corrected) [figure 2 ].. one - way analysis of variance shows brain regions with voxel - mirrored homotopic connectivity differences across the three groups. brain regions with significant differences in vmhc mni : montreal neurological institute ; vmhc : voxel - mirrored homotopic connectivity., there were no significant correlations between vmhc in the visual region and panss positive score (partial correlation coefficient [pr ] = 0.010, p = 0.957), negative score (pr = 0.006, p = 0.972), and general score (pr = 0.063, p = 0.725), and between vmhc in the sensorimotor region and panss positive score (pr = 0.349, p = 0.043), negative score (pr = 0.025, p = 0.887), and general score (pr = 0.121, p = 0.495). in schizophrenia patients with incomplete remission, we also found no significant correlations between vmhc in the visual region and panss positive score (pr = 0.051, p = 0.707), negative score (pr = 0.086, p = 0.530), and general score (pr = 0.158, p = 0.246), and between vmhc in the sensorimotor region and panss positive score (pr = 0.087, p = 0.526), negative score (pr = 0.051, p = 0.708), and general score (pr = 0.004, p = 0.976). the three groups showed no significant differences in age (one - way anova, f = 0.249, p = 0.780) or gender (chi - square test, = 0.040, p = 0.980). the complete remission group and the incomplete remission group exhibited no significant differences in the dosage of antipsychotic agent (two - sample t - test, t = 0.051, p = 0.960) or the disease duration (two - sample t - test, t = 0.443, p = 0.659). the differences in vmhc among the three groups are shown in figure 1 and table 2. the vmhc was significantly different in the visual region (inferior occipital and fusiform gyri) and the sensorimotor region (paracentral lobule) across the three groups (p < 0.05, fdr corrected). pair - wise comparisons in the post hoc roi - based analysis showed that the vmhc of the visual and sensorimotor regions in schizophrenia patients with complete remission and incomplete remission was lower than that in healthy controls ; however, there was no significant difference between the two patient subgroups (p < 0.05, bonferroni corrected) [figure 2 ]. one - way analysis of variance shows brain regions with voxel - mirrored homotopic connectivity differences across the three groups. brain regions with significant differences in vmhc mni : montreal neurological institute ; vmhc : voxel - mirrored homotopic connectivity. in schizophrenia patients with complete remission, there were no significant correlations between vmhc in the visual region and panss positive score (partial correlation coefficient [pr ] = 0.010, p = 0.957), negative score (pr = 0.006, p = 0.972), and general score (pr = 0.063, p = 0.725), and between vmhc in the sensorimotor region and panss positive score (pr = 0.349, p = 0.043), negative score (pr = 0.025, p = 0.887), and general score (pr = 0.121, p = 0.495). in schizophrenia patients with incomplete remission, we also found no significant correlations between vmhc in the visual region and panss positive score (pr = 0.051, p = 0.707), negative score (pr = 0.086, p = 0.530), and general score (pr = 0.158, p = 0.246), and between vmhc in the sensorimotor region and panss positive score (pr = 0.087, p = 0.526), negative score (pr = 0.051, p = 0.708), and general score (pr = 0.004, p = 0.976). in this study, we found that vmhc in the visual and sensorimotor regions was lower in schizophrenia patients with complete remission and incomplete remission compared to normal controls ; however, there was no significant difference between the two patient subgroups. our findings suggest that changes in the interhemispheric functional connectivity are unrelated to the disease state and may, therefore, serve as a trait marker of schizophrenia. dysconnectivity in the sensorimotor and visual pathways has been consistently reported in patients with schizophrenia, and it has been associated with behavioral and psychological symptoms of the disease. a prior study has shown that patients with schizophrenia exhibited reduced vmhc in the occipital lobes, thalamus, and cerebellum compared to normal controls, and the vmhc aberrations in the sensorimotor regions are associated with the symptoms of schizophrenia. a previous study also found that vmhc was decreased in the precuneus, precentral gyrus, superior temporal gyrus, occipital gyrus, and cerebellum in patients with first - episode and drug - naive schizophrenia, and vmhc reduction in the precentral gyrus was associated with positive symptoms. chang. reported that the altered vmhc in the default mode network, inferior frontal gyrus, and cerebellum was associated with auditory hallucination symptoms, and aberrant vmhc in the sensorimotor region has been found only in schizophrenia patients experiencing nonauditory hallucination. consistent with previous findings, we found aberrant vmhc in the sensorimotor and visual - related pathways in patients with schizophrenia. in regard to the association between vmhc and psychotic symptoms, previous studies have uniformly demonstrated correlations between vmhc and schizophrenia symptoms, but the brain regions associated with symptoms are not consistently reported. in this study, we compared the vmhc differences between schizophrenia patients with complete remission and those with incomplete remission. we found no vmhc difference between the two patient groups, suggesting that reduced vmhc may be a trait marker of schizophrenia. first, patients with first - episode and drug - naive schizophrenia were not available in this study ; therefore, our findings need to be confirmed by future studies investigating interhemispheric connectivity differences between patients with first - episode and drug - naive schizophrenia and recovered patients after medication administration. second, most of the enrolled patients were chronic and had followed long - term medication regimens, which may affect our results. finally, the criteria to distinguish between complete and incomplete remission were whether patients had experienced psychotic symptoms in the past 3 months. however, the time for determining clinical outcomes was relatively short, which may weaken the applicability of our study 's inferences. therefore, a long - term follow - up study should be adopted in the future study. in conclusion, interhemispheric functional connectivity in the sensorimotor and visual processing pathways was reduced in patients with schizophrenia. more importantly, the reduction of interhemispheric functional connectivity was unrelated to the remission state of the disease ; therefore, it may serve as a trait marker of schizophrenia. this study was supported by the grants from the national basic research program of china (973 program, no. 2011cb707801) ; the natural science foundation of china (no. 81501451, no. this study was supported by the grants from the national basic research program of china (973 program, no. 2011cb707801) ; the natural science foundation of china (no. 81501451, no. | background : previous studies have demonstrated interhemispheric functional connectivity alterations in schizophrenia. however, the relationship between these alterations and the disease state of schizophrenia is largely unknown. therefore, we aimed to investigate this relationship using voxel - mirrored homotopic connectivity (vmhc) method.methods:this study enrolled 36 schizophrenia patients with complete remission, 58 schizophrenia patients with incomplete remission and 55 healthy controls. the vmhc was calculated based on resting - state functional magnetic resonance imaging data. differences in vmhc among three groups were compared using one - way analysis of variance. a brain region with a significant difference in vmhc was defined as a region of interest (roi), and the mean vmhc value in the roi was extracted for the post hoc analysis, i.e., pair - wise comparisons across the three groups.results:vmhc in the visual region (inferior occipital and fusiform gyri) and the sensorimotor region (paracentral lobule) showed significant differences among the three groups (p < 0.05, a false discovery rate method corrected). pair - wise comparisons in the post hoc analysis showed that vmhc of the visual and sensorimotor regions in schizophrenia patients with complete remission and incomplete remission was lower than that in healthy controls (p < 0.05, bonferroni corrected) ; however, there was no significant difference between the two patient subgroups.conclusions:interhemispheric functional connectivity in the sensorimotor and visual processing pathways was reduced in patients with schizophrenia, but this reduction was unrelated to the disease state ; thus, this reduction may serve as a trait marker of schizophrenia. |
tachycardia, tachypnea, fever or hypothermia and leukocytosis or leukopenia are the hallmarks of systemic inflammatory response syndrome (sirs). although sirs is commonly associated with infectious etiologies, it also occurs in patients with noninfectious conditions, including trauma, burns, pancreatitis, anaphylaxis, adrenal insufficiency, pulmonary embolism, myocardial infarction, massive hemorrhage, and following cardiopulmonary bypass [2 - 4 ]. we describe a case of sirs associated with air embolism following the removal of a central line catheter. a 65-year - old male with adult immune deficiency syndrome (cd4cell count, 90), chronic obstructive pulmonary disease and hepatitis - related cirrhosis was admitted for a transjugular intrahepatic porto - systemic shunt procedure for recurrent bleeding from esophageal varices. the following afternoon, an internal jugular sheath used to gain access to the vena cava during the procedure was pulled in anticipation of discharge. approximately 20 min later, and against medical advice, the patient went to the bathroom, where he collapsed while attempting to defecate. vital signs showed tachycardia (heart rate, 96 beats / min), tachypnea (28 breaths / min) and arterial blood pressure of 170/100 mmhg. a physical examination revealed an unconscious man with a weak gag reflex, with sluggishly reactive, 3 mm pupils and who was able to withdraw to pain. a diagnosis of venous air embolism was made and the patient was taken to a hyperbaric chamber for treatment with 100% oxygen at 2.5 atm for 90 min. upon removal from the hyperbaric chamber, the patient 's blood pressure was 58/40 mmhg with a heart rate of 105 beats / min, and aggressive volume and vasopressor (norepinephrine) resuscitation was initiated. a pulmonary artery was inserted approximately 20 hours after the air embolism episode. as shown in fig. 1, initial hemodynamic measurements showed an elevated cardiac output at 8.2 l / min and a low systemic peripheral resistance of 460 dynes / s / cm. at that time the patient developed hematemesis and profuse bleeding both from a tongue laceration and from the internal jugular puncture site. the patient was transfused several units of packed red blood cells, fresh frozen plasma, platelets and cryoprecipitate. bleeding had stopped and volitional movement had returned to all extremities by the next morning. over the next 24 hours, the patient 's hemodynamic parameters normalized and vasopressor support was discontinued. forty - eight hours later the patient was weaned off the ventilator without difficulty and the antibiotics were discontinued. air embolism is defined as the entry of air into the vasculature, and it can occur during the insertion or removal of central venous catheters. for air to enter the venous circulation, there must be both a direct communication between the atmosphere and a noncollapsed vein and a pressure gradient favoring the passage of air into the circulation. the patient described in the present report met these criteria by having a patent lumen from skin to the central vein formed by the internal jugular sheath and by developing a pressure gradient while taking a deep inspiration immediately before or after a valsalva maneuver. moreover, this patient exhibited symptoms compatible with arterial air embolism, implying that a large portion of air sucked into the central veins found its way into the left ventricle and the systemic circulation. the pfo provided the mechanism by which venous air passed into the systemic circulation, a condition defined as ' paradoxical ' air embolism. a noteworthy aspect of this case was the patient 's physiological response to the acute embolic event. embolization of large quantities of air into the right ventricle usually results in pulmonary hypertension, a phenomenon that appears to be related to the release of endothelin-1 from the pulmonary vascular endothelium. the rapid increase in pulmonary artery pressure leads to right ventricular decompensation, to decreased left ventricular preload, and to a rapid decline in cardiac output with profound hypotension. these mechanisms may have been present immediately after the entry of air into the patient 's circulation, but a few hours after the episode of air embolism the patient 's hemodynamic and clinical condition (elevated cardiac output, decreased systemic vascular resistance, tachypnea, fever and diffuse intravascular coagulation) was compatible with the diagnosis of sirs. the rapidity of the patient 's recovery, as well as the lack of positive cultures, suggest that sepsis was not the cause of his hemodynamic decompensation, although this possibility can not be totally ruled out. instead, we hypothesize that entry of air into the systemic circulation through the pfo, a condition present in approximately 30% of the population, must have triggered the release of inflammatory mediators that resulted in sirs. perhaps owing to the episode of air embolism being a single, self - limited event, the systemic response in this patient was relatively short lived and resulted in no permanent organ dysfunction. inflammatory states associated with systemic air embolism have been described in animal models. air can form microscopic bubbles that disrupt microvascular flow, resulting in platelet aggregation and the release of plasminogen - activator inhibitor. this mechanism has been implicated as a trigger to the cascade of cytokines thought to be causative agents of sirs, among them interleukin-1 and tumor necrosis factor. the host response to these cytokines may include diffuse endovascular injury, microvascular thrombosis, organ ischemia, multiorgan dysfunction, and death. animal studies suggest that agents such as heparin and lidocaine attenuate the thrombo - inflammatory response of the endothelium to luminal air. in conclusion, the removal of an internal jugular vein sheath introducer in this patient, coupled with a deep inspiratory maneuver, allowed the passage of a clinically significant amount of air from the venous circulation to the systemic circulation through a pfo. the interaction of air with systemic arterial endothelium may have triggered the release of endothelium - derived cytokines, resulting in the physiologic response of sirs. air embolization can occur following the removal of a central venous catheter a patent foramen ovale (pfo) allows air to pass from the venous to the arterial circulation (paradoxical air embolism) air may trigger the release of cytokines by the arterial endothelium resulting in the development of the systemic inflammatory response syndrome (sirs) changes in hemodynamic parameters following the removal of the right internal jugular vein introducer (time = 0). there was an initial rise in blood pressure and in heart rate at the time of air entry into the circulation. this was followed by severe hypotension, which was treated with intravenous (i.v.) norepinephrine. at the time of insertion of a pulmonary artery catheter, approximately 20 hours after the removal of the introducer, | we describe a case of systemic inflammatory response syndrome associated with air embolism following the removal of a central line catheter, coupled with a deep inspiratory maneuver. the presence of a patent foramen ovale allowed the passage of a clinically significant amount of air from the venous circulation to the systemic circulation. the interaction of air with the systemic arterial endothelium may have triggered the release of endothelium - derived cytokines, resulting in the physiologic response of systemic inflammatory response syndrome. |
medicinal herbs have been used in folk medicine for millennia. simply, in recent times, scientific study of their effects has flourished. nevertheless, some of them can cause adverse effects or have the potential to interact with other medications ; moreover, there is little information on the potential risk to health of such herbs. based on their long - term use by humans, one might expect herbs used in traditional medicine to have low toxicity. it is known that green plants in general are a primary source of antimutagens as well as natural toxic agents, and many plants contain cytotoxic and genotoxic substances. recent investigations have revealed that many plants used as food or in traditional medicine have mutagenic effects and cytotoxic and genotoxic effects in vitro and in vivo assays [47 ]. this raises concern about the potential mutagenic or genotoxic hazards resulting from the long - term use of such plants. many plants contain mutagenic and/or carcinogenic substances [8, 9 ] and their use has been correlated with high rate of tumor formation in some human populations [8, 1013 ]. cupularia viscosa g. et g., dittrichia viscosa greuter) (compositae) ; common name, sticky fleabane is a perennial weed that is found in most of the mediterranean basin [1416 ]. i. viscosa has been used for years in folk medicine for its antiinflammatory, antipyretic, antiseptic, and antiphlogistic activities [18, 19 ], and in the treatment of diabetes. aqueous extracts of i. viscosa exhibit antifungal activity in vitro [21, 22 ] and it has been demonstrated that some of its organic solvent extracts are antibacterial. provided evidence for the antifungal activity in plant extracts made with organic solvents, including methanol, ethanol, ethyl acetate, acetone, chloroform, and n - hexane. in addition, this herb has been used in spanish folk medicine for treating gastroduodenal disorders. i. viscosa has antiulcerogenic effects, causes abortion [16, 27, 28 ], prevents zygote implantation in mammals, prevents growth of pathogenic fungi, has a strong antioxidant activity. i. viscosa contains some pharmacologically active compounds [32, 33 ] including sesquiterpenes, sesquiterpenes acids, azulenes, lactones, flavonoids, and essential oils which are isolated and identified in its leaves. currently, there is no published data on the cytotoxicity and genotoxicity of i. viscosa leaf extracts. the purpose of this study is to investigate cytotoxic and genotoxic effects of i. viscosa leaf extracts using the allium test. ethyl methanesulfonate (ems) (cas no : m-0880) was purchased from sigma (sigma chemical co., st louis, mo). i. viscosa specimens were collected from vicinities of ske - kuadas / aydn (turkey) during flowering (november 2007). zkan eren, botanist, department of biology ; university of adnan menderes and voucher specimen was deposited at the herbarium of department of biology, adnan menderes university. the extracts were prepared according to the traditional use in turkey (decoction) and we used in this study crude extracts of i. viscosa leaves. studying with crude extracts is appropriate because traditional medicinal herbs are generally used as crude extracts. the i. viscosa leaves were rinsed with water, dried in a ventilated oven at 55c for 24 h and subsequently milled to a fine powder by ground into fine powder using a kitchen blender. the powder was placed in small plastic bags (100 g each) and stored at 4c until use. the extract was prepared by boiling 20 g powdered plant material mixed with 200 ml distilled water for covered beaker (10% stock solution) for 5 min and, cooled to room temperature for 10 min. stock solution was diluted with distilled water to 2.5 mg / ml, 5 mg / ml, and 10 mg / ml concentrations. small bulbs (1.52.0 cm in diameter) of the common onion, a. cepa, (2n = 16) were purchased at a local supermarket. prior to initiating the test, the outer scales of the bulbs and the dry bottom plate, a series of six bulbs were placed in tap water (ph 7.3) for 48 h and then onion roots were treated with the leaves extracts at 2.5 mg / ml, 5 mg / ml, and 10 mg / ml concentrations of i. viscosa. the test tubes were kept in an incubator at 22 1c and the test samples were changed daily at the same time. several of the newly formed root tips were then cut from each bulb and examined for any visible morphological abnormalities. the bulbs with satisfactory root lengths (22.5 cm) were used in the study, while those with exceptionally long or short roots were discarded (on average 2 - 3 bulbs). tap water (ph 7.3) was used as a negative control [35, 36 ] and ethyl methanesulfonate (ems, 2.10 m) used as a positive control mutagen. ems has been used in a wide variety of biological test systems in studies of mutation effects [3739 ]. ems induces dna damage by a direct mechanism, acting at various sites as a monofunctional ethylating agent of nucleotides. after 24 h of exposure, several root tips were removed from the bulbs, fixed in 3 : 1 (v / v) ethanol : glacial acetic acid and stored overnight at 4c. the next day they were placed in 70% (v / v) aqueous alcohol and refrigerated until used. an average of five slides was made for each bulb using five roottips which hydrolyzed in 1 n hydrochloric acid (hcl) for 3 min and microscope slides were prepared by squashing the stained root tips in 2% (w / v) acetic orcein. five slide was prepared per bulb, and each slide was examined using olympus bx51 at a total magnification of 40 10. the following parameters were used for determination of cytotoxicity and genotoxicity : (i) the mitotic index (mi) was calculated as the ratio between the number of mitotic cells and the total number of cells scored and expressed as percentage and (ii) chromatin aberrations (stickiness, breaks and polar deviation) were used as endpoints for determination of cytogenetic effects and micronuclei (mnc) were scored in interphase cells per 1000 cells (after 72 h of exposure to the i. viscosa leaf extract samples, the root lengths were measured and used as an index of general toxicity. the results for mitotic index and root length are expressed as percent of the negative and positive control. visible morphological modifications, such as changes in root consistency and color as well as the presence of swelling (c - tumors), hooks or twists in the roots were also observed. data on physicochemical parameters, root length, root growth, and mitotic index and chromosomal aberrations were compared using analysis of variance (anova) to confirm the variability of the data and validity of results. differences between corresponding controls and exposure treatments were considered statistically significant at p <.05. the levels of the physicochemical parameters (root number and root length) are presented in table 1. this results show that all tested concentrations of i. viscosa leaf extracts caused significant inhibition in the growth of roots in comparison to negative control and positive control. the inhibition of root number and root length was greater with increasing concentrations of i. viscosa leaf extracts. measured average root length is 3.58 cm in negative control and 2.74 cm in positive control. however, average root length in 10 mg / ml treatment group was decreased significantly compared to that of the negative control (2.82 cm) (table 1). the root morphology was nearly normal during the negative control treatment, but at 2.5 mg / ml inula leaf extract, the roots appeared slightly yellow and at 5 mg / ml inula leaf extract, the roots appeared a slightly brown. at 10 mg / ml inula leaf extract, the roots morphology showed an obvious difference in its appearance in that it turned to brownish in colour. with the objective of investigating the possible mechanism involved in root growth inhibition, i. viscosa leaf extracts provoked strong inhibition of the mitotic index, where a statistically significant difference in relation to the control and the decrease in the mitotic index was positively correlated with increasing concentration of the i. viscosa leaf extracts (table 2). i. viscosa leaf extracts induced chromosome and cytological alterations both in treatment and control groups. an analysis of chromosome aberrations showed that most of the fragments detected in the different treatments were of chromosome type (figure 1(a)). the occurrence of chromosome fragments allows observation of statistically significant differences at i. viscosa leaf extracts. in addition to the chromosome fragments, sticky metaphase and polar deviations (wrong directions of chromosome movement) were also observed (figures 1(b) and 1(c)). in general, it was possible to observe an increase of different abnormalities as the i. viscosa leaf extracts concentration increased. in allium test, a strong toxic effect of i. viscosa leaf extract was observed, supported by great occurrence of sticky metaphases, leading to cellular death (mitotic index decrease). a statistically significant increase in total aberrant cells (p <.05) (aberrant cells include chromosome breaks, stickiness and polar deviation) as compared with the negative control (table 3), however, the highest value of aberrant cells is shown by the positive control. statistical analysis showed that the genotoxic activities of the i. viscosa leaf extracts induced micronuclei in the root tip meristem cells of a. cepa. micronucleus formation in 1000 cells per slide (mnc value) was also increased in extract concentrations compared with negative and positive control, which is statistically significant (p <.05) (figure 3(a)). the increase occurred in the positive control, inula1 and inula2 with respect to the negative control, and not in inula3 which is expected since mi is extremely low. in addition, cells with membrane damage (figure 1(d)), binucleated cells (figure 1(e)), and nucleus damage (figures 1(g) and 1(h)) were found in various frequencies. also, apoptotic cells (figure 1(f)) were detected in the group treated with i. viscosa leaf extract. moreover, ghost cells were detected in 10 mg / ml i. viscosa leaf extract treatment (figure 2(a)). in this study, toxic effects of i. viscosa leaf extract was evaluated by analyzing root growth and root morphology. the higher i. viscosa extracts caused an inhibition of root growth and there was a statistically significant difference between control groups. in addition, the i. viscosa extracts induced slightly yellow, slightly brown and brownish in coloration in roots. cyto- and genotoxicity were estimated by observing cytological parameters such as the mitotic index and number of chromosome abnormalities, including chromosome breaks, stickiness, and polar deviations. the mitotic index (mi) of a. cepa meristematic cells treated with the ems was significantly decreased (1.924% in comparison to negative control). significant inhibition in the onion roots treated with the i. viscosa extracts (3.200%, 1.984% and 0.088% compared to the negative control) (table 2). the decline of mi below 22% in comparison to negative control can have lethal impact on the organism, while a decrease below 50% usually has sublethal effects and is called cytotoxic limit value. mi measures the proportion of cells in the m - phase of the cell cycle and its inhibition could be interpreted as cellular death or a delay in the cell proliferation kinetics. reduction in the mitotic activity could be due to inhibition of dna synthesis or a blocking in the g2 phase of the cell cycle, preventing the cell from entering mitosis. mitodepressive effects of some herbal extracts, including the ability to block the synthesis of dna and nucleus - proteins, were reported earlier [47, 48 ]. several other herbal extracts have been reported to inhibit mitosis [7, 49, 50 ]. the decreased mi in a. cepa roots treated with i. viscosa leaf extracts is probably due to either disturbances in the cell cycle or chromatin dysfunction induced by an external factor, in this case, i. viscosa extracts- dna interactions. the results herein suggest that the tested i. viscosa leaf extracts concentrations have inhibitory, mito - depressive effects on root growth and cell division of a. cepa and it can prevent dna synthesis and the reduction in number of the dividing cells in roots produced by the cytotoxic effects of compounds found in i. viscosa leaf extracts. the observation of sticky metaphase reinforces the hypothesis of the toxic effect of i. viscosa leaf extracts. metaphases with sticky chromosome, loses their normal appearance, and they are seen with a sticky surface, causing chromosome agglomeration. stickiness has been attributed to the effect of pollutants and chemical compounds on the physical - chemical properties of dna, protein or both, on the formation of complexes with phosphate groups in dna, on dna condensation or on formation of inter- and intra chromatid cross links [5256 ]. chromosomal aberrations (ca) are changes in chromosome structure resulting from a break or exchange of chromosomal material. most of the ca observed in cells are lethal, but there are many related aberrations that are viable and that can cause genetic effects, either somatic or inherited. the presence of chromosome fragments is an indication of chromosome breaks, and can be a consequence of anaphase / telophase bridges [58, 59 ]. the induction of chromosome breaks, disturbances on microtubule assembly and cellular death can be related. our results showed induction of chromosome type of aberration in the cells treated with the i. viscosa leaf extracts. somehow the i. viscosa leaf extracts not only interfere with the cell cycle, but also affect chromatin organization or dna replication, causing chromosome breaks. frequencies of total chromosome aberrations increased significantly upon exposure to i. viscosa leaf extracts which indicate clastogenic activity (table 3). these results are in line with the results of many research groups that examined the effects of different medicinal herbs [7, 60, 61 ]. i. viscosa leaf extracts significantly induced the formation of mnc in a. cepa root cells at 2.510 mg / ml concentrations. frequencies of mnc increased in 2.5 mg / ml and 5 mg / ml i. viscosa leaf extract. however, mnc frequency decreased in a. cepa roots treatment at the highest i. viscosa leaf extract concentration (10 mg / ml), due to high cytotoxicity. the frequency of cells with micronuclei is a good indicator of the cytogenetic effects of tested chemicals. micronuclei (mn) often results from the acentric fragments or lagging chromosomes that fail to incorporate into the daughter nuclei during telophase of the mitotic cells and can cause cellular death due to the deletion of primary genes [62, 63 ]. recent studies have suggested mnc - induced effect of various plant extracts. in our recent study, we report mnc - induced effect of lavandula stoechas aqueous extract and ecballium elaterium fruit juices on a. cepa root tip meristematic cells [7, 64 ]. aqueous extract treatment on a. cepa root tip meristematic cells.akinboro and bakare reported mnc formation by treatment of some psychotria species extracts on a. cepa root tip meristematic cells. in this study, membrane damage cells was observed in groups treated with 5 mg / ml and 10 mg / ml i. viscosa leaf extracts. these results show that i. viscosa leaf extracts over certain concentrations may cause cytotoxicity as they cause membrane damage. maoz and neeman evaluated effects of i. viscosa extract on chitin synthesis in dermatophytes and candida albicans. they demonstrated that i. viscosa extract inhibited the growth of dermatophytes and c. albicans and caused a significant decline in chitin content. chemically speaking, chitin is closely related to chitosan (a more water soluble derivative of chitin). it is also closely related to cellulose in that it is a long unbranched chain of glucose derivates which replaces chitin in plants. in addition, it is needed to clarify whether the decline of cellulose is due to direct inhibition of the membrane enzyme, cellulose synthase, or due to damage of the whole membrane. the occurrence of binucleated cells is the result of prevention of cytokinesis or cell plate formation. microtubules have been implicated in cell plate formation and i. viscosa leaf extracts can be one of the involved factors, resulting in inhibition of cytokinesis. similar inhibition of cytokinesis cells were also reported by kaushik, borah and talukdar, and gmrgen.. on the other hand, ghost cells have been observed in various frequencies in this study for the first time (figure 2). ghost cell is a dead cell in which the outline remains visible, but whose nucleus and cytoplasmic structures are not stainable. it is a possibility that substances in the high concentrations (10 mg / ml) of i. viscosa leaf extract leading to nucleus damage and prevention of cytoplasmic structures resulted in ghost cells. in addition, i. viscosa leaf extracts induced dna damage and cell death and/or apoptosis in various frequencies in this study. this study shows for the first time the induction of cell death and/or apoptosis caused by high concentrations (5 mg / ml and 10 mg / ml). the cell death was induced by high concentrations of such as toxin, stress, heavy metals, chemicals and other. the authors suggested that cells undergo death after moderate stress. in this way, in this paper demonstrated that i. viscosa leaf extract induced cytogenetic alterations (cytoplasmic shrinkage, nuclear condensation, dna fragmentation, membrane blebbing, cytoskeleton alterations and appearance of apoptotic bodies) aid mainly cell death in root tips of a. cepa (figures 1(d), 1(f), 1(g), and 1(h)). the aim of this study is to determine the features of cell death in root tips of a. cepa induced by i. viscosa leaf extracts. finally, we conclude that when applied in high doses, i. viscosa leaf extract shows cytotoxic and genotoxic activity. we used in this study crude extracts of i. viscosa leaves. studying with crude extracts however, working with crude extracts also means working with complex mixtures of biologically active compounds. some of these compounds can be cytotoxic and/or genotoxic ; others can be cytoprotective and/or antigenotoxic. the results of this study suggest that, although i. viscosa has beneficial effects as a medicinal herb, it can cause serious problems and damage on cells when used improperly. | i. viscosa has been used for years in folk medicine for its anti - inflammatory, antipyretic, antiseptic, and paper antiphlogistic activities. in this study, cytotoxic and genotoxic effects of i. viscosa leaf extracts on the root meristem cells of allium cepa have been examined. onion bulbs were exposed to 2.5 mg / ml, 5 mg / ml, and 10 mg / ml concentrations of the extracts for macroscopic and microscopic analysis. tap water has been used as a negative control and ethyl methanesulfonate (ems) (2 102 m) has been used as a positive control. the test concentrations have been determined according to doses which are recommended for use in alternative medicine. there has been statistically significant (p <.05) inhibition of root growth depending on concentration by the extracts when compared with the control groups. all the tested extracts have been observed to have cytotoxic effects on cell division in a. cepa. i. viscosa leaf extract induces the total number of chromosomal aberrations and micronuclei (mnc) formations in a. cepa root tip cells significantly when compared with control groups. also, this paper shows for the first time the induction of cell death, ghost cells, cells with membrane damage, and binucleated cells by extract treatment. these results suggest the cytotoxic and genotoxic effects of the i. viscosa leaf extracts on a. cepa. |
the normal prostate luminal cells, which are the histological origin of most prostate malignancies, are physically separated from the stroma by the basal cells and basement membrane (bm). basal cells are joined by intercellular junctions and adhesion molecules, constituting a continuous sheet encircling luminal cells 1 - 2. the bm is composed of type iv collagen, laminins, and other molecules, forming a continuous lining surrounding and attaching to the basal cell layer 3 - 4. the epithelium is normally devoid of blood vessels and lymphatic ducts, and totally relies on the stroma for its metabolic and even survival needs. because of these structural relationships (fig 1), the disruption of both the basal cell layer and the bm is a pre - requisite for prostate tumor invasion or metastasis. it is a commonly held belief that human prostate carcinogenesis is a multistage process, progressing sequentially from normal to hyperplasia, to prostatic intraepithelial neoplasia (pin), and to invasive or metastatic stages 5 - 8. progression from pin to invasion is believed to be triggered by overproduction of proteolytic enzymes primarily by cancer cells, which cause degradation of the bm 9 - 10. these theories are consistent with results of studies in tissue cultures and animal models, whereas are hard to interpret the following critical facts : our previous studies revealed that some healthy men between 19 and 29 years old had a spectrum of proliferative lesions, including hyperplasia, pin, and incipient adenocarcinoma 11 - 13. recent studies detected a dna phenotype that is identical to that of invasive prostate cancer in some healthy men, and in morphologically normal prostate tissues adjacent to prostate cancer 14 - 17. a vast majority of pin express high levels of proteolytic enzymes, while only 10 - 30% of untreated pin progress to invasive lesions during patients ' lifetime 18 - 21. unfortunately, none of the current approaches could predict which pin lesions will progress 22 - 25. the only established approach to monitor pin progression is repeat biopsy 22 - 25, which is costly and painful. together, these facts argue that alternative pathways may exist, because if the linear model of tumor progression and invasion is universally applicable, the normal prostate tissues of healthy subjects should not harbor malignant structural abnormalities. since over 90% of prostate cancer related mortality result from invasion - related illness, and the incidence of pin could be up to 16.5%-25% in prostate biopsies 24 - 28, there is an urgent need to uncover the intrinsic mechanism of tumor invasion. promoted by the fact that the basal cell layer is the sole source of tumor suppressor p63 and maspin 29 - 32, and that degradation of basal cell layers is a pre - requisite for tumor invasion, our initial study examined the physical integrity of basal cell layers in 50 patients with co - existing pre - invasive and invasive prostate tumors. of 2,047 ducts and acini examined, 197 were found to harbor focal disruptions (the absence of basal cells resulting in a gap greater than the combined size of at least 3 basal cells) in their basal cell layers. the frequency of focal basal cell layer disruptions (fbcld) varied from none in 22 (44%) cases to over 1/3 of the ducts or acini with fbcld in 17 (34%) cases (table 1) 33. of the 17 cases with a high frequency of fbcld, the basal cell layer had several unique alterations that were not or rarely seen in their morphologically similar counterparts in other cases 33 - 44 : a. significantly reduced expression of tumor suppressor p63 : in sections double immunostained for p63 and ck34e12, an average of 87% of the basal cells in non - disrupted layers expressed both molecules, while only 59% of the basal cells in focally disrupted layers had p63 expression (fig 3 ; table 2). b. significantly reduced expression of proliferating cell nuclear antigen (pcna):in sections double immunostained for pcna and ck4e12, an average of 74% of the normal basal cells showed pcna expression, but only 51% of basal cells in disrupted basal layers were pcna positive (fig. 4 ; table 3). c. significantly elevated apoptosis and degeneration : of 78 ducts and acini with fbcld examined, 59 (75.6%) harbored apoptotic basal cells, compared to 9 (11.5%) in 78 similar structures with intact basal layers. under high magnification, basal cells near fbcld often had cytological signs of degeneration, including nuclear swelling, shrinkage, fragmentation, or rod - like structures of fused basal cells (fig. d. significantly elevated leukocyte infiltration : in sections double immunostained for ck 34e12 and leukocyte common antigen (lca), most ducts and acini with fbcld showed leukocyte infiltration, but most ducts and acini with non - disrupted layers had no leukocyte infiltration (table 4). e. a total loss of the expression of all basal cell phenotypic markers : in addition to focal alterations, the entire basal cell layer in some ducts and acini showed degenerative changes. these basal cell layers were morphologically distinct, surrounding pin or normal - appearing duct or acinar clusters (fig 7). all the basal cells, however, lacked the expression of basal cell specific markers (fig 8). in contrast, tumor cells in pin and normal - appearing duct or acinar clusters had enlarged nuclei and nucleoli. in addition, these basal cell layers were devoid of expression of pcna, in contrast to normal basal cells and associated tumor cells, which were strongly positive for pcna (fig. ducts or acini with altered basal cell layers had a significantly higher frequency of mast cell infiltration (fig 9 ; table 5). together, these findings suggest that the basal cell layers in these patients have significantly higher degenerative alterations. as the basal cell layer is the sole source of several tumor suppressors 29 - 32, degenerated basal cells are very likely to have impaired or reduced functions. in contrast to degenerative alterations in basal cells, luminal cells overlying fbcld showed several proliferative alterations that were not seen in their adjacent counterparts distant from the disruptions : a. significantly elevated cell proliferation : in sections double immunostained for basal cell phenotypic and proliferation - related markers, these clusters had a significantly higher proliferation index than their counterparts without fbcld, and most proliferating cells were located near fbcld (fig.10). of 78 ducts and acini with fbcld examined, 47 had clusters of multiple proliferating cells, compared to 8 in 78 morphologically similar ducts or acini with non - disrupted basal cell layers (table 5). b. significantly higher expression of malignancy- and tumor invasion - related molecules : elevated expression of prostate specific antigen (psa) and alpha - methylacyl - coa racemase (amacr), are consistently seen in cells overlying fbcld (fig.11. a & b), and also in normal appearing ducts that lacked the expression of basal cell phenotypic markers (fig. in contrast, adjacent cells within the same duct, and adjacent ducts with intact basal cell layers were largely negative (fig 11). c. physical continuity with, and morphological resemblance to, invasive prostate cancer : a vast majority of these normal appearing clusters were adjacent to, or blended within, invasive cancers. in some cases, cells overlying fbcld had significantly enlarged nuclei and nucleoli, and were in physical continuity with, or morphologically similar to, their adjacent invasive counterparts (fig 12). in some cases, multiple epithelial cell nests appeared to be budding from the same acinus or duct (fig.13). these budding cells had a higher proliferation and were similar to adjacent invasive cancer cells. the only difference was that budding cells had residual basal cells (fig 13, thin arrows). d. significantly elevated expression of tumor invasion - related genes : in studies of gene expression profiling, cell clusters overlying fbcld consistently showed significantly higher expression of cell proliferation, apoptosis, angiogenesis, immuno - response, and stem cell related genes (fig 14 ; table 6). the above alterations were consistently seen in all 17 cases with a high frequency of fbcld, while were only seen in 1 (9.1%) of the 11 cases with a low frequency of fbcld, and in none of the 22 cases with non - disrupted basal cell layers. together, these findings suggest that the physical and functional status of the basal cell layer significantly impact the biological presentation of associated epithelial cells. these findings also suggest that fbcld may represent a trigger factor for prostate tumor progression and invasion. to our best knowledge, the most likely reasons are : 1 the enzyme theory has dominated the direction of researches in the field, and the roles of basal cells have been ignored ; 2 these alterations could be seen only by double immunohistochemistry to simultaneously elucidate both basal cells and other markers. based on the above findings, we have proposed that these normal appearing duct or acinar clusters represent a population of maturation arrested tumor progenitors derived from monoclonal proliferation of genetically damaged primitive stem cells at early stages of the prostate morphogenesis probably by trauma, radiation, inflammation, or other factors. these clusters retain the potential for unlimited cell proliferation or multi - lineage differentiation, and could progress directly to invasive lesions through two different pathways : (1) clonal in situ transformation (cist), and (2) multi - potential progenitor mediated budding (mpmb). these pathways are likely to contribute to early onset of prostate cancer at young ages, and to biologically and clinically more aggressive prostate tumors. our hypothesized main steps are the followings : at the early stage of prostate morphogenesis, the prostate of these patients exposed to external or internal insults, such as radiation, carcinogens, localized trauma, inflammation, or other factors, which caused permanent damages in the dna structures of some primitive stem cells. localized dna structural damages caused the inactivation of, or defects, in basal cell renewal - related genes, which impaired the basal cell replenishment process to replace the aged or injured basal cells, resulting in a. localized dna structural damages also caused the inactivation of, or defects in, apoptosis-, or cell cycle control related genes in the luminal cell population, which allow these cells to escape from programmed death, to continuously proliferate, and to generate their own vascular structures. deregulated proliferation in epithelial cells and impaired self - renewal in basal cells resulted in the overstretch and disassociation of the basal cell layer and the bm, which lead to focal breakdown and degeneration of these two structures. the degradation products of degenerated basal cells and the diffusible molecules of the overlying luminal cells function as self - epitopes to attract migration and infiltration of immunoreactive cells (irc) or auto - antibodies into the affected sites. the direct physical contact between irc and degenerated basal cells results in the discharge of the digestive enzymes from irc, leading to the physical destruction of altered basal cell layers and the local basement membrane, resulting in a focal disruption in these structures. since the ep is normally devoid of both blood vessels and lymphatic ducts, and the basal cell layer is the sole source of several tumor suppressors, a fbcld in a given basal cell layer could lead to several focal alterations, including : a) a loss or reduction of tumor suppressors and the paracrine inhibitory functions, which allow the luminal cells to undergo elevated proliferation 45 - 49. b) alterations in the permeability for oxygen or growth factors, which selectively triggers the exit of stem or progenitor cells from quiescence, and favor proliferation of cells overlying fbcld 50 - 52. c) the exposure of luminal cells to different cytokines, which facilitates vasculogenic mimicry and tumor angiogenesis 53 - 54. d) the physical contact between luminal and stromal cells augments the expression of stromal mmp, facilitating epithelial - mesenchymal transition (emt) and cell motility 55 - 57. e) the physical contact between luminal and immunoreactive cells directly cause genomic or cellular damages that trigger a cascade reaction of malignant transformation 58 - 63. these alterations could individually or collectively trigger elevated proliferation in luminal cells near fbcld, which leads to the enlargement of fbcld and stretching - out of the residual basal cells. eventually, the entire basal cell layer becomes dissociated or degenerated (as those shown in figs. 7 - 13), which facilitates progression of these clusters directly to invasive prostate lesions through two different mechanisms : a) clonal in situ transformation (cist), in which the entire luminal cell population of these clusters completely lose their surrounding structures and transform directly to a new microenvironment that favors cell motility and epithelial - mesenchymal transition. although these cells might not possess all the properties of invasive cancer cells, the changed microenvironment may act as a second hit to trigger a cascade reaction of malignant transformation that rapidly alters the genetic and biochemical profiles of these cells. b) multi - potential progenitor mediated budding, in which a subset of cell clusters overlying fbcld retain the properties of primitive stem cells. thus, they are able to constantly produce new cells and their own vascular structures through budding (as those shown in fig.13), representing the source for biologically and clinically more aggressive prostate tumors. the above events may occur and progress immediately after the external or internal insults, leading to development of prostate cancer at young ages. on the other hand, cells of these normal appearing duct or acinar clusters may become maturation - arrested after a few cycles of cell divisions, and remain idle until a new insult 64, representing bad seeds for bad crops at later ages. based on our own and other findings, we had proposed that prostate tumor invasion is triggered by localized degeneration of the basal cells and leukocyte infiltration induced auto - immunoreactions, which selectively favor monoclonal proliferation and invasion of progenitor or stem cells overlying fbcld 43. our hypothesis differs from the traditional theories of tumor invasion in 5 aspects : (1) the stage of tumor invasion, (2) the cellular origin of invasive lesions, (3) the significance of immunoreactive cells, (4) the significance of stromal cells, and (5) the potential approaches for interventions and prevention of tumor invasion. our new hypothesis is the expansion of our previous one with the following new points of views : a. the preservation of large clusters of genetically damaged stem or progenitor cells : our previous hypothesis proposes that cell clusters overlying fbcld represent tumor stem or progenitor cells, which undergo a series of immunohistochemical and morphologic changes, and finally transform into invasive lesions 43. our current hypothesis suggests that it is also possible that multiple genetically damaged primitive stem or progenitor cells within the same site may form large duct or acinar clusters that harbor the same genetic defects. these clusters may be formed immediately after external or internal insults during the early stage of morphogenesis, and progress rapidly, leading to early onset of prostate cancer at young ages. these clusters could also become maturation arrested at patients ' early ages, while they retain the potential for unlimited proliferation and multi - lineage differentiation, representing bad seeds for bad crops at later ages. b. direct transformation of the entire duct or acinar cluster into invasive lesions : our previous hypothesis proposes that invasive cells are derived exclusively or preferentially from monoclonal proliferation of stem or progenitor cells overlying fbcld 43. our current hypothesis suggests that, in addition to monoclonal proliferation, it is possible that the entire duct or acinar cluster may directly transform into invasive lesions after the disappearance of all surrounding basal cells and the basement membrane. c. angiogenesis by genetically altered tumor stem cells : our previous hypothesis proposes that a subset of luminal cell clusters overlying fbcld are in direct physical continuity with vascular- or lymphatic duct - like structures, which allows them to progress directly to metastasis 43. our current hypothesis further suggests that some normal appearing duct or acinar clusters may retain genetically damaged primitive stem cells that could manufacture their own blood vessels or lymphatic ducts, which directly lead to metastasis. scientifically, it could lead to a new direction to explore novel approaches for early detection, intervention, and prevention of prostate tumor invasion. for example, as non - disrupted basal cell layers have significant inhibitory functions on epithelial cell growth, the development of therapeutic agents to stimulate basal cell growth or regeneration may provide a more effective approach for treatment and prevention of prostate cancer invasion. clinically, it could potentially bring the following benefits : better recognition of these clusters may avoid misdiagnosis and facilitate early interventions, which may significantly improve prognosis. double immunohistochemical staining to assess the physical integrity of the basal cell layer, or an quantitative measurement of basal cell degeneration - related molecules in the blood or biopsies, may facilitate early identification of individuals at greater risk to develop invasive lesions. as genetic alterations not only define the scope and extent of, but also precede, both biochemical and morphological alterations, elucidation of the genetic profile of these normal appearing duct or acinar clusters may lead to the identification of the specific molecules that trigger the initiation of prostate carcinogenesis, progression, and invasion. our technical approaches of assessing the physical and functional status of the basal cells may be used as a more reliable alternative for repeat biopsy to monitor prostate tumor progression and invasion. more importantly, our hypothesis may be also applicable to progression and invasion of other epithelium derived tumors. | it is a commonly held belief that prostate carcinogenesis is a multi - stage process and that tumor invasion is triggered by the overproduction of proteolytic enzymes. this belief is consistent with data from cell cultures and animal models, whereas is hard to interpret several critical facts, including the presence of cancer in healthy young men and cancer dna phenotype in morphologically normal prostate tissues. these facts argue that alternative pathways may exist for prostate tumor invasion in some cases. since degradation of the basal cell layer is the most distinct sign of invasion, our recent studies have attempted to identify pre - invasive lesions with focal basal cell layer alterations. our studies revealed that about 30% of prostate cancer patients harbored normal appearing duct or acinar clusters with a high frequency of focal basal cell layer disruptions. these focally disrupted basal cell layers had significantly reduced cell proliferation and tumor suppressor expression, whereas significantly elevated degeneration, apoptosis, and infiltration of immunoreactive cells. in sharp contrast, associated epithelial cell had significantly elevated proliferation, expression of malignancy - signature markers, and physical continuity with invasive lesions. based on these and other findings, we have proposed that these normal appearing duct or acinar clusters are derived from monoclonal proliferation of genetically damaged stem cells and could progress directly to invasion through two pathways : 1) clonal in situ transformation (cist) and 2) multi - potential progenitor mediated budding (mpmb). these pathways may contribute to early onset of prostate cancer at young ages, and to clinically more aggressive prostate tumors. |
triple - negative breast cancer (tnbc) is defined by the absence of estrogen receptor (er), progesterone receptor (pgr), and human epidermal growth factor receptor 2 (her2) expression, and has a highly aggressive nature. accordingly, recurrence usually develops between 1 and 3 years after the initial diagnosis and most deaths occur within 5 years. despite poor prognosis, tnbc is characterized by molecular mechanisms that have potential as therapeutic targets, including abnormalities in dna repair mechanisms, and a propensity toward neoangiogenesis. dna repair defects are characteristic of brca mutant cancers, which are present in 25% of patients with tnbc and confer sensitivity to dna damaging agents. bevacizumab is a target therapy for vascular endothelial growth factor, which suppresses tumor growth by inhibiting neoangiogenesis. moreover, in a large - scale clinical trial of metastatic tnbc patients, bevacizumab improved overall response rates (orr) and progression - free survival (pfs) in combination with conventional chemotherapy. accordingly, the combination of bevacizumab, paclitaxel, and carboplatin (bpc) has been approved for patients with progressive metastatic nonsmall cell lung cancers (nsclc), and significant improvements in survival rates have been demonstrated. in this report, we describe the successful use of bpc therapy in the treatment of metastatic tnbc in a woman with a brca2 germline mutation. complete remission (cr) was achieved without additional treatment and the patient remained disease - free after 5 years. our institutional review board considered the approval was not required for this case report since written informed consent was obtained from the patient. in june 2009, a 34-year - old woman was diagnosed with cancer of the right breast during her 28th week of pregnancy. her family included several members with breast cancer and 1 with prostate cancer, suggesting likely genetic dispositions. mastectomy was performed during the 29th week of pregnancy and axillary lymph nodes were resected. subsequent pathological examinations of the surgical specimen revealed an invasive ductal carcinoma of 28 20 mm with the diagnostic characteristics ly+, v+, nuclear grade 2, lymph node metastasis (level i 1/10), er - negative, pgr - negative, and her2-negative. the fetus was developing normally immediately before surgery, with appropriate - for - date fetal weight of 1552 g at 29 weeks of gestation. in august 2009, a caesarean section was performed at 35 gestational weeks and a male with no physical or neurological abnormalities was delivered (birth weight 2311 g and apgar scores of 7 and 8 at 1 and 5 min, respectively). although temporary artificial respiration was required, the infant 's development was normal and he was discharged from the hospital at 45 days after delivery. abdominal computed tomography scans of the patient were performed immediately after delivery, and multiple low - density masses were detected in both liver lobes (figure 1). subsequent abdominal magnetic resonance imaging indicated the presence of liver metastases of the breast cancer (figures 2 and 3). thus, a multidisciplinary discussion involving breast surgeons, hepato - biliary - pancreatic surgeons, oncologists, obstetricians, radiologists, pathologists, and nursing staff was held to formulate a management plan for the patient, and systemic therapy containing bevacizumab was prescribed. although the patient accepted the decision, bevacizumab had not been approved in japan for the treatment of breast cancer at that time (august 2009). because our hospital is licensed only to provide medical care that is underwritten by the national healthcare system therefore, the patient was referred to a facility that was not underwritten by the national healthcare system, and bpc therapy was initiated on august 27, 2009. upon commencement of treatment, lung, liver, kidney, and heart functions were all within normal limits, and the patient 's eastern cooperative oncology group performance status was 0. contrast - enhanced computed tomography images from before the start of bevacizumab / paclitaxel / carboplatin therapy in 2009. low - density masses are visible in both liver lobes : (a) low - density masses in s7, (b) low - density masses in s2 and s7, and (c) low - density masses in s8 (arrows indicate lesions). contrast - enhanced computed tomography images from after the completion of bevacizumab / paclitaxel / carboplatin therapy in 2014. metastatic lesions were replaced by visible cavitations in s7 (arrows indicate cavitations ; (a), which subsequently disappeared (b and c). magnetic resonance (mr) etoxybenzyl - mri images of hepatocytes ; low - intensity masses are visible in the same region shown in the computed tomography image ; low - intensity masses in s7 (a) and low - intensity masses in s2 and s8 (b). t2-weighted image ; high - intensity masses are visible in s7 and on the surface of the left lobe of the liver (c). therapy comprised intravenous administration of 6 courses of bpc over a 28-day cycle, and 2 mg / kg bevacizumab was administered on days 1 and 8, followed by 4 mg / kg on day 15. paclitaxel (80 mg / mm) was administered on days 1, 8, and 15, and carboplatin (area under the curve, 2.0 mg / ml per min) was administered on days 1, 8, and 15. neoplastic lesions had disappeared after completion of the sixth course of bpc and cavitation was observed in s7 of the metastatic lesions. side effects during therapy included grade 3 leukopenia, grade 1 peripheral neuropathy, general lethargy, and diarrhea, but no fever occurred during therapy. in 2014, the patient consented to direct genetic sequencing of the brca2 gene and a 6781delg mutation was identified. after completion of the sixth course of bpc therapy, no other additional treatment was given. at the most recent follow - up in 2014 (5 years after the start of treatment), no recurrence of liver lesions (figures 2 and 4) or new metastases to any other area were observed (figure 5) and the patient reported a good quality of life. magnetic resonance images from after the completion of bevacizumab / paclitaxel / carboplatin therapy in 2014. metastatic lesions were replaced by visible cavitations in s7 (arrows indicate cavitations ; a) and subsequently disappeared (b). full - length positron emission tomography image showing the absence of metastatic lesions. in 2009, when we began treating the present patient, it was accepted that conventional chemotherapy was less effective for metastatic tnbc than for other metastatic breast cancers, and resulted in worse prognosis. all treatment policies, including surgical approaches for liver metastases, were discussed by the multidisciplinary team. although no studies of bpc therapy for breast cancer were available, subgroup analyses in small - scale studies of platinum agents and targeted therapies indicated efficacy in the treatment of metastatic tnbc. moreover, the clinical safety of this regimen was sufficiently demonstrated in patients with metastatic and recurrent nsclc. the effects of platinum agents in brca mutant breast cancers have been examined in several reports that were published after completion of the present bpc regimen. these indicate that whereas platinum agents are not effective in all cases of tnbc, good results have been achieved in with the presence of brca1 mutations. because brca mutations are present in only a small proportion of breast cancer cases, relatively small numbers of these patients have been included in previous analyses. however, preoperative chemotherapy with cisplatin alone reportedly resulted in pathologic complete response rates of 90% and 100%, and a recent prospective study of cisplatin monotherapies demonstrated a preoperative pathologic complete response rate of 61% in 107 brca1-mutant patients. in another study of metastatic breast cancer patients with brca1 germline mutations, treatment with cisplatin alone yielded an orr of 80% and a complete response rate of 45%, even though 50% of patients had received prior chemotherapy. moreover, a previous study showed that anthracyclines achieved better results among patients with brca2-related breast cancers than among those with brca1-related breast cancers. although the data from this study were limited, brca1 and brca2 have similar dna repair functions and comparable ovarian cancer treatment effects were observed in patients with brca1 and brca2 mutations. in contrast, poly(adp - ribose) polymerase was specifically effective against brca2-related breast cancers. taken together, these studies suggest that brca1- and brca2-related breast cancers respond similarly to certain treatment regimens. previous analysis of 585 patients with metastatic tnbc from the athena study showed a response rate of 49%, a complete response rate of 10%, pfs of 7.2 months, and a 1-year survival rate of 60% after combined treatments with bevacizumab and taxane agents. moreover, in a meta - analysis of the e2100, avado, and ribbon trials, addition of bevacizumab to initial chemotherapies for tnbc patients led to improved median pfs (8.1 months), response rates (42%), and 1-year survival (71%) compared with chemotherapy alone. small - scale studies also report superior results using 3-agent approaches to tnbc, in which platinum agents were added to molecular targeted therapies and conventional chemotherapies as in the present case. specifically, in a phase ii clinical trial of preoperative chemotherapy comprising carboplatin, bevacizumab, and docetaxel for tnbc patients, a pathological complete response rate of 42% (n = 19) and a clinical response rate of 96% (n = 43) were achieved. another phase ii clinical trial of combined carboplatin, bevacizumab, and nab - paclitaxel treatments for patients with metastatic tnbc achieved a median pfs of 9.2 months, a clinical benefit rate of 94%, and a response rate of 85%, further indicating the benefits of bevacizumab co - treatments for patients with tnbc. the bpc regimen is reportedly effective in cases of progressive, metastatic, and recurrent nsclc, and although no previous studies report the effects of this regimen in breast cancer patients, platinum agents have been effective in patients with brca mutations. finally, the reported effectiveness of bevacizumab in combination with taxane and platinum agents for the treatment of tnbc cases provides further evidence of the beneficial effects of platinum agents. it is unclear whether the long - term cr (5 years after the start of treatment) in our patient reflects the efficacy of bevacizumab, the platinum agent, or the taxane agent, and to what extent synergistic effects are responsible. in a previous study, 4 brca1-positive metastatic breast cancer patients survived for 50 to 62 months after treatment with cisplatin, although progression occurred after 28 to 36 months in these patients, and additional chemotherapy was administered. in the present patient, cavitation in the liver appeared after the disappearance of metastases, potentially indicating the effects of bevacizumab. however, in the aforementioned meta - analysis of e2100, avado, and ribbon trials, no patients achieved a long - term (5-year) cr with first - line treatment alone, and second - line therapies were administered upon disease progression. moreover, median overall survival was 20 months among patients treated with first - line chemotherapy plus bevacizumab for liver metastases from her2-negative breast cancers. although surgical treatment was not selected for the present patient, recent retrospective studies support surgical approaches for patients with liver metastases of breast cancers, and indicate better outcomes of surgical treatments than chemotherapy alone. hence, surgery can achieve survival benefits and prolonged remission in patients with good responses to systemic therapy. accordingly, surgery may be considered in the event of recurrent liver metastasis in the present patient. no previous studies have investigated the use of bpc for the treatment of patients with tnbc associated with brca1/2 germline mutations. however, the present encouraging case warrants clinical studies of the efficacy of bpc therapy in this population. | abstracttriple - negative breast cancer (tnbc) is aggressive, with high risk of visceral metastasis and death. a substantial proportion of patients with tnbc is associated with brca mutations, implying that these tumors are sensitive to dna - damaging agents. we report successful treatment of a metastatic tnbc in a woman with a brca2 germline mutation using combined bevacizumab / paclitaxel / carboplatin (bpc) therapy. the patient was pregnant and had liver metastases, and a complete clinical response was sustained for approximately 5 years. mastectomy was performed during the 29th week of pregnancy, and the baby was later delivered by caesarean section. subsequently, multiple metastases in both liver lobes were detected using computed tomography and magnetic resonance imaging and the patient was treated with a bpc regimen, which led to complete disappearance of metastatic lesions in the liver. no additional treatment was provided, and after 5 years the patient consented to direct sequencing of brca2 and a 6781delg mutation was identified. at the most recent (5-year) follow - up, the patient was alive with good quality of life and no evidence of metastases.this finding suggests that bpc therapy might be considered a good therapeutic option for the treatment of metastatic tnbc in a woman with a brca2 germline mutation. |
unexpected findings were made during studies of the cell invasion mechanism of the protozoan parasite trypanosoma cruzi. this process was originally assumed to be similar to the entry mechanism of many bacterial pathogens, which mobilize the actin cytoskeleton of host cells in a phagocytosis - like process (galan and bliska, 1996). surprisingly, however, no polymerized actin was detected around recently formed t. cruzi containing intracellular vacuoles, and invasion was actually significantly enhanced by disruption of host cell microfilaments (tardieux., 1992). images of the invasion process in fibroblasts revealed a gradual accumulation of host cell lysosomes at the parasite entry site, and progressive fusion of these lysosomes with the plasma membrane as invasion proceeded (tardieux., 1992) these findings led to the suggestion that the cortical actin cytoskeleton, similar to what is observed in regulated exocytosis (trifar., 1992), acts as a barrier for lysosome recruitment and fusion, and trypomastigote entry. subsequent studies showed that fusion of lysosomes with the plasma membrane is required for t. cruzi entry into several cell types, and that the process is triggered by elevations in intracellular free ca concentration ([ca]i) induced by the parasite (burleigh and andrews, 1998). (a) phase - contrast image of a trypomastigote in the process of entering a hela cell. (b) immunofluorescence image of the same cell shown in panel a stained with antibodies against human lamp-1. the arrow points to the extracellular portion of the parasite, not yet surrounded by lamp-1containing membranes. the green line represents lysosomal membranes that are gradually incorporated into the vacuole, the small arrows indicate the direction of lysosome movement, and the large arrows indicate the direction of parasite movement. in addition to [ca]i transients, t. cruzi trypomastigotes also trigger camp elevation in host cells (rodrguez., 1999). this event also appears to play a key role in the invasion mechanism, as trypomastigote entry is markedly reduced in cells treated with adenylyl cyclase inhibitors (rodrguez., 1999). interestingly, camp elevation enhances ca - regulated exocytosis in several cell types (morgan., 1993 ; rodrguez., 1999). although the mechanisms responsible for this effect are not completely understood, facilitated vesicular transport / docking at the plasma membrane and removal of the physical barrier posed by the actin cytoskeleton may be involved (heuser, 1989 ; morgan, 1995). these findings reinforce the view that rearrangements in the cortical actin cytoskeleton of host cells are essential for the recruitment and fusion of lysosomes required for t. cruzi invasion. this series of intriguing similarities between the t. cruzi cell invasion process and ca - regulated exocytosis raised the possibility that the parasites might be taking advantage of a previously unsuspected property of conventional lysosomes, namely the capacity for mobilization to the cell periphery and fusion with the plasma membrane. as discussed below, a direct investigation of the ability of conventional lysosomes to exocytose in response to ca generated several lines of evidence in support of this view. a particularly intriguing finding, also further discussed below, is the involvement of ca - regulated lysosomal exocytosis in the repair of plasma membrane lesions. t. cruzi trypomastigotes trigger [ca]i elevation in host cells by an ip3-mediated pathway leading to ca mobilization from intracellular stores (rodrguez., 1996). this signaling process requires a parasite cytosolic serine peptidase, oligopeptidase b (opdb) (burleigh., 1997). interestingly, opdb - null trypomastigotes, although markedly deficient in both signaling and invasion, still show a residual capacity to elevate [ca]i and to enter host cells (caler., 2000). it is thus conceivable that the opdb - independent signaling activity is a consequence of direct permeabilization of the host cell plasma membrane by the parasite. indeed, earlier studies showed that trypomastigotes secrete a hemolytic protein with the capacity to form discrete channels on lipid bilayers (andrews and whitlow, 1989). it remains to be investigated if the ca influx triggered by the t. cruzi hemolysin plays a role in triggering a lysosome - mediated plasma membrane repair process, which would then be subverted by the parasite for gaining access to the intracellular environment. although the concept of regulated exocytosis of conventional lysosomes is relatively new, secretory properties have long been associated with lysosome - related organelles. as reviewed extensively elsewhere (marks and seabra, 2001 ; blott and griffiths, 2002), the regulated secretory compartments of several specialized cells have many properties in common with lysosomes. the most widely recognized examples are found in hemopoietic cells : -granules from platelets, azurophil granules from neutrophils, lytic granules from cytotoxic lymphocytes, and mast cell granules have acidified lumens and contain acidic hydrolases and lysosomal membrane markers. secretory lysosomes, were also shown to be accessible to tracers trafficking through the endocytic pathway (stinchcombe and griffiths, 1999). osteoclasts, which also belong to the hemopoietic lineage, show a dramatic reorganization of the lysosomal compartment, with translocation of lysosomal glycoproteins to the ruffled border membrane and secretion of lysosomal enzymes at the site of bone resorption (mostov and werb, 1997). there are, however, several examples of cells with secretory lysosomes that do not belong to the hemopoietic lineage. melanosomes, the melanin - containing granules that are transferred from melanocytes to keratinocytes, share several characteristics with conventional lysosomes, in spite of the existence of unique biogenetic steps (marks and seabra, 2001 ; raposo., 2001). in pulmonary alveolar type ii cells, the lysosome - related lamellar bodies are responsible for the ca - regulated secretion of surfactant (ashino., 2000). the acrosome of mammalian spermatozoa, another ca - regulated exocytic compartment, has also been considered to be a modified lysosome, owing to its acidic lumen containing a full set of acidic hydrolases (tulsiani., 1998). in addition, recent observations revealed an intriguing overlap between markers for lysosomes and weibel - palade bodies, the regulated secretory granules of endothelial cells (tulsiani., 1998). in response to injury, endothelial cells are activated by inflammatory mediators, such as thrombin or histamine, triggering exocytosis of weibel - palade bodies and release of von willebrand factor. interestingly, von willebrand factor, an adhesive protein involved in primary hemostasis, is also secreted by the lysosome - related -granules of megakayocytes and platelets (wagner, 1993). therefore, it is clear that a capacity for ca - regulated exocytosis is commonly associated with lysosomal properties, and that this occurs independently of cell lineage. the acidic lumenal ph of lysosomes and lysosome - related organelles may play a central role in the processing of specific secretory products, as suggested by the low ph requirement for the synthesis and polymerization of melanin in melanosomes (marks and seabra, 2001). the pathology of a group of autosomal recessive diseases of humans and mice that includes the hermansky - pudlak (hps) and chediak - higashi (chs) syndromes is usually attributed to the dysfunction of specialized lysosome - related organelles, mainly because of the common symptoms of oculocutaneous albinism (caused by the defective transfer of melanosomes to keratinocytes) and prolonged bleeding (caused by impaired secretion of platelet clotting mediators). however, it is important to note that there is ample evidence indicating that conventional lysosomes are also affected in these diseases. the most striking example is chs, caused by mutations in lyst in the mouse (beige) and chs1 in humans. although the function of the very large (> 400 kd) cytosolic protein encoded by lyst / chs1 remains elusive, it is clear that all cells in beige mice and chs human patients show an abnormal enlargement of lysosomes (ward., 2000). severe immunodeficiency, which is usually responsible for early death in humans, has been attributed to defects in the exocytosis of cytotoxic t lymphocyte lytic granules (baetz., 1995), and to delayed major histocompatibility complex (mhc) class ii due to the severity of chs in humans, not much information is available on additional lysosome - related disorders in these patients. in this respect, analysis of the symptoms associated with hps has been more informative and revealed many intriguing features. analysis of the various mouse models of hps has led to the identification of a series of genes clearly involved in organelle biogenesis and membrane traffic events (marks and seabra, 2001). because this syndrome is less severe than chs and more heterogeneous in clinical presentation, a number of intriguing symptoms have been reported. in addition to hypopigmentation and blood clotting defects, human hps patients (and the mice, in several instances) show a series of additional abnormalities, not all obviously related to specialized secretory lysosomes. importantly, accumulation of partially degraded proteolipids (ceroid / lipofuscin) is observed in the lysosomes of many different cell types, and secretion of lysosomal enzymes into the urine, a phenomenon normally observed in male mice, is greatly reduced. massive amounts of ceroid accumulate in the epithelium of kidney proximal tubule cells and other cell types of hps patients, a process proposed to be a consequence of defective secretion of lysosomal contents (swank., 1998). hps patients also develop a serious restrictive lung fibrosis, and occasional granulomatous hemorragic colitis, granulomatous gengivitis, and cardiomyopathy (swank., 1998). the pulmonary fibrosis has been attributed to the intracellular ceroid deposition in the lung, although it is conceivable that defective secretion of surfactant by the lysosome - related lamellar bodies is also involved. no obvious explanation, however, has yet been offered for the colitis, gengivitis, and cardiomyopathy observed in hps patients. in light of the recent evidence discussed below, it is tempting to speculate that these symptoms might be, at least in part, related to defects in lysosome - mediated repair of plasma membrane lesions. regulated secretion has generally been considered to be a specialization of only a few cell types. it therefore came as a surprise when a large component of ca - regulated exocytosis was detected in a variety of cells previously believed to only be capable of constitutive secretion, such as fibroblasts and epithelial cells (chavez. membrane capacitance measurements revealed a 2030% increase in the surface area of cho and 3t3 fibroblasts after elevation in [ca]i (coorsen., 1996). in cho cells, the diameter of the ca - regulated exocytic vesicles detected by capacitance measurements was estimated to be between 0.4 and 1.5 m in diameter (ninomiya., 1996), a size consistent with the dimensions of lysosomes in these cell types. interestingly, even in cells such as pc-12 and adrenal chromaffin cells, which contain classical ca - regulated secretory granules, an additional population of exocytic vesicles with distinct properties was detected by electrophysiological methods (xu. such studies reinforced the growing perception that most cell types contain a population of vesicles that can be mobilized for fusion with the plasma membrane upon elevation in [ca]i. detailed studies performed with normal rat kidney (nrk) cells in our laboratory identified these vesicles as conventional lysosomes (rodrguez., 1997). upon stimulation with 1 m ca, the lumenal domain of lysosomal membrane glycoproteins is exposed on the plasma membrane, and lysosomal contents are released extracellularly. in contrast, no significant increase in the traffic of early endosomal markers to the cell surface is observed under the same conditions. furthermore, only the lysosomally processed form of cathepsin d is secreted in response to ca, reinforcing the conclusion that mature lysosomes, and not biosynthetic carrier vesicles, are involved in this exocytic process (rodrguez., 1997). recent findings from our laboratory revealed that synaptotagmin (syt) vii, a ubiquitously expressed member of the syt family of ca - binding proteins, is a powerful tool for modulating ca - dependent exocytosis of lysosomes. syts are transmembrane proteins with a short amino terminus ectodomain, a single transmembrane region, and two highly conserved, independently folding ca - binding c2 domains (c2a and c2b) homologous to the c2 regulatory region of protein kinase c (sudhof and rizo, 1996). syt i, the most extensively studied isoform, is present on the membrane of synaptic vesicles in neurons, where it was proposed to function as a ca sensor for rapid exocytosis. genetic studies in mice, drosophila, and caenorhabditis elegans demonstrated that syt i ablation or mutation strongly decreases the ca dependency of neurotransmitter release (nonet., 1993 ; diantonio and schwarz, 1994 ; geppert., 1994). several syt isoforms have been described to date, some of which appear to be more abundantly expressed in brain (li., 1995). syt vii, however, was shown by hybridization studies to be expressed at significant levels on most mouse tissues (ullrich and sudhof, 1995). consistent with this ubiquitous pattern of expression, recent work from our laboratory showed that syt vii is localized on the membrane of lysosomes in nrk (martinez., 2000) and other cell types (caler., 2001). several lines of evidence indicate that the c2a domain plays a central role in the mechanism by which syts regulate ca - triggered exocytosis. antibodies generated against the syt i c2a domain, and recombinant peptides containing the syt i c2a domain, were reported to inhibit ca - triggered exocytosis when introduced into neuronal cells (schiavo., 1998). similarly, the c2a domain of syt vii or antibodies against this domain block ca - triggered exocytosis of lysosomes in permeabilized nrk cells (martinez., 2000). importantly, inhibition of lysosomal exocytosis was only observed in the presence of the syt vii c2a domain, and not the c2a domain of the exclusively neuronal isoform syt i. consistent with our previous evidence indicating that common molecular mechanisms regulate lysosomal exocytosis and cell entry by t. cruzi, the recombinant syt vii c2a domain and anti syt vii c2a antibodies also specifically inhibited host cell invasion by trypomastigotes (caler., 2001). the existence of a ubiquitous pathway for ca - regulated lysosomal exocytosis raised the question of what could be its physiological role in mammalian cells. interestingly, a series of studies in the last decade concluded that the repair of lesions on the plasma membrane of animal cells involves the ca - regulated delivery of intracellular membrane to wound sites. ca influx through plasma membrane disruptions triggers a high rate of vesicular exocytosis at the wound site, an event that is required for membrane resealing (mcneil and steinhardt, 1997). the mechanism by which exocytosis promotes resealing is still unclear, but it may involve the reduction in plasma membrane tension known to result from intracellular membrane delivery (togo., 2000). although this series of studies strongly suggested that a ubiquitous form of ca - regulated exocytosis was a necessary and rate - limiting step in plasma membrane repair (mcneil and steinhardt, 1997), the exact nature of the intracellular vesicles involved was not clear. in sea urchin eggs, resealing of plasma membrane disruptions was initially proposed to be mediated by cortical granules (bi., 1995), interestingly, yolk granules from eggs of several species contain acidic hydrolases, in addition to being accessible to tracers chased through the endocytic pathway (wall and meleka, 1985). labeling experiments using the dye fm-143 also implicated the endosomal / lysosomal pathway in the repair of injured endothelial cells and fibroblasts, although the vesicular population involved was not directly identified (miyake and mcneil, 1995). the capacity of conventional lysosomes to respond to [ca]i by fusing with the plasma membrane suggested that lysosomes might correspond to the exocytic vesicles involved in cell resealing. a recent study generated several lines of evidence in support of this view (reddy. lumenal epitopes of the lysosomal glycoprotein lamp-1 readily appear on the cell surface of wounded cells (fig. 2), in a process strictly dependent on the presence of extracellular ca (reddy., 2001). wounding in the presence of the inhibitory syt vii c2a peptides or antibodies inhibits both the surface appearance of lamp-1 and resealing of the plasma membrane. furthermore, microinjection of agglutinating antibodies directed against the cytosolic tail of lamp-1 (bakker., 1997) also impairs resealing, directly implicating lysosomes in the plasma membrane repair process (reddy., 2001). (a) a wounded nrk cell, containing cytosolic texas red dextran, is shown next to the track left on the coverslip by scratching the monolayer. (b and c) sequential confocal z - sections through the bottom (b) and middle (c) of a wounded 3t3 fibroblast. in all images, the green / yellow punctate staining corresponds to the lumenal epitope of lamp-1, recognized by a monoclonal antibody on the surface of nonpermeabilized cells. plasma membrane disruption appears to be a frequent event in mechanically active tissues (mcneil, 2002). previous studies in rodents showed that cells from the skin, gastrointestinal tract, and muscle are frequently injured, as indicated by the incorporation of membrane - impermeant molecules into their cytosol (mcneil, 2002). interestingly, the frequency of wounding in rat skeletal muscle cells was reported to increase proportionally with exercise (mcneil and khakee, 1992). primary skin fibroblasts, when moving in culture, also suffer frequent and rapidly reversible plasma membrane disruptions, during retraction of the trailing edge (chen, 1981). such lesions, believed to be caused by rupture of the focal adhesions formed between fibroblasts and the substrate, also occur extensively in the fibroblast - collagen - matrix model of wound contraction (lin., 1997). this is a model developed to study the regulation of wound contraction, a critical step in the healing of cutaneous lesions (grinnell, 2000). in this system, skin fibroblasts embedded in a three - dimensional matrix of polymerized collagen attached to a substrate (an environment that mimics the granulation tissue secreted by fibroblasts in cutaneous wounds) develop extensive stress fibers and strong focal adhesions. the tension generated by the fibroblasts under these conditions is believed to represent the force responsible for the contraction and closure of cutaneous wounds in vivo. this contraction event can be reproduced in an accelerated time scale when the collagen - embedded fibroblast matrices are mechanically detached from the substrate. after release, the floating matrix condenses rapidly, a result of isometric tension generated by the fibroblasts. during this contraction event, the majority of the fibroblasts contained in the matrix suffer synchronous plasma membrane disruptions, followed by rapid, ca - dependent resealing (lin., 1997). this system provided a very sensitive and quantitative assay for investigating the role of lysosomes in the repair of plasma membrane injuries suffered by primary cells. when human foreskin fibroblast - collagen - matrices were released from the substrate in the presence of agents that inhibit lysosomal exocytosis, release of the lysosomal enzyme -hexosaminidase was inhibited while leakage of cytosolic enzyme lactate dehydrogenase was enhanced. sustained inhibition of lysosomal exocytosis in this system caused significant loss in cell viability, strongly suggesting that this ca - regulated process has a physiological role in the maintenance of plasma membrane integrity. cells from mechanically challenged tissues probably suffer repeated cycles of plasma membrane wounding and repair. recent studies examining sequentially wounded cells found that brefeldin a, although not affecting repair of the initial wound, inhibits resealing of the second wound (togo., 1999). these results were interpreted as reflecting the involvement of distinct types of intracellular vesicles in the initial and subsequent waves of exocytosis repair. although this possibility can not be ruled out, these findings are also consistent with the involvement of lysosomes located at distinct cellular sites. it is conceivable that the initial wound could trigger an exocytic response from lysosomes located in the close proximity of the plasma membrane, while mobilizing an additional population from inside the cell to replace it. because brefeldin a also affects the morphology and functional properties of lysosomes (lippincott - schwartz., 1991), it might inhibit replenishing of the peripheral lysosomal population, and subsequent cycles of exocytosis repair. future studies using cells impaired in lysosomal exocytosis should be useful in clarifying this interesting issue. the findings discussed above illustrate well the enormous potential that the study of host pathogen interactions has for uncovering novel, surprising aspects of the physiology of animal cells. the investigation of a highly unusual form of host cell invasion by a trypanosome led to the discovery of ca - regulated lysosomal exocytosis, a widespread process with a central role in the mechanism of plasma membrane repair. these fundamental findings, in turn, significantly advanced our understanding of the strategy used by t. cruzi to invade host cells. it now appears that this parasite subverts a very basic housekeeping process for its own advantage : contact with the parasite triggers a repair response by host cell lysosomes, which are then hijacked for formation of the intracellular vacuole. exocytosis - mediated membrane repair is believed to represent a primitive form of secretion (mcneil and steinhardt, 1997) ; it is an intriguing idea that encounters with microbes may have played a key role in the selection of lysosomes for this role (reddy., 2001). | studies of the cell invasion mechanism of the parasite trypanosoma cruzi led to a series of novel findings, which revealed a previously unsuspected ability of conventional lysosomes to fuse with the plasma membrane. this regulated exocytic process, previously regarded mostly as a specialization of certain cell types, was recently shown to play an important role in the mechanism by which cells reseal their plasma membrane after injury. |
historically, because of visibility issues and the complicated relationship between the liver and its vasculature, hepatectomy has presented a challenge to the surgeon. laparoscopy for liver resection was first documented in the early 1990s, proving to be as safe as conventional open hepatectomy while retaining oncologic integrity. however, laparoscopy has limitations in transection, mobilization, and the ability to control bleeding. to overcome some of these shortcomings, robot - assisted approaches have been devised and implemented that broaden visualization from 2 dimensions to 3 dimensions and increase range of motion to 360 via the endowrist (intuitive surgical inc., sunnyvale, california). although minimally invasive approaches to surgery are known to decrease postoperative pain scores and length of hospitalization (loh), with the rising costs of health care, controversy continues to surround discussions of these approaches. the focus of this study is to evaluate the role of minimally invasive techniques in liver surgery as compared with a conventional open approach. we compared data related to operative time, perioperative complications, loh, surgical margins, mortality rates, and cost analysis to assess differing approaches. this is the first systematic review to include analysis of the robotic approach, reflecting trends in modern surgery. two independent reviewers conducted a systematic search of pubmed and embase on articles published until august 2013. the following medical subject headings were used to locate articles : liver robotic, hepatic robotic, hepatic laparoscopic, hepatectomy laparoscopic, and hepatectomy open. the inclusion criteria for articles were as follows : (1) articles comparing conventional open liver resection with either a laparoscopic or robotic approach ; (2) controlled clinical trials, multicenter studies, or randomized controlled trials ; (3) studies that reported outcomes of intraoperative and postoperative outcomes, including total operative time, estimated blood loss (ebl), loh, surgical margins, postoperative complications, and postoperative mortality rates ; and (4) studies that reported a measure of variance (standard error, standard deviation, or confidence interval [ci ]). the references of articles included in the analysis were manually searched for additional articles for inclusion. excluded from analysis were articles on resection of colorectal cancer with synchronous liver metastasectomy and articles not published in english. in instances in which research groups reported findings using shared patient populations, the earliest publication by that research group was included for analysis. the results from the 2 independent reviews were compared for accuracy, with disagreement resolved by consensus. to achieve completeness and to assemble the most representative patient database, series with limited sample sizes the primary outcomes of interest in this study were total operative time, ebl, loh, surgical margins, perioperative complications, and postoperative mortality rates. a cost analysis was included as a secondary outcome of interest. for continuous outcomes, mean net changes were calculated as primary outcomes, whereas for categorical outcomes, odds ratios (ors) were calculated to examine the treatment effect. dersimonian and laird random - effects models were used to pool mean net changes or ors across the studies. the presence of heterogeneity was assessed with the cochran q test, and the extent of heterogeneity was quantified with the i index. to assess publication bias, the begg rank correlation test was used to examine the asymmetry of the funnel plot, and the egger weighted linear regression test was used to examine the association between the mean effect estimate and its variance. in addition, sensitivity analyses were conducted by excluding each study in turn to evaluate its relative influence on the pooled estimates. all analyses were conducted using stata software, version 10 (statacorp, college station, texas). two independent reviewers conducted a systematic search of pubmed and embase on articles published until august 2013. the following medical subject headings were used to locate articles : liver robotic, hepatic robotic, hepatic laparoscopic, hepatectomy laparoscopic, and hepatectomy open. the inclusion criteria for articles were as follows : (1) articles comparing conventional open liver resection with either a laparoscopic or robotic approach ; (2) controlled clinical trials, multicenter studies, or randomized controlled trials ; (3) studies that reported outcomes of intraoperative and postoperative outcomes, including total operative time, estimated blood loss (ebl), loh, surgical margins, postoperative complications, and postoperative mortality rates ; and (4) studies that reported a measure of variance (standard error, standard deviation, or confidence interval [ci ]). the references of articles included in the analysis were manually searched for additional articles for inclusion. excluded from analysis were articles on resection of colorectal cancer with synchronous liver metastasectomy and articles not published in english. in instances in which research groups reported findings using shared patient populations, the earliest publication by that research group was included for analysis. the results from the 2 independent reviews were compared for accuracy, with disagreement resolved by consensus. to achieve completeness and to assemble the most representative patient database, series with limited sample sizes the primary outcomes of interest in this study were total operative time, ebl, loh, surgical margins, perioperative complications, and postoperative mortality rates. a cost analysis was included as a secondary outcome of interest. for continuous outcomes, mean net changes were calculated as primary outcomes, whereas for categorical outcomes, odds ratios (ors) were calculated to examine the treatment effect. dersimonian and laird random - effects models were used to pool mean net changes or ors across the studies. the presence of heterogeneity was assessed with the cochran q test, and the extent of heterogeneity was quantified with the i index. to assess publication bias, the begg rank correlation test was used to examine the asymmetry of the funnel plot, and the egger weighted linear regression test was used to examine the association between the mean effect estimate and its variance. in addition, sensitivity analyses were conducted by excluding each study in turn to evaluate its relative influence on the pooled estimates. all analyses were conducted using stata software, version 10 (statacorp, college station, texas). eight hundred seventy - three abstracts were identified, 867 of which were obtained via searches of 2 databases, with an additional 6 retrieved through manual searches of references. the final set of articles undergoing analysis was attained using the preferred reporting items for systematic reviews and meta - analyses (figure 1). of the 873 abstracts identified, 55 underwent full - text review and 49 articles are included in this meta - analysis, with 3 comparing laparoscopic liver resection with robotic hepatectomy and 46 comparing laparoscopic liver resection with a conventional open approach (table 1). preferred reporting items for systematic reviews and meta - analyses flowchart showing literature search and study selection. studies selected for meta - analysis lh = laparoscopic hepatectomy ; oh = open hepatectomy ; rh = robotic hepatectomy. the 49 articles analyzed represent a total of 3702 patients, with sample sizes ranging from 17 to 400 patients. the distribution of the patients was as follows : 60 in the robotic group, 1901 in the laparoscopic group, and 1741 in the open group. baseline patient demographic data, including sex, age, and body mass index, were well matched among groups (table 2) distribution of resection type by the 40 articles mentioning this characteristic is listed in table 3. demographic characteristics bmi = body mass index ; crc = colorectal cancer ; fnh = focal nodular hyperplasia ; hcc = hepatocellular carcinoma ; lh = laparoscopic hepatectomy ; oh = open hepatectomy ; rh = robotic hepatectomy. forty - six publications reported total operative length, with similar results among groups (figure 2a). the mean total operative time was 203.6 minutes, 203.9 minutes, and 234.8 minutes for the laparoscopic, open, and robotic groups, respectively. forest plots and pooled analyses of mean difference in length of hospitalization (a), operative time (b), estimated blood loss (c), and odds ratio of postoperative complications (d). there was no difference between minimally invasive approaches, and there was a statistically significant increase in blood loss in laparotomy cases as compared with laparoscopy cases, with a pooled net mean change of 152.0 ml (95% ci, 103.3200.8 ml) (figure 2b). the total number of conversions in the laparoscopic group was 106, which represents a 5.68% conversion rate to open surgery. in the robotic group, 9 cases required conversion to open surgery, representing a 15% conversion rate. laparoscopy showed a significantly higher rate of negative surgical margins (pooled or 1.06) as compared with laparotomy (pooled or 1.01). forty - four studies reported loh. as compared with patients undergoing the laparoscopic approach, those undergoing a conventional open approach had a significantly longer loh (pooled mean difference, 2.22 days ; 95% ci, 1.782.66 days) (figure 2c). postoperative morbidity, including wound infection, biliary leakage, pleural effusion, bleeding, fluid collection, incisional hernia formation, renal failure, and ascites or cirrhotic decompensation, was reported by 47 articles. for total postoperative complications, minimally invasive approaches showed similar results with a rate significantly lower than that of the open group (or, 0.49 ; 95% ci, 0.420.57) (figure 2d). specifically, minimally invasive approaches had lower rates of wound infections (or, 0.39 ; 95% ci, 0.220.68), incisional hernias (or, 0.20 ; 95% ci, 0.060.67), and ascites and cirrhotic decompensation events (or, 0.50 ; 95% ci, 0.290.87) than the open group. forty studies reported data on postoperative mortality rates. there were no statistically significant differences between laparotomy and minimally invasive approaches for rates of both in - hospital mortality (or, 1.01 ; 95% ci, 0.671.54) and postoperative mortality within 30 days of discharge (or, 0.88 ; 95% ci, 0.411.88). four studies reported cost differences between minimally invasive approaches and conventional approaches, with three comparing laparotomy with laparoscopy and one comparing a robotic approach with an open approach. these studies showed a nonsignificant trend of higher total operative costs of $ 334.10 (95% ci, $753.50$1421.60) for minimally invasive approaches. these researchers found a trend of higher total hospital costs in patients undergoing the conventional open approach of $ 3223 (95% ci, $474$692). of note, one additional article normalized cost values for both total operative costs and total hospital costs and was subsequently not included in the statistical analysis. eight hundred seventy - three abstracts were identified, 867 of which were obtained via searches of 2 databases, with an additional 6 retrieved through manual searches of references. the final set of articles undergoing analysis was attained using the preferred reporting items for systematic reviews and meta - analyses (figure 1). of the 873 abstracts identified, 55 underwent full - text review and 49 articles are included in this meta - analysis, with 3 comparing laparoscopic liver resection with robotic hepatectomy and 46 comparing laparoscopic liver resection with a conventional open approach (table 1). preferred reporting items for systematic reviews and meta - analyses flowchart showing literature search and study selection. studies selected for meta - analysis lh = laparoscopic hepatectomy ; oh = open hepatectomy ; rh = robotic hepatectomy. the 49 articles analyzed represent a total of 3702 patients, with sample sizes ranging from 17 to 400 patients. the distribution of the patients was as follows : 60 in the robotic group, 1901 in the laparoscopic group, and 1741 in the open group. baseline patient demographic data, including sex, age, and body mass index, were well matched among groups (table 2) distribution of resection type by the 40 articles mentioning this characteristic is listed in table 3. demographic characteristics bmi = body mass index ; crc = colorectal cancer ; fnh = focal nodular hyperplasia ; hcc = hepatocellular carcinoma ; lh = laparoscopic hepatectomy ; oh = open hepatectomy ; rh = robotic hepatectomy. forty - six publications reported total operative length, with similar results among groups (figure 2a). the mean total operative time was 203.6 minutes, 203.9 minutes, and 234.8 minutes for the laparoscopic, open, and robotic groups, respectively. forest plots and pooled analyses of mean difference in length of hospitalization (a), operative time (b), estimated blood loss (c), and odds ratio of postoperative complications (d). lh = laparoscopic hepatectomy ; oh = open hepatectomy. regarding ebl, 44 studies reported this variable. there was no difference between minimally invasive approaches, and there was a statistically significant increase in blood loss in laparotomy cases as compared with laparoscopy cases, with a pooled net mean change of 152.0 ml (95% ci, 103.3200.8 ml) (figure 2b). the total number of conversions in the laparoscopic group was 106, which represents a 5.68% conversion rate to open surgery. in the robotic group, 9 cases required conversion to open surgery, representing a 15% conversion rate. laparoscopy showed a significantly higher rate of negative surgical margins (pooled or 1.06) as compared with laparotomy (pooled or 1.01). forty - four studies reported loh. as compared with patients undergoing the laparoscopic approach, those undergoing a conventional open approach had a significantly longer loh (pooled mean difference, 2.22 days ; 95% ci, 1.782.66 days) (figure 2c). postoperative morbidity, including wound infection, biliary leakage, pleural effusion, bleeding, fluid collection, incisional hernia formation, renal failure, and ascites or cirrhotic decompensation, was reported by 47 articles. for total postoperative complications, minimally invasive approaches showed similar results with a rate significantly lower than that of the open group (or, 0.49 ; 95% ci, 0.420.57) (figure 2d). specifically, minimally invasive approaches had lower rates of wound infections (or, 0.39 ; 95% ci, 0.220.68), incisional hernias (or, 0.20 ; 95% ci, 0.060.67), and ascites and cirrhotic decompensation events (or, 0.50 ; 95% ci, 0.290.87) than the open group. there were no statistically significant differences between laparotomy and minimally invasive approaches for rates of both in - hospital mortality (or, 1.01 ; 95% ci, 0.671.54) and postoperative mortality within 30 days of discharge (or, 0.88 ; 95% ci, 0.411.88). eight studies included cost analyses and discussion on this outcome. of these, one was excluded because it was out of scope. four studies reported cost differences between minimally invasive approaches and conventional approaches, with three comparing laparotomy with laparoscopy and one comparing a robotic approach with an open approach. these studies showed a nonsignificant trend of higher total operative costs of $ 334.10 (95% ci, $753.50$1421.60) for minimally invasive approaches. these researchers found a trend of higher total hospital costs in patients undergoing the conventional open approach of $ 3223 (95% ci, $474$692). of note, one additional article normalized cost values for both total operative costs and total hospital costs and was subsequently not included in the statistical analysis. this meta - analysis of 3702 patients over a 14-year period yielded 49 pertinent studies showing minimally invasive approaches for hepatectomy to be as safe and efficacious as conventional laparotomy, with similar total operative times. minimally invasive approaches afford shorter loh, decreased ebl, and decreased postoperative morbidity. specifically, these approaches resulted in fewer incisional hernias, wound infections, and ascites or cirrhotic decompensation events and retained oncologic integrity. all approaches to liver resection resulted in similar mortality rates. in terms of cost, minimally invasive approaches required nearly the same amount of money in the operating room as the conventional approach but saved money over the entire loh. favorable operative outcomes, such as decreased ebl and lower rates of postoperative morbidity, lend credence to increased implementation of minimally invasive approaches. bile leaks and massive hemorrhages are two important perioperative considerations in hepatic surgery owing to the unique anatomic structure of the liver, with minimally invasive approaches showing decreased intraoperative blood loss and equitable postoperative bile leak rates. the observed lower ebl is likely multifactorial, owing to both hepatic vein tamponade from pneumoperitoneum and improved dissection via field magnification. furthermore, higher ebl and consequent blood transfusions are associated with increased postoperative morbidity, helping explain the lower rates of postoperative morbidity observed in this study. when immediate postoperative deaths were excluded, nonsignificant differences were found between laparoscopic hepatectomy and open hepatectomy for overall survival and for disease - free survival by all research groups except one. kandil found no difference in overall survival (p =.818) but found a significant difference in disease - free survival, with 100% 3-year survival in laparoscopic hepatectomy patients versus 71.4% survival in open hepatectomy patients (p =.03). of note, the operative indication for this research group was neuroendocrine metastasis, whereas the indications for the remaining groups were primarily hepatocellular carcinoma or colorectal cancer metastases (table 2). perhaps a survival advantage exists in this population of patients ; however, further studies are needed to establish the potential validity of this relationship. a focus of debate regarding implementation of minimally invasive surgery centers on cost. in comparing total operative costs and total hospital costs among groups, studies found that although operative costs were higher for laparoscopic groups, their hospitalization costs were lower because of shorter loh, which is intimately tied to postoperative morbidity, as well as decreased intensive care unit admission rates. only 1 article assessed comparative costs between robotic and conventional open approaches, finding increased operating room costs with the robotic approach. however, without discussion of total hospital costs, no conclusions can be drawn from that study regarding the potential financial tradeoff gained by implementing robotic intervention. further studies including the economic impact of minimally invasive surgery are needed to advance this discussion. minimally invasive approaches to surgery afford the surgeon increased visibility and the patient decreased loh, improved cosmesis, and decreased postoperative pain. colorectal metastases are a leading indication for hepatectomy, for which a majority of patients need repeat hepatectomy. minimally invasive approaches not only better facilitate reoperations in this patient population but also allow for simultaneous operations in colorectal cancer patients with synchronous hepatic metastases. although this study is comprehensive and is the most current evaluation of approaches to liver resection, there are several limitations and shortcomings to our study. first, the included studies are nonrandomized, retrospective studies, making them of moderate quality with increased selection bias. also contributing to selection bias was patient selection by the surgeon, wherein healthier patients more fit for surgery were more likely to undergo minimally invasive options, leading to more favorable postoperative outcomes. furthermore, patients selected for laparoscopic surgery may have had more easily resectable tumors, possibly contributing to their relative increase in negative margins. intimately linked to minimally invasive surgical outcomes is both the surgeon 's experience with the procedure and the volume of cases to which each care center is accustomed, neither of which was included in these studies, thereby prohibiting subanalysis. specifically, significant heterogeneity in reporting of resection outcomes, positive and negative versus r0r1, prevents subanalysis of this outcome. although 873 citations were initially identified, an overwhelming majority of these were out of scope, focusing on tangential topics relating to liver donations, radiofrequency ablation, and tumor staging. moreover, although these articles may have marginally touched on some of our primary outcomes, they neglected to contain data pertinent to this study. furthermore, patient overlap by research groups led to the exclusion of 5 articles from analysis, totaling 488 patient experiences that are not represented. the only statistically significant difference noted between minimally invasive approaches was a roughly 10% lower conversion rate to open surgery in the laparoscopic group as compared with the robotic group. with only 3 comparative studies including a robotic group, the ability to accurately ascertain any relationship to the robotic group further comparative studies that include robotic approaches are needed. at present, the limited volume is likely because of the financial investment and operative training required to implement robots into common surgical practice. to our knowledge, this review represents the largest, most current analysis of outcomes related to minimally invasive approaches to hepatectomy, with minimally invasive approaches showing improved postoperative morbidity, retained oncologic integrity, and potentially decreased economic burden to the health care system. furthermore, future research comparing the robotic approach with the laparoscopic approach, as well as assessing the cost associated with each approach, is warranted. | background : the aim of this study is to compare the safety and efficacy of conventional laparotomy with those of robotic and laparoscopic approaches to hepatectomy.database:independent reviewers conducted a systematic review of publications in pubmed and embase, with searches limited to comparative articles of laparoscopic hepatectomy with either conventional or robotic liver approaches. outcomes included total operative time, estimated blood loss, length of hospitalization, resection margins, postoperative complications, perioperative mortality rates, and cost measures. outcome comparisons were calculated using random - effects models to pool estimates of mean net differences or of the relative risk between group outcomes. forty - nine articles, representing 3702 patients, comprise this analysis : 1901 (51.35%) underwent a laparoscopic approach, 1741 (47.03%) underwent an open approach, and 60 (1.62%) underwent a robotic approach. there was no difference in total operative times, surgical margins, or perioperative mortality rates among groups. across all outcome measures, laparoscopic and robotic approaches showed no difference. as compared with the minimally invasive groups, patients undergoing laparotomy had a greater estimated blood loss (pooled mean net change, 152.0 ml ; 95% confidence interval, 103.3200.8 ml), a longer length of hospital stay (pooled mean difference, 2.22 days ; 95% confidence interval, 1.782.66 days), and a higher total complication rate (odds ratio, 0.5 ; 95% confidence interval, 0.420.57).conclusion : minimally invasive approaches to liver resection are as safe as conventional laparotomy, affording less estimated blood loss, shorter lengths of hospitalization, lower perioperative complication rates, and equitable oncologic integrity and postoperative mortality rates. there was no proven advantage of robotic approaches compared with laparoscopic approaches. |
in 2006, takahashi and yamanaka demonstrated that differentiated cells can be converted into induced pluripotent stem cells (ipscs) by the expression of four transcription factors - oct4, sox2, klf4 and c - myc - which have been termed yamanaka factors. from the perspective of basic cell biology, somatic cell reprogramming has radically altered our thinking on the plasticity of cell states. in addition, the derivation of ipscs from numerous normal and diseased cell sources has enabled the generation of patient - specific stem cells for eventual use in cell therapy and regenerative medicine. a number of alternatives and refinements to the original four - factor reprogramming method have been devised over the years. these have included ectopic expression of alternative reprogramming factors, such as nanog and lin28, manipulation of pathways that act as barriers to reprogramming, such as p53 and p21, transient expression of reprogramming proteins to avoid stable genetic modification, and inclusion of chemical inhibitors that increase the efficiency of the reprogramming process. however, reprogramming largely remains dependent on the delivery and exogenous expression of one or more of the original yamanaka factors. in a recent issue of cell stem cell, morrisey and colleagues report that ipscs can be generated solely through the expression of a set of mirnas, thereby avoiding all original yamanaka factors for the first time. this breakthrough is destined to expand our understanding of the pathways that drive reprogramming. using lentivirus - based expression of the mir-302/367 cluster to reprogram both mouse and human cells, morrisey and colleagues show that mirna - based reprogramming proceeds faster than with standard four - factor reprogramming. consistent with this finding, pluripotency genes such as sox2, nanog and rex1 are upregulated earlier in fibroblasts expressing the mir-302/367 cluster than in fibroblasts transduced with the four transcription factors. using a mouse line expressing a reporter gene with the oct4 promoter driving green fluorescent protein, the authors also show that the endogenous oct4 locus is reactivated to a greater extent following mirna expression than without mirna expression. this rapid induction of endogenous pluripotency genes in the majority of target cells results in a significantly more efficient reprogramming process, up to two orders of magnitude higher than standard four - factor reprogramming. the authors report that the mirna - based approach can reprogram up to 10% of the input cells, although this could be an overestimation as only morphological criteria, and not pluripotency marker expression, were used to quantify the efficiency of reprogramming of human fibroblasts. several mirna families are expressed exclusively and at high levels in embryonic stem cells (escs). esc - specific mirnas, such as the mir-290 and mir-302 families, are directly regulated by the pluripotency factors oct4, sox2 and nanog and are thus integrated into the core pluripotency network. these mirna families have important roles in esc self - renewal and pluripotency, as knocking out either of the two key enzymes in mirna biogenesis (dicer and dgcr8) leads to defects in esc proliferation and differentiation. inhibition of dicer and dgcr8 also decreases reprogramming efficiency, indicating that mirna biogenesis is essential to robust reprogramming. the mir-302/367 cluster used by morrisey and colleagues is composed of five mirnas, four of which (mir-302a - d) have the same seed sequence - a seven base pair stretch of nucleotides that determines target specificity. the mir-302 family belongs to a subset of mirnas referred to as esc - specific cell - cycle - regulating (escc) mirnas that regulate the g1-s transition and can rescue the cell cycle defect of dgcr8-null escs. another set of esc - specific mirnas, the mir-290 family, has been shown to substitute for c - myc expression during reprogramming. recent work has revealed that the mir-302 and mir-290 family members both target many genes in several pathways, such as cell cycle regulation (cdk1na, rbl2) and epithelial - mesenchymal transitions (rhoc and tgfbr2). many of these target genes are functionally important, as inhibiting them individually using sirnas or chemical inhibitors can also enhance four - factor reprogramming. the seed sequence of the other mirnas used in the new report, mir-367, is different from that of the mir-302 family. importantly, exclusion of mir-367 from the mirna cocktail abrogated reprogramming. in fact, without mir-367, endogenous oct4 is never activated, suggesting that this mirna either directly or indirectly regulates oct4 expression. given the wide variety of cellular processes targeted by these mirnas, it is likely that simultaneous suppression of multiple targets is key to their reprogramming ability (figure 1). genes involved in cell cycle progression, epithelial - mesenchymal transition (emt) and epigenetic regulation are among genes that are targeted by the mir-302/367 cluster, but there are probably multiple important targets yet to be determined. inhibition of hdac2 by vpa, in conjunction with the mirnas, is likely to enable reprogramming by promoting the activation of pluripotency genes. although mirna - based reprogramming did not require expression of any yamanaka factors, reprogramming of mouse cells with the mirnas did require the use of valproic acid (vpa), a histone deacetylase (hdac) inhibitor. interestingly, vpa and other hdac inhibitors have previously been shown to enhance four - factor reprogramming. using fibroblasts derived from hdac2 null mice, the authors demonstrate that the effects of vpa are entirely dependent on the presence of this protein. moreover, human fibroblasts express lower levels of hdac2 than mouse fibroblasts, which may explain why mirna - based reprogramming of human cells does not require vpa. as in all standard retro- and lentiviral - based reprogramming methods, exogenous lentiviral mirna expression is eventually silenced in the resulting ipscs, when the endogenous pluripotency genes become reactivated. because mirnas primarily act as repressors of gene expression through mrna degradation or inhibition of translation, it will be interesting to know how the endogenous pluripotency factors become activated during this process. in differentiated somatic cells these factors are kept silent by a combination of dna and histone methylations ; therefore, the mirnas must somehow prompt the removal of these repressive chromatin marks. a plausible scenario might be one in which the mirnas target the enzymes that maintain these epigenetic marks. inhibition of hdacs, which seem to be essential for mirna - based reprogramming, at least in mice, may then shift the balance towards histone acetylation and transcriptional activation. even then, how reprogramming is initiated in the absence of any strong transcriptional activator remains unresolved. apart from the fascinating biological questions raised by this report, mirna - based reprogramming has important practical implications. alternative methods of mirna delivery, such as transfections of mirna mimics, are worth pursuing as a way to generate ipscs with no genomic integrations. although there are non - integrating methods of delivering the yamanaka factors, the low efficiency of these approaches has hampered their wide adoption. as mirna - based reprogramming seems robustly efficient, even non - integrating methods such as mirna transfection may generate appreciable numbers of ipsc clones. if rapid and efficient reprogramming by transient mirna delivery becomes a reality, routine ips derivation for future clinical applications may rely entirely on this method. esc : embryonic stem cell ; hdac : histone deacetylase ; ipsc : induced pluripotent stem cell ; mirna : microrna ; vpa : valproic acid. | embryonic stem cell specific micrornas (mirnas) have previously been shown to enhance the efficiency of transcription - factor - based reprogramming. however, whether reprogramming could be achieved entirely by mirnas remained unclear. a recent report shows that the expression of the mir-302/367 cluster of mirnas can directly reprogram somatic cells without the use of any transcription factors. this new method raises interesting questions about the mechanisms of reprogramming and is likely to facilitate the generation of induced pluripotent stem cells for potential future clinical use. |
an efficient, stable and scalable hybrid photoelectrode for visible - light - driven h2 generation in an aqueous ph 9.2 electrolyte solution is reported. the photocathode consists of a p - type si substrate layered with a ti and ni - containing composite film, which acts as both a protection and electrocatalyst layer on the si substrate. the film is prepared by the simple drop casting of the molecular single - source precursor, [{ ti2(oet)9(nicl)}2 ] (tinipre), onto the p - si surface at room temperature, followed by cathodic in situ activation to form the catalytically active tini film (tinicat). the p - si|tinicat photocathode exhibits prolonged hydrogen generation with a stable photocurrent of approximately 5 ma cm2 at 0 v vs. rhe in an aqueous ph 9.2 borate solution for several hours, and serves as a benchmark non - noble photocathode for solar h2 evolution that operates efficiently under neutral alkaline conditions. |
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sarcomas of penis are very uncommon, representing less than 5% of malignant tumors in this area. ks is the most common sarcoma of the penis and the second one is leiomyosarcoma. primary presentation of ks on penis is not common, but more often observed in aids patients, whose lesions are " aggressive form ", and only approximately 2 - 3% cases have shown penile ks lesions as first manifestation of disease. the patient was 47 years old male, suffered from itchy penile papules which the first one had started to grow 5 years ago. the first manifestation of his disease had appeared by a violet sub - coronal papule (about 5 mm in diameter). during the last three months the lesion had extended rapidly and appeared multiple lesions similar to the first lesion, around the coronal region. he did n't have any history of fever, weight loss, or mucosal involvement. he had unprotected extra marital sex with a female partner but 2 years after initiation of lesions. the patient did n't have a history of chemotherapy, radiotherapy, hiv infection, dermatologic disorders and disease and medications with immune suppression. in his past medical history, there was no report of any kind of disease or surgery, but only he underwent surgical remove of right urethral stone, 7 years ago. on examination, there were popular indurate glandular and sub - coronal lesions which some of them were crusted. no inguinal or iliac nodes were palpable and other sites of skin and mucosal surface had not involved by tumor (figure 1, 2). laboratory tests include : cell blood count (cbc), haemoglobin, urine analysis and urine culture were normal. serology of hiv, hbv, hcv tests were negative in two times but the hhv8 was detected in tissue by pcr method in biopsy sample. wedge biopsy of the lesions had shown a tumor which was composed of spindle cells, around the blood spaces and ectatic capillaries. fibrotic connective tissue with hemosiderin deposition and mild infiltration of lymphoplasmacytic inflammatory cells had enveloped spindle cells and blood spaces. the abdominopelvic ultrasonography had shown renal stone in the middle calix of left kidney with mild fullness and other findings were normal. computed topographic scan (ct scan) of thorax, abdomen and pelvic were normal too. classic forms have often occurred in elderly patients, black equatorial africans, patients with lymphoma or immune deficiencies. primary kaposi sarcoma of penis often has seen in hiv infected patients and in patients with competent immune system, it has occurred in elderly patients. according to some studies, primary presentation of ks on penis is reddish - purple to bluish nodules. other types of lesions such as papules, plaques and wart like lesions are less common [4 - 9 ]. ks is a proliferative disease, has characterized by angiogenesis, endothelial spindle cell growth (ks cells), inflammatory cell infiltration, and edema. these lesions have reflected immune dysregulation characterized by cd8 + t - cell activation, production of th1 cytokines, and angiogenic factors. treatment needs to be individualized, based on the patient 's clinical and immunologic status. highly active anti - retroviral treatment (haart) results in clinical improvement of ks lesions and prolongation of time to treatment failure anti - ks activity of haart appears to reflect immune system reconstitution and, to a lesser extent, suppression of hiv replication. hiv protease inhibitors are also potent anti - angiogenic molecules that could affect ks pathogenesis. localized, cutaneous ks lesions could be treated using radiation, laser, cryotherapy, or intralesional injections of antineoplastic medicines. cytotoxic chemotherapy has indicated for patients who have not responded to haart and patients with life - threatening or visceral disease. in some studies, palliative excision of lesions of ks on penis without chemotherapy after oncologic consult and evaluation of the patient 's condition, we planed radiotherapy for our patient. | primary kaposi sarcoma of penis is very rare. we will introduce a 47 years old male patient referred to our clinic from dermatology service, in this report. the patient suffered from itchy penile papules around coronal region. the lab tests had revealed a negative serology of hiv but tissue pcr was positive for human herpesvirus-8 (hhv8). histological findings were compatible with kaposi sarcoma. primary kaposi sarcoma of penis is rare but could occur in hiv negative patients. |
receptacle consisting of the basal and the distal portions, 125~130 m long ; the basal portion composed of two superposed one celled layers, forming basally a blackish foot, 75 m long ; cell i 30 m long (including the foot), 10~13 m thick, brownish or blackish translucent, cylindrical ; cell ii 20~40 m long, 15 m thick, blackish, posteriorly more deeply blackened, separated by oblique septae from cell i and from the stalk - cell of the perithecium, connected by a horizontal septum from the distal portion of the receptacle ; the distal portion of the receptacle 70~75 m long, almost wholly blackened (in young thallus), composed of three superposed layers ; cell iii and iv consisting of third and fourth layers of receptacle, one - celled, cell v and vi consisted of the distal layer, broader than length. appendages produced on the distal end of the receptacle, filamentous, hyaline, basally blackened in young individuals, 90~100 m long. perithecium consisting of perithecium proper and the stalk - cells ; perithecium proper composed of four layers, second and third layers deeply blackish brown, mostly stout, straight, slightly inflated towards the basal portion, tapering gradually to the blunt symmetrical apex, 180~193 m long, 35~50 m thick ; the stalk cells composed of two layers, the basal cell 2 times longer than the subbasal cells, hyaline, tapering gradually to the blackish basal portion, cylindrical, 70 m long, 30 m thick, the subbasal cells of the perithecium stalk composed of two cells arranged horizontally, slightly darkened, shorter than the basal cells, nearly diametrical, slightly darker than the basal cells, 35 m long, 15 m thick. ochthephilum) (coleoptera, staphylinidae).host species in korea : ochthephilum densipenne (sharp)distribution : brazil, korea and west indies.specimen examined : a valley near temple hwaeom, mt. lee, na, lim, park, l - y-1597, 1598, 1599 and 1600. host species in korea : ochthephilum densipenne (sharp) distribution : brazil, korea and west indies. lee, na, lim, park, l - y-1597, 1598, 1599 and 1600. this species is closely related to corethromyces crytobii, c. brasilianus and c. shazawae, parasites of the beetles of the genus ochthephilum. all of them have elongated, slender perithecium proper and blackish receptacle (thaxter, 1896, 1908, 1931). however, c. purpurascens is well distinguished from c. shazawae in having the deeply blackish brown perithecium proper inflated gradually from the rounded apex to the subbasal portion, whereas in the latter it has a slightly brownish perithecium proper elongated cylindrically. this species is also different from c. crytobii and c. brasilianus in having only the wholly hyaline appendages on the distal end of the receptacle, whereas in two species of the latter they have both together two kinds of the hyaline and blackish appendages. according to the figures of the thaxter (1900), this species has the perithecium being sometimes distinctly inflated basally and the wholly hyaline appendages. authors determine consistently the korean specimens with c. purpurascens because of the former 's morphological characters of this fungus. receptacle consisting of the basal and the distal portion ; receptacle proper with stout or slender stalk, 130~160 m long, 65~70 m thick ; the basal portion composed of five cells and the insertion cell ; cell i bent anteriorly, tapering downwards foot, broadening upwards, producing the blackish foot on the tip of the basal portion, 35~40 m long, 20~30 m thick ; cell ii broadening upward, more or less elongated, connected obliquely with the basal cell of perithecial stalk, 30~35 m long, 40 m thick ; cell iii slightly longer than broad, 40~45 m long, 25~35 m thick ; cell iv isodiametric, 35~40 m long ; cell v nearly as long as cell iv, obtriangular, 20 m long, 5 m thick ; the insertion cell darkened, constricted, 5~7 m long, 15 m thick ; the distal portion of receptacle composed of two appenages arranged anterior - posteriorly ; the basal cells secondarily divided, giving rise to many branchlets ; the basal cell of outer appendages stout larger than those of inner appendages, 15 m long, 8~10 m thick. antheridia formed on lower parts of branchlets of the inner appendage filamentous, 10 m long, 5 m thick. perithecium consisting of the perithecium proper and the stalk cells ; perithecium ovate or nearly ellipsoidal, half - free, tapering gradually upwards with subapical blackening and prominent rounded posterior lips, 100~120 m long, 55~60 m thick ; the stalk of perithecium composed of a large basal cell and one or three (rarely) subbasal cells, the basal cell (cell vi) 20~30 m long, 20~30 m thick. host genus : philonthus (coleoptera, staphylinidae).host species in korea : philonthus longicornis stephensdistribution : france, holland, korea and poland.specimens examined : swamp upo, gyeongnam prov., lee, na, lim, park, l - y-1283 - 1 and 1283 - 2 host genus : philonthus (coleoptera, staphylinidae). host species in korea : philonthus longicornis stephens distribution : france, holland, korea and poland. lee, na, lim, park, l - y-1283 - 1 and 1283 - 2 three species of the genus labulbenia were reported on korean representatives of the genus philonthus. they are l. philonthi (lee and na, 1998), l. stenolophi (lee and na, 1998) and l. barbara (this paper). l. barbara described by middelheok (1943), balazuc (1974) and majewski (1994) has the elliptical, narrow, elongated perithecia that 4/5 freed from the receptacle, whereas in the present materials it has ovate, stout, inflated perithecia that half freed from the receptacle. the outer appendages of the former are blackened anteriorly, however, they are slightly blackened anteriorly or hyaline in the korean materials. although some morphological differences between these specimens mentioned above are noticed, authors determine consistently the korean specimens with l. barbara because of the biological specialization with respect to host and the morphological characters of this fungus. receptacle bright yellowish, consisting of the basal and the distal portions ; the basal portion cylindrical, tapering towards the end, forming a blackish foot at the end, composed of five cells and the insertion cell, 210~215 m long, 60~70 m in diameter ; cell i about 2 times longer than broad, 55~60 m long, 30~35 m thick ; cell ii larger than the other cells, 60~70 m long, 40~45 m thick ; cell iii isodiametric, longer than broad, 35~45 m long, 25~30 m thick ; cell iv nearly isodiametric, 40~42 m long, 30~35 m thick ; cell v small, oval, 20 m long, 10~13 m thick. the insertion cell dark flated, 10 m long, 10~13 m thick ; the distal portion of the receptacle composed of two branches arranged anterior - posteriorly ; the outer appendage once ramified on the subbasal cell, the basal cell nearly equal or slightly larger than the subbasal cell, the basal and the subbasal cells slightly blackish yellow ; two branchlets hyaline exception the basal cell of outer branchlet, 235~250 m long ; the inner appendage third or fourth ramified on the basal cell, which is bearing the sterile or the fertile branchlets ; the sterile branchlets extremely longer than the fertile, exceeding the apex of perithecium, 110~150 m long ; the fertile branchlets bearing antheridia on the distal portions, slightly darkened, not exceeding the apex of perithecium, 70 m long. perithecium composed of the perithecium proper and the stalk ; perithecium ellipsoidal, cylindrical, blackish brown yellow, half free from the receptacle on its posterior side, blackish and slightly constricted in the subapical part ; the posterior lip - cell forming a rounded termination of the apex ; the stalk of perithecium consisting of two or three subbasal cells and a large basal cell, each cells connected laterally under the septum of the cell of the receptacle, the basal cell, 30~35 m long, 25~30 m thick. antheridia cylindrical, tapering towards the distal end, 18~20 m long, 3~5 m thick. host genus : ansodactylus and harpalus (coleoptera, carabidae)host species in korea : harpalus tinctulus batesdistribution : finland, korea, u s a. and u s s r.specimens examined : gosiri, hancheonmyeon, hwasun, jeonnam prov. lee, na, lim, park, 2003, l - y-2009 - 1, 2009 - 2 and 2009 - 3 host genus : ansodactylus and harpalus (coleoptera, carabidae) host species in korea : harpalus tinctulus bates distribution : finland, korea, u s a. and u s s r. specimens examined : gosiri, hancheonmyeon, hwasun, jeonnam prov., lee, na, lim, park, 2003, l - y-2009 - 1, 2009 - 2 and 2009 - 3 the present species is closely related to l. filifera and l. polyphaga, but it differs in the following features ; 1) the outer appendages of receptacle in the former are much shorter than those of l. filifera and the apex of the perithecium is terminated slightly hyaline towards the distal end and the subapical portion is slightly constricted, whereas in the latter it is rounded, darkened towards the distal end and the subapical portion is inflated. 2) the inner appendages of receptacle in l. compressa are exceeding the apex of perithecium, whereas in l. polyphaga they are not exceeding it. antheridia of this species illustrated by thaxter (1893) are bearing on the lateral side of subapical cell of inner fertile appendages and in huldn 's materials (1983) they are bearing on the apical cells. in the present specimens they agreed with the huldn 's materials. receptacle consisting of the basal and the distal portions, 125~130 m long ; the basal portion composed of two superposed one celled layers, forming basally a blackish foot, 75 m long ; cell i 30 m long (including the foot), 10~13 m thick, brownish or blackish translucent, cylindrical ; cell ii 20~40 m long, 15 m thick, blackish, posteriorly more deeply blackened, separated by oblique septae from cell i and from the stalk - cell of the perithecium, connected by a horizontal septum from the distal portion of the receptacle ; the distal portion of the receptacle 70~75 m long, almost wholly blackened (in young thallus), composed of three superposed layers ; cell iii and iv consisting of third and fourth layers of receptacle, one - celled, cell v and vi consisted of the distal layer, broader than length. appendages produced on the distal end of the receptacle, filamentous, hyaline, basally blackened in young individuals, 90~100 m long. perithecium consisting of perithecium proper and the stalk - cells ; perithecium proper composed of four layers, second and third layers deeply blackish brown, mostly stout, straight, slightly inflated towards the basal portion, tapering gradually to the blunt symmetrical apex, 180~193 m long, 35~50 m thick ; the stalk cells composed of two layers, the basal cell 2 times longer than the subbasal cells, hyaline, tapering gradually to the blackish basal portion, cylindrical, 70 m long, 30 m thick, the subbasal cells of the perithecium stalk composed of two cells arranged horizontally, slightly darkened, shorter than the basal cells, nearly diametrical, slightly darker than the basal cells, 35 m long, 15 m thick. ochthephilum) (coleoptera, staphylinidae).host species in korea : ochthephilum densipenne (sharp)distribution : brazil, korea and west indies.specimen examined : a valley near temple hwaeom, mt. lee, na, lim, park, l - y-1597, 1598, 1599 and 1600. host species in korea : ochthephilum densipenne (sharp) distribution : brazil, korea and west indies. lee, na, lim, park, l - y-1597, 1598, 1599 and 1600. this species is closely related to corethromyces crytobii, c. brasilianus and c. shazawae, parasites of the beetles of the genus ochthephilum. all of them have elongated, slender perithecium proper and blackish receptacle (thaxter, 1896, 1908, 1931). however, c. purpurascens is well distinguished from c. shazawae in having the deeply blackish brown perithecium proper inflated gradually from the rounded apex to the subbasal portion, whereas in the latter it has a slightly brownish perithecium proper elongated cylindrically. this species is also different from c. crytobii and c. brasilianus in having only the wholly hyaline appendages on the distal end of the receptacle, whereas in two species of the latter they have both together two kinds of the hyaline and blackish appendages. according to the figures of the thaxter (1900), this species has the perithecium being sometimes distinctly inflated basally and the wholly hyaline appendages. authors determine consistently the korean specimens with c. purpurascens because of the former 's morphological characters of this fungus. receptacle consisting of the basal and the distal portion ; receptacle proper with stout or slender stalk, 130~160 m long, 65~70 m thick ; the basal portion composed of five cells and the insertion cell ; cell i bent anteriorly, tapering downwards foot, broadening upwards, producing the blackish foot on the tip of the basal portion, 35~40 m long, 20~30 m thick ; cell ii broadening upward, more or less elongated, connected obliquely with the basal cell of perithecial stalk, 30~35 m long, 40 m thick ; cell iii slightly longer than broad, 40~45 m long, 25~35 m thick ; cell iv isodiametric, 35~40 m long ; cell v nearly as long as cell iv, obtriangular, 20 m long, 5 m thick ; the insertion cell darkened, constricted, 5~7 m long, 15 m thick ; the distal portion of receptacle composed of two appenages arranged anterior - posteriorly ; the basal cells secondarily divided, giving rise to many branchlets ; the basal cell of outer appendages stout larger than those of inner appendages, 15 m long, 8~10 m thick. antheridia formed on lower parts of branchlets of the inner appendage filamentous, 10 m long, 5 m thick. perithecium consisting of the perithecium proper and the stalk cells ; perithecium ovate or nearly ellipsoidal, half - free, tapering gradually upwards with subapical blackening and prominent rounded posterior lips, 100~120 m long, 55~60 m thick ; the stalk of perithecium composed of a large basal cell and one or three (rarely) subbasal cells, the basal cell (cell vi) 20~30 m long, 20~30 m thick. host genus : philonthus (coleoptera, staphylinidae).host species in korea : philonthus longicornis stephensdistribution : france, holland, korea and poland.specimens examined : swamp upo, gyeongnam prov., august 10, 1996, coll. lee, na, lim, park, l - y-1283 - 1 and 1283 - 2 host genus : philonthus (coleoptera, staphylinidae). host species in korea : philonthus longicornis stephens distribution : france, holland, korea and poland. specimens examined : swamp upo, gyeongnam prov., august 10, 1996, coll. lee, na, lim, park, l - y-1283 - 1 and 1283 - 2 three species of the genus labulbenia were reported on korean representatives of the genus philonthus. they are l. philonthi (lee and na, 1998), l. stenolophi (lee and na, 1998) and l. barbara (this paper). l. barbara described by middelheok (1943), balazuc (1974) and majewski (1994) has the elliptical, narrow, elongated perithecia that 4/5 freed from the receptacle, whereas in the present materials it has ovate, stout, inflated perithecia that half freed from the receptacle. the outer appendages of the former are blackened anteriorly, however, they are slightly blackened anteriorly or hyaline in the korean materials. although some morphological differences between these specimens mentioned above are noticed, authors determine consistently the korean specimens with l. barbara because of the biological specialization with respect to host and the morphological characters of this fungus. receptacle bright yellowish, consisting of the basal and the distal portions ; the basal portion cylindrical, tapering towards the end, forming a blackish foot at the end, composed of five cells and the insertion cell, 210~215 m long, 60~70 m in diameter ; cell i about 2 times longer than broad, 55~60 m long, 30~35 m thick ; cell ii larger than the other cells, 60~70 m long, 40~45 m thick ; cell iii isodiametric, longer than broad, 35~45 m long, 25~30 m thick ; cell iv nearly isodiametric, 40~42 m long, 30~35 m thick ; cell v small, oval, 20 m long, 10~13 m thick. the insertion cell dark flated, 10 m long, 10~13 m thick ; the distal portion of the receptacle composed of two branches arranged anterior - posteriorly ; the outer appendage once ramified on the subbasal cell, the basal cell nearly equal or slightly larger than the subbasal cell, the basal and the subbasal cells slightly blackish yellow ; two branchlets hyaline exception the basal cell of outer branchlet, 235~250 m long ; the inner appendage third or fourth ramified on the basal cell, which is bearing the sterile or the fertile branchlets ; the sterile branchlets extremely longer than the fertile, exceeding the apex of perithecium, 110~150 m long ; the fertile branchlets bearing antheridia on the distal portions, slightly darkened, not exceeding the apex of perithecium, 70 m long. perithecium composed of the perithecium proper and the stalk ; perithecium ellipsoidal, cylindrical, blackish brown yellow, half free from the receptacle on its posterior side, blackish and slightly constricted in the subapical part ; the posterior lip - cell forming a rounded termination of the apex ; the stalk of perithecium consisting of two or three subbasal cells and a large basal cell, each cells connected laterally under the septum of the cell of the receptacle, the basal cell, 30~35 m long, 25~30 m thick. antheridia cylindrical, tapering towards the distal end, 18~20 m long, 3~5 m thick. host genus : ansodactylus and harpalus (coleoptera, carabidae)host species in korea : harpalus tinctulus batesdistribution : finland, korea, u s a. and u s s r.specimens examined : gosiri, hancheonmyeon, hwasun, jeonnam prov. lee, na, lim, park, 2003, l - y-2009 - 1, 2009 - 2 and 2009 - 3 host genus : ansodactylus and harpalus (coleoptera, carabidae) host species in korea : harpalus tinctulus bates distribution : finland, korea, u s a. and u s s r. specimens examined : gosiri, hancheonmyeon, hwasun, jeonnam prov. lee, na, lim, park, 2003, l - y-2009 - 1, 2009 - 2 and 2009 - 3 the present species is closely related to l. filifera and l. polyphaga, but it differs in the following features ; 1) the outer appendages of receptacle in the former are much shorter than those of l. filifera and the apex of the perithecium is terminated slightly hyaline towards the distal end and the subapical portion is slightly constricted, whereas in the latter it is rounded, darkened towards the distal end and the subapical portion is inflated. 2) the inner appendages of receptacle in l. compressa are exceeding the apex of perithecium, whereas in l. polyphaga they are not exceeding it. antheridia of this species illustrated by thaxter (1893) are bearing on the lateral side of subapical cell of inner fertile appendages and in huldn 's materials (1983) they are bearing on the apical cells. in the present specimens they agreed with the huldn 's materials. | three species of the laboubeniales from korea are described. they are new to the mycological flora of korea. corethromyces purpurascens on several parts of ochthephilum densipenne, laboulbenia barbara on the metasternum of philonthus longicornis and l. compressa on the elytra of harpalus tinctulus were found. |
the main objectives of modern phacoemulsification (phaco) technology are the reduction of ultrasound (us) power and improved efficiency.1 interrupted phaco modes, improved pump systems, chopping techniques, and vacuum - assisted phaco have reduced the amount of energy needed to remove a cataract and, therefore, the potential associated risks.1,2 however, us power required for traditional or longitudinal phaco continues to be a risk factor for endothelial cell loss and tissue damage.3 through the use of improved phaco technologies, recent studies on cataract surgery have demonstrated a reduction in corneal endothelial cell loss after phaco.411 in an attempt to increase us efficiency during phaco cataract surgery, torsional phaco technology was introduced in 2005. in traditional phaco, the longitudinal movement of the phaco tip pushes nuclear fragments away with each forward stroke, although interrupted us has the supposed advantage of allowing retraction of the pushed nuclear fragments back toward the phaco tip. furthermore, due to the jackhammer effect, the emulsifying power of longitudinal phaco is only effective during the forward movement of the tip. conversely, the lateral tip movement of torsional phaco shears the lens material while moving in both directions without a repellent force. it also uses a lower frequency (32 mhz) than traditional phaco (40 mhz), allowing for 20% energy conservation. as such, torsional technology represents a significant improvement in the emulsifying efficiency of us during phaco.12,13 torsional us can also be mixed, intermittently, with a quantum of longitudinal phaco. this is commercially known as ozil intelligent phaco (ip) (alcon laboratories, fort worth, tx, usa), whereby the quantum is liberated at a threshold vacuum in a duration ranging from 10 to 20 milliseconds. ip aims to maximize torsional us efficacy by keeping the nuclear fragments in the ideal shearing plane.14 in addition to torsional technology, transversal us technology is a new form of phaco. the transversal us model, ellips (abbott medical optics, santa ana, ca, usa), was developed to reduce the repulsion that commonly occurs with longitudinal phaco. ellips simultaneously generates lateral and longitudinal motions at a working frequency of 28 khz ; thus, the phaco tip moves in an elliptical motion, promoting an optimization of efficiency by emulsifying lens matter in more than one direction. moreover, the accompanying longitudinal component adds the jackhammering effect to transversal us efficiency, constantly maintaining the nuclear fragments at the ideal shearing plane in front of the phaco tip.15 the transversal technology can also be used with a straight or curved tip, and the ellips model can work in a continuous or hyperpulse mode with different duty cycles.16 recently, ellips fx (abbott medical optics), a further hardware and software modification on the previous version, has been developed for a working frequency of 38 khz (a 45% increase on the previous version) and a stroke length three times larger (longitudinal and lateral stroke length ratio is 1:1) than the previous version. existing studies have experimentally compared torsional and transversal phaco systems in terms of vacuum, surge, and thermal effect.1517 furthermore, christakis and braga - mele5 recently compared the clinical outcomes obtained after the same surgeon performed cataract surgery with phaco, using three different systems : whitestar signature ellips fx (transversal), infiniti ozil ip (torsional), and stellaris (bausch and lomb, rochester, ny, usa) (longitudinal). these authors evaluated the phaco needle time, chatter, followability, the anterior chamber stability, the level of corneal edema, and the 1-day postoperative visual acuity. however, to date, no comparisons between torsional and transversal phaco have been reported in terms of corneal endothelial changes. hence, the aim of this study was to compare and evaluate the corneal morphological changes, including endothelial modifications, as an indicator of safety for cataract surgery through a 2.2 mm corneal incision. in this study, this study included 139 consecutive cataractous eyes of 82 patients undergoing cataract surgery through a 2.2 mm corneal incision. the inclusion criteria were patients of 45 years or older and the presence of a senile cataract graded as nuclear color (nc) 24, according to the lens opacities classification system iii (locsiii).18 the exclusion criteria were patients with glaucoma, corneal opacities, cornea guttata, abnormal iris, macular degeneration or retinopathy, previous posterior segment surgery, a corneal endothelial cell density (ecd) of less than 1900 cell / mm, neuro - ophthalmic disease, or a history of ocular inflammation. the study adhered to the tenets of the declaration of helsinki, and the study was approved by the local ethical committee. one of the two different phaco platforms was assigned to be used with each eye included in this study. accordingly, two groups were differentiated : group i included eyes undergoing phaco via the whitestar signature platform using the ellips fx transversal continuous ultrasound mode with the 0.9 mm curved microtip, and group ii included eyes undergoing phaco via the infiniti platform using the ozil ip torsional phaco with the 0.9 mm/45 kelman tip. preoperatively, all patients had a full ophthalmological examination, including evaluation of the refractive status, uncorrected and corrected distance visual acuity testing (snellen optotypes and decimal notation), slit lamp examination, and goldmann applanation tonometry and fundoscopy. besides these basic clinical tests, corneal endothelial cell analysis and central corneal thickness (cct) measurements were performed (em-3000 ; tomey, erlangen, germany). corneal endothelial analysis included the measurement and recording of the ecd, the coefficient of variation (cv), and the hexagonality. patients were also evaluated during the follow - up at 1 day and 1 month after surgery. at 1 day after surgery, intraocular pressure and the integrity of the anterior segment were evaluated. all surgeries were performed by the same surgeon (aa), using a standard mini - incision of the sutureless micro - coaxial phaco technique. in all cases, a corneal incision of 2.2 mm after injection of the cohesive ophthalmic viscoelastic device (healon gv ; abbott medical optics) in the anterior chamber, a curvilinear rhexis of 55.5 mm was performed. after hydrodissection, nucleus disassembly was started using the quick - chop technique, impaling the curved phaco - tip in the central part of the nucleus (11.5 mm). this was followed by vertical chopping of the nucleus with the tobias neuhann chopper (geuder gmbh, heidelberg, germany). the incision size change was tested using the tsuneoka micro - coaxial incision gauge (asico, westmont, il, usa) after completion of the cortical cleaning and prior to intraocular lens (iol) implantation. one - piece acrylic iols (tecnis 1 zcb00 ; abbott medical optics) were implanted in all cases. the wound seal was confirmed at the end of the surgery by a negative seidel test. settings for the infiniti platform (torsional) were as follows : the power was set at 100% linear torsional continuous us amplitude ; the ip power delivery option was activated to produce a 10 millisecond longitudinal phaco pulse when 95% vacuum was reached ; the flow rate was 28 ml / min, with dynamic rise of + 1 ; the vacuum was set at 350 mmhg ; and the bottle height was 108 cm. settings for the whitestar signature platform (transversal) were as follows : the power of the ellips fx was set at 100% linear continuous transversal us ; the aspiration rate was set at 28 and 26 ml / min before and after occlusion, respectively ; vacuum parameters were set at a maximum of 320 mmhg ; the chamber stabilization environment (case) vacuum was set at 280 mmhg ; case time was set at 500 milliseconds for nc 2 and at 8001000 milliseconds for nc 3 and 4 ; and the bottle height was 67 cm. this included : using moxifloxacin hcl 0.5% (vigamox ; alcon laboratories) eye drops three times per day for two weeks ; rimexolone 1% (vexol ; alcon laboratories) eye drops three times per day for 1 week, then twice per day for the following 2 weeks ; tobramycin - dexamethasone (tobradex ; alcon laboratories) eye ointment once at bedtime for 2 weeks. spss statistics software package (v 19.0) for windows (ibm corporation, armonk, ny, usa) was used for statistical analysis. normality of all data samples was first evaluated by means of the kolmogorov - smirnov test. when parametric analysis was possible, the student t - test for paired data was used for comparisons between preoperative and postoperative examinations, and the student t - test for unpaired data was used for the comparison between groups. when parametric analysis was not possible, the wilcoxon rank - sum test was applied to assess the significance of differences between preoperative and postoperative data, and the mann whitney test was applied for the comparison between groups, in all cases using the same level of significance (p < 0.05). before the start of the study and according to our previous experience, we estimated that a sample size of 64 eyes per group would be required to detect a mean difference of 10 cell / mm between both groups, with a standard deviation of 20. we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research. this study included 139 consecutive cataractous eyes of 82 patients undergoing cataract surgery through a 2.2 mm corneal incision. the inclusion criteria were patients of 45 years or older and the presence of a senile cataract graded as nuclear color (nc) 24, according to the lens opacities classification system iii (locsiii).18 the exclusion criteria were patients with glaucoma, corneal opacities, cornea guttata, abnormal iris, macular degeneration or retinopathy, previous posterior segment surgery, a corneal endothelial cell density (ecd) of less than 1900 cell / mm, neuro - ophthalmic disease, or a history of ocular inflammation. the study adhered to the tenets of the declaration of helsinki, and the study was approved by the local ethical committee. one of the two different phaco platforms was assigned to be used with each eye included in this study. accordingly, two groups were differentiated : group i included eyes undergoing phaco via the whitestar signature platform using the ellips fx transversal continuous ultrasound mode with the 0.9 mm curved microtip, and group ii included eyes undergoing phaco via the infiniti platform using the ozil ip torsional phaco with the 0.9 mm/45 kelman tip. preoperatively, all patients had a full ophthalmological examination, including evaluation of the refractive status, uncorrected and corrected distance visual acuity testing (snellen optotypes and decimal notation), slit lamp examination, and goldmann applanation tonometry and fundoscopy. besides these basic clinical tests, corneal endothelial cell analysis and central corneal thickness (cct) measurements were performed (em-3000 ; tomey, erlangen, germany). corneal endothelial analysis included the measurement and recording of the ecd, the coefficient of variation (cv), and the hexagonality. patients were also evaluated during the follow - up at 1 day and 1 month after surgery. at 1 day after surgery, intraocular pressure and the integrity of the anterior segment all surgeries were performed by the same surgeon (aa), using a standard mini - incision of the sutureless micro - coaxial phaco technique. in all cases, a corneal incision of 2.2 mm after injection of the cohesive ophthalmic viscoelastic device (healon gv ; abbott medical optics) in the anterior chamber, a curvilinear rhexis of 55.5 mm was performed. after hydrodissection, nucleus disassembly was started using the quick - chop technique, impaling the curved phaco - tip in the central part of the nucleus (11.5 mm). this was followed by vertical chopping of the nucleus with the tobias neuhann chopper (geuder gmbh, heidelberg, germany). the incision size change was tested using the tsuneoka micro - coaxial incision gauge (asico, westmont, il, usa) after completion of the cortical cleaning and prior to intraocular lens (iol) implantation. one - piece acrylic iols (tecnis 1 zcb00 ; abbott medical optics) were implanted in all cases. the wound seal was confirmed at the end of the surgery by a negative seidel test. settings for the infiniti platform (torsional) were as follows : the power was set at 100% linear torsional continuous us amplitude ; the ip power delivery option was activated to produce a 10 millisecond longitudinal phaco pulse when 95% vacuum was reached ; the flow rate was 28 ml / min, with dynamic rise of + 1 ; the vacuum was set at 350 mmhg ; and the bottle height was 108 cm. settings for the whitestar signature platform (transversal) were as follows : the power of the ellips fx was set at 100% linear continuous transversal us ; the aspiration rate was set at 28 and 26 ml / min before and after occlusion, respectively ; vacuum parameters were set at a maximum of 320 mmhg ; the chamber stabilization environment (case) vacuum was set at 280 mmhg ; case time was set at 500 milliseconds for nc 2 and at 8001000 milliseconds for nc 3 and 4 ; and the bottle height was 67 cm. this included : using moxifloxacin hcl 0.5% (vigamox ; alcon laboratories) eye drops three times per day for two weeks ; rimexolone 1% (vexol ; alcon laboratories) eye drops three times per day for 1 week, then twice per day for the following 2 weeks ; tobramycin - dexamethasone (tobradex ; alcon laboratories) eye ointment once at bedtime for 2 weeks. spss statistics software package (v 19.0) for windows (ibm corporation, armonk, ny, usa) was used for statistical analysis. normality of all data samples was first evaluated by means of the kolmogorov - smirnov test. when parametric analysis was possible, the student t - test for paired data was used for comparisons between preoperative and postoperative examinations, and the student t - test for unpaired data was used for the comparison between groups. when parametric analysis was not possible, the wilcoxon rank - sum test was applied to assess the significance of differences between preoperative and postoperative data, and the mann whitney test was applied for the comparison between groups, in all cases using the same level of significance (p < 0.05). before the start of the study and according to our previous experience, we estimated that a sample size of 64 eyes per group would be required to detect a mean difference of 10 cell / mm between both groups, with a standard deviation of 20. we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research. the total number of eyes included in this study was 150 of 88 patients (group i, 85 eyes ; group ii, 65 eyes). six patients (11 eyes : six in group i and five in group ii) did not complete the postoperative follow - up examinations and were excluded from the study. therefore, the data used for the statistical analysis included 139 eyes of 82 patients : 79 eyes in group i and 60 eyes in group ii. the mean age sd was 65.3 7.0 and 64.0 7.6 years in groups i and ii, respectively. no statistically significant differences in age were detected between groups (mann whitney test : p = 0.29). according to the locsiii, in group i, 29 eyes presented an nc 2 cataract, 41 eyes presented an nc 3 cataract, and nine eyes presented an nc 4 cataract. in group ii, a similar distribution of cataract types, according to the locsiii, was found : 23 eyes with an nc 2 cataract, 30 eyes with an nc 3 cataract, and seven eyes with an nc 4 cataract. intraoperatively, we observed improved followability with less chattering of free nuclear fragments in both systems. the mean size of the main incision was 2.24 0.06 mm in both groups, with no statistically significant differences between them (mann whitney test : p = 0.75). on the first postoperative day, the slit lamp examination showed mild stromal corneal folds in both groups with complete resolution at an average of 7 days, ranging from 4 to 10 days. table 1 summarizes the corneal endothelial changes for groups i and ii, occurring 1 month after surgery. as shown, we found significant changes in the three parameters analyzed (ecd, cv, and hexagonality) in both groups (wilcoxon test : p 0.02) 1 month after surgery, except for the hexagonality in group ii. the mean postoperative decrease in ecd was 40.0 37.5 cell / mm and 45.3 31.7 cell / mm in groups i and ii, respectively. no statistically significant differences in ecd were found between the groups preoperatively (mann whitney test : p = 0.80) and postoperatively (mann whitney test : p = 0.98). in relation to preoperative values (figure 1), group i showed a mean endothelial cell loss of 1.7% 1.6%, whereas group ii showed a loss of 2.0% 1.4% (mann whitney test : p = 0.29). regarding cv, no statistically significant differences were found between groups preoperatively (mann whitney test : p = 0.82) and postoperatively (mann whitney test : p = 0.96). likewise, hexagonality did not differ significantly between groups before (mann whitney test : p = 0.35) and after (mann whitney test : p = 0.97) surgery. the preoperative cct was 534.5 29.3 m and 536.9 26.2 m in groups i and ii, respectively (mann whitney test : p = 0.40). the postoperative cct was 562.6 33.7 m (increased by 28.1 23.6 m) and 560.9 29.9 m (increased by 24.0 24 m) in groups i and ii, respectively (mann whitney test : p = 0.68). thus, the postoperative increase in cct was similar in both groups (mann whitney test : p = 0.10). the central pachymetry change with surgery was statistically significant in both groups (wilcoxon test : p < 0.01). intraoperatively, we observed improved followability with less chattering of free nuclear fragments in both systems. the mean size of the main incision was 2.24 0.06 mm in both groups, with no statistically significant differences between them (mann whitney test : p = 0.75). on the first postoperative day, the slit lamp examination showed mild stromal corneal folds in both groups with complete resolution at an average of 7 days, ranging from 4 to 10 days. table 1 summarizes the corneal endothelial changes for groups i and ii, occurring 1 month after surgery. as shown, we found significant changes in the three parameters analyzed (ecd, cv, and hexagonality) in both groups (wilcoxon test : p 0.02) 1 month after surgery, except for the hexagonality in group ii. cell / mm and 45.3 31.7 cell / mm in groups i and ii, respectively. no statistically significant differences in ecd were found between the groups preoperatively (mann whitney test : p = 0.80) and postoperatively (mann whitney test : p = 0.98). in relation to preoperative values (figure 1), group i showed a mean endothelial cell loss of 1.7% 1.6%, whereas group ii showed a loss of 2.0% 1.4% (mann whitney test : p = 0.29). regarding cv, no statistically significant differences were found between groups preoperatively (mann whitney test : p = 0.82) and postoperatively (mann whitney test : p = 0.96). likewise, hexagonality did not differ significantly between groups before (mann whitney test : p = 0.35) and after (mann whitney test : p = 0.97) surgery. the preoperative cct was 534.5 29.3 m and 536.9 26.2 m in groups i and ii, respectively (mann whitney test : p = 0.40). the postoperative cct was 562.6 33.7 m (increased by 28.1 23.6 m) and 560.9 29.9 m (increased by 24.0 24 m) in groups i and ii, respectively (mann whitney test : p = 0.68). thus, the postoperative increase in cct was similar in both groups (mann whitney test : p = 0.10). the central pachymetry change with surgery was statistically significant in both groups (wilcoxon test : p < 0.01). technological advances in phaco have made cataract removal safer and more efficient by reducing phaco energy and duration. several studies have confirmed and reported the superiority of torsional phaco over longitudinal phaco in terms of needle time, chatter, followability, corneal edema, and corneal endothelium.1,5,712,19,20 however, to date, only one study has compared longitudinal, torsional, and transversal phaco.5 in that study, the clinical outcomes with three different phaco platforms were evaluated : whitestar signature ellips fx (transversal), infiniti ozil ip (torsional), and stellaris (longitudinal). those authors found that the torsional machine had less phaco needle time, less chatter, better followability, and yielded less central corneal edema than the transversal or longitudinal machines. however, they did not compare the effect of using the platforms in terms of the corneal endothelium. the aim of the current study was to compare and evaluate the corneal morphological changes, including endothelial modifications, as an indicator of safety for cataract surgery through a 2.2 mm corneal incision. because of different algorithms adopted by the two platforms, the current comparative analysis did not measure the effective phaco time, cumulative dissipated energy, phaco time, or balanced salt solution consumption. georgescu demonstrated that a comparable unoccluded flow vacuum of 60 ml / min would correspond with approximately 220 mmhg and 120 mmhg for the infiniti and whitestar signature platforms, respectively. our comparative analysis preferred the use of independent and more clinically significant parameters, such as wound stretch, corneal edema, endothelial cell loss, pleomorphism, and central pachymetry. the function of followability is to bring free nuclear fragments close to the phaco tip for continuous and smoothly progressive emulsification and aspiration. fluidic models have shown that the longitudinal movement repels lens material from the tip, whereas the rotational motion of torsional us reduces the repulsion of the lens fragments, thus improving followability of nuclear material into the phaco tip.20 longitudinal phaco tip movement repels lens matter with every stroke. conversely, lateral movement reduces repulsion of the lens fragments, thus improving followability during phaco. in transversal phaco, nuclear fragments are exposed to shearing stress and the jackhammering effect caused by the transversal and longitudinal motion, respectively, but without the repulsion seen in longitudinal phaco. furthermore, with the ellips fx, the longitudinal us could not be separated from the transversal component. with the ozil ip, the longitudinal component might be switched off or set to an automatic activation when a preset threshold vacuum was reached. for this reason, a better followability was expected in the torsional group than the transversal group. however, both systems showed improved and comparable followability. georgescu demonstrated that the postocclusion surge (pos) was significantly worse for the infiniti platform than for the whitestar signature platform. however, this could only be clinically important in extreme experimental conditions, such as using 60 ml / min of flow, a 550 mmhg vacuum, and a bottle height of 60 cm. pos is often at its worst when dealing with a broken capsule and corneal endothelial damage.17 according to the settings used in our study, we expected the pos to be negligible. indeed, no pos was noticed during surgery in either of our groups. regarding corneal incision size before iol implantation the minimal wound stretch found in group i may be attributable to the compliant nature of the sleeve and the microvoid between the phaco tip and incision. hence, the transversal movement of the phaco tip did not induce wound stress or burn. it should be noted that the new, ultrasleeve 21 g tip was used with the ellips fx in our study. this use was off - label, due to its commercial unavailability at the beginning of the study. in concordance with this, jun stated that the smaller incision phaco of torsional us may decrease the risk for wound burn in eyes with denser cataracts. furthermore, li have recently demonstrated that, in the context of torsional phaco, a micro - coaxial, 2.2 mm incision may cause slightly less damage than the 2.8 mm standard incision. therefore, considering the twist movement of the phaco tip in torsional us and considering that the mini - flare tip and ultra - sleeve used in group ii were designed to fit 2.2 mm and 2.4 mm incisions, the minimal wound stretch found in this group seems to be a coherent finding. finally, the enhanced followability with the torsional phaco mode reduced the additional hand - piece movements to reacquire the nuclear fragments, minimizing the stretch in the incision.20 the mean endothelial cell loss in the current study was 1.7% and 2.0% in groups i and ii, respectively. these values were better than those obtained in other studies using the torsional technology.8,9 this may be attributable to the use of an improved technology, the different surgical technique, the ophthalmic viscoelastic device used, and the different classification system adopted (locs iii vs locs ii). one study found an endothelial cell loss of 7.2% 4.6% using the torsional phaco technology through a 2.75 mm clear corneal incision,9 yet, in our study, no significant differences between groups were observed in endothelial cell loss, cv, and hexagonality, confirming the presence of similar postoperative levels of polymegathism and pleomorphism in both groups. therefore, the same minimal level of endothelial damage seems to be induced with both phaco technologies. it has been postulated that a longer phaco time22,23 and higher us power22 are associated with endothelial cell loss, although some studies failed to prove these correlations.9,24,25 reuschel found a statistically significant difference in us energy and time between the torsional and longitudinal phaco, but there was no significant correlation between these parameters and endothelial cell loss. in addition, the cell loss was not significantly different between the two groups.9 in the current study, a more significant endothelial cell loss would have been expected in the ellips fx group, since the longitudinal movement component of the tip was constantly present, yet this finding was not obtained. this suggests that we have approached the critical value for safe phaco modality with the currently available torsional and transversal phaco technology, beyond which no significant improvements in endothelial cell protection can be observed. we also found a minimal, but statistically significant increase of 28.1 and 24.0 m in cct in groups i and ii, respectively. the difference between the groups in this minimal level of corneal edema did not reach statistical significance. however, christakis and braga - mele5 found that torsional technology yielded less central corneal edema than the transversal and longitudinal technologies, corresponding to a smaller increase in the mean corneal thickness (torsional, 5% ; transversal, 10% ; longitudinal, 12%). one of the main factors accounting for this discrepancy with our findings may be the difference in the nuclear cataract grade, which was nc 2.0 0.8 in christakis and braga - mele s study.5 ellips fx transversal and ozil ip torsional phaco platforms are safe for performing cataract surgery, inducing minimal corneal thickness and endothelial changes. in terms of followability, the effect on the endothelium and the corneal incision, and the induced corneal thickness increase, both technologies seem to be equivalent for extraction of an nc 24 (locs iii) cataract through a 2.2 mm incision. future optical coherence tomography studies are needed to compare the effects of torsional and transversal phaco types on the corneal incision. in addition, the potential benefits and applicability of these technologies in eyes with a compromised endothelium should be further evaluated. | purposethis paper compares and evaluates the corneal morphological changes occurring after cataract surgery through a 2.2 mm corneal incision. we use two platforms for comparison and evaluation, transversal and torsional phacoemulsification.patients and methodsthis study includes 139 consecutive cataractous eyes (nuclear color 24, according to the lens opacities classification system iii [locsiii ]) of 82 patients undergoing cataract surgery through a 2.2 mm corneal incision. two different phacoemulsification platforms were used and assigned randomly : we used the whitestar signature system with the ellips fx transversal continuous ultrasound (us) mode for group i (mean age : 65.33 6.97 years), and we used the infiniti system with the ozil intelligent phaco (ip) torsional us mode for group ii (mean age : 64.02 7.55 years). the corneal endothelium and pachymetry were evaluated preoperatively and at 1 month postoperatively. incision size changes were also evaluated.resultsall surgeries were uneventful. before intraocular lens implantation, the mean incision size was 2.24 0.06 mm in both groups (p = 0.75). in terms of corneal endothelial cell density, neither preoperative (i vs ii : 2304.1 122.5 cell / mm2 vs 2315.6 83.1 cell / mm2, p = 0.80) nor postoperative (i vs ii : 2264.1 124.3 cell / mm2 vs 2270.3 89.9 cell / mm2, p = 0.98) differences between the groups were statistically significant. the mean endothelial cell density loss was 1.7% 1.6% and 2.0% 1.4% in groups i and ii, respectively. furthermore, no significant differences between groups i and ii were found preoperatively (p = 0.40) and postoperatively (p = 0.68) in central pachymetry. with surgery, the mean increase in central pachymetry was 28.1 23.6 m and 24.0 24.0 m in groups i and ii, respectively (p = 0.1).conclusionellips fx transversal and ozil ip torsional phacoemulsification modes are safe for performing cataract surgery, inducing minimal corneal thickness and endothelial changes. |
he had undergone a caldwell - luc operation 30 years ago, and had no history of medical disease. the right nasal cavity was not visible because of bulging from the inferior meatus, which almost reached the septum. images of the paranasal sinus revealed a 6 5-cm - sized well - demarcated cystic mass filling the right maxillary sinus and nasal cavity (fig. 1 a and c). the bulging mass led to erosion of the posterior wall of the maxillary sinus and pterygoid plate causing anatomical deformity and sphenoid sinusitis (fig. endoscopic decompression and sphenoid sinusotomy were performed, considering it to be a patient of postoperative maxillary cyst with sphenoid sinusitis. the sphenoid sinus ostium was located using an image - guided system (fiagon gmbh, berlin, germany), and massive bleeding was noted during the widening of the ostium. the estimated blood loss was about 1500 cc and the bleeding was temporarily controlled by nasal packing using rapid rhino (smith & nephew, austin, tx) and merocel (medtronic, mystic, ct). since right facial swelling was gradually progressive, we performed immediate angiography, which revealed a thrombosed large pseudoaneurysm feeding from the right i m a (fig. materials used for embolization were n - butylcyanoacrylate (33%) and 3 8 mm platinum detachable coils (covidien, axium, plymouth, mn). (a) axial and (b) coronal images of paranasal sinus computed tomography. the bulging mass shows a homogenous density occupying right maxillary sinus with erosion of the posterior wall of maxillary sinus and sphenoid sinusitis (arrow). a 6 5-cm - sized, gadolinium - enhanced mass with internal cystic change (arrow) can be seen and the lesion is well demarcated with smooth margins. blood circulation of right internal carotid artery and external carotid artery was checked before embolization. embolization of right i m a (arrow) was done using n - butylcyanoacrylate and platinum coils. in this patient, since the postoperative maxillary cyst was huge and bulging onto adjacent structures, normal anatomic structure was altered, and the course of the i m a was also displaced. the diagnosis of pseudoaneurysm can be confirmed by duplex ultrasonography, computed tomography angiography, or conventional angiography. in this patient, even though the bleeding was temporarily controlled by nasal packing, immediate computed tomography angiography was performed because of progressive facial swelling. several studies showed that the success and complication rates are quite comparable between surgical ligation and embolization. although embolization is a minimally invasive technique, there are many possible complications including skin ischemia, temporary hemiparesis, temporary monocular visual field loss, monocular blindness, peripheral facial nerve paralysis, and cerebral infarction. therefore, the application of embolization should depend on the anatomical factors, patient preference, and availability of experienced interventional staff. in this patient, immediate embolization was successfully performed, and there were no complications after 6 months. in review of the literature, the pseudoaneurysms occurred following a postoperative delay of 2 days to 4 months (table 1). it is clinically significant that we did not require a transfusion despite encountering uncontrolled massive bleeding after the compact nasal packing, since we immediately performed an intervention procedure. another point of clinical significance is that this is the first patient of pseudoaneurysm occurring in i m a after ess. therefore, this patient highlights the importance of the application of immediate embolization in the management of pseudoaneurysm occurring due to intractable bleeding after ess. previously reported patients of pseudoaneurysm after endoscopic sinus surgery ica, internal carotid artery ; pva, polyvinyl alcohol ; rbc, red blood count ; spa, sphenopalatine artery ; u, unit. | abstractpseudoaneurysm is defined as blood leaking out of a vessel that does not have true 3 arterial walls like a true aneurysm, and is susceptible to rupture. only 4 patients of pseudoaneurysm after endoscopic sinus surgery have been reported so far in english literature. recently, the authors encountered a pseudoaneurysm in the internal maxillary artery after endoscopic sinus surgery, which was immediately and successfully managed with endovascular embolization. there was no bleeding or complications 6 months after the embolization. |
the development of type 2 diabetes is characterized by chronic insulin resistance and a progressive fall in -cell compensation for insulin resistance (14). previous studies (510) have demonstrated that calorie restriction reduces insulin resistance, improves glucose tolerance, and delays or prevents the onset of type 2 diabetes. severe, short - term calorie restriction has been shown to improve insulin sensitivity and enhance -cell function (11,12). however, little is known about the relationship between spontaneous calorie intake and long - term changes in -cell function under free - living conditions. in this report, we examine this relationship using data from a cohort of nondiabetic hispanic women with recent gestational diabetes mellitus (gdm) who were prospectively observed postpartum for up to 12 years in the university of southern california (usc) gdm cohort study. briefly, pregnant hispanic women referred to the los angeles county women s hospital for management of gdm between august 1993 and march 1995 were asked to participate in the usc gdm cohort study if they of the following criteria : 1) gestational age between 28 and 34 weeks ; 2) no current or prior insulin therapy ; 3) all fasting serum glucose concentrations 140 mg / dl (> 7.8 mmol / l), at which time they were referred for pharmacological treatment. women who were pregnant at the time of a scheduled follow - up visit were studied at least 4 months after pregnancy and at least 1 month after the completion of breast - feeding. all participants gave written informed consent for participation in the study, which was approved by the institutional review board of the usc and the los angeles county+usc medical center. for the present report, we analyzed data from the 62 members of the cohort who had baseline ogtt and ivgtt results without an indication of diabetes and returned to undergo at least one additional ogtt and ivgtt. for each follow - up assessment, women came to the clinical research center on two separate days, at least 48 h apart, after 812 h of overnight fasting and at least 3 days on an unrestricted diet. on day 1, a bia and ogtt were performed (15). on day 2, glucose was measured by glucose oxidase (beckman glucose analyzer ii ; beckman coulter, brea, ca). insulin was measured by a radioimmunoassay (novo pharmaceuticals, danbury, ct) that measured insulin and proinsulin with intraobserver and interobserver coefficients of variation of 140 mg / dl (> 7.8 mmol / l), at which time they were referred for pharmacological treatment. women who were pregnant at the time of a scheduled follow - up visit were studied at least 4 months after pregnancy and at least 1 month after the completion of breast - feeding. all participants gave written informed consent for participation in the study, which was approved by the institutional review board of the usc and the los angeles county+usc medical center. for the present report, we analyzed data from the 62 members of the cohort who had baseline ogtt and ivgtt results without an indication of diabetes and returned to undergo at least one additional ogtt and ivgtt. for each follow - up assessment, women came to the clinical research center on two separate days, at least 48 h apart, after 812 h of overnight fasting and at least 3 days on an unrestricted diet. on day 1, a bia and ogtt were performed (15). on day 2, glucose was measured by glucose oxidase (beckman glucose analyzer ii ; beckman coulter, brea, ca). insulin was measured by a radioimmunoassay (novo pharmaceuticals, danbury, ct) that measured insulin and proinsulin with intraobserver and interobserver coefficients of variation of 75 min / week vigorous activity for adults (24), and no women met the recommendation of > 150 min / week moderate activity (24). correlations between baseline total calorie intake and baseline anthropometric and metabolic outcomes were weak and were not statistically significant. baseline characteristics and rates of change during a median of 8.0 years of follow - up for the 62 women with gdm data are median (25th, 75th percentiles), unless otherwise indicated. the median duration of follow - up was 8.0 years (interquartile range 4.510.8 years) with a median of five sets of ogtts, ivgtts, and bias per participant over the follow - up period. during follow - up, 41 women in the cohort (66%) experienced a deterioration of glucose tolerance over time, and diabetes developed in 27 of the women (44%), as determined by american diabetes association criteria (28). fourteen of the women (23%) experienced one or more additional pregnancies during follow - up, and 5 of them had gdm during these pregnancies. no significant quadratic trends of change over follow - up time were observed for any metabolic traits (p > 0.14). measures of adiposity, fasting glucose, 2-h glucose, fasting insulin, and moderate and total pa increased significantly over time (all p 0.10). table 2 presents associations between baseline total calorie intake and rates of change in metabolic traits over time. in the unadjusted analysis, higher total calorie intake at baseline was significantly associated with a faster decline in si and di (p = 0.029 and p = 0.027, respectively). although statistically insignificant, higher total calorie intake at baseline was associated with a faster increase in adiposity, and fasting and 2-h glucose levels, and a faster decrease in 2-h insulin levels and airg. adjustment for baseline age, bmi, parity, levels of pa, and baseline value of the trait had little impact on the results ; higher baseline calorie intake remained significantly associated with a faster decline in si (p = 0.035) and di (p = 0.021). although calorie intake did not change significantly over time among the entire cohort, an increase in calorie intake over time was positively associated with an increase in bmi, weight, and total body fat mass over time (p = 0.032, p = 0.024, and p = 0.025, respectively) after adjusting for baseline characteristics. change in calorie intake was not significantly associated with rates of change in metabolic outcomes (all p > 0.11). higher baseline calorie intake remained significantly associated with a faster decline in si (p = 0.05) and di (p = 0.034) after further adjusting for changes in calorie intake, bmi, levels of pa, and additional pregnancies during follow - up (adjusted-2). sensitivity analysis excluding the six women who met the who recommended level of pa did not change the conclusion. associations between baseline calorie intake and rates of change in morphometric and metabolic traits during a median of 8.0 years of follow - up for the 62 women with gdm log transformation was applied prior to calculating annualized rates of change. we tested whether the association between baseline calorie intake and rates of decline in si and di differed by whether or not 1) women experienced a deterioration in glucose tolerance, 2) diabetes developed, or 3) women had additional pregnancies during follow - up, using interaction tests in the mixed - effects models. interaction test results were not significant for deterioration of glucose tolerance and additional pregnancies (all p > 0.55) and were of only borderline significance for the development of diabetes during follow - up for si (p = 0.06 for si, p = 0.79 for di). further stratified analysis by follow - up diabetes status showed that the inverse association between baseline calorie intake and rates of decline in si and di was a little stronger for women in whom diabetes developed at follow - up (= 0.066 for si, = 0.035 for di) than for women who did not developed diabetes (= 0.033 for si, and = 0.016 for di). figure 1 depicts the covariate (from adjusted-2) adjusted geometric means for si, di, and fasting and 2-h glucose levels over the follow - up years when the cohort was divided at the median of the total baseline calorie intake (2,091 kcal / day). on average, the high - calorie group (median calorie intake 3,013 kcal / day) had a threefold faster decline in si (0.06 vs. 0.02 units / year, p = 0.10) and a 17% faster decline in di (810 vs. 692 units / year, p = 0.042) compared with the low - calorie group (median calorie intake 1,526 kcal / day). the high - calorie group also tended to have a faster increase in fasting glucose levels (0.08 vs. 0.06 units / year) and 2-h glucose levels (0.35 vs. 0.16 units / year) than the low - calorie group, although these differences did not reach statistical significance (p > 0.21). adjusted geometric means (sem) of si (a), -cell function (di) (b), ogtt fasting glucose level (c), and ogtt 2-h glucose level (d) over the follow - up years for women who had baseline total calorie intake above (high calories, open square, n = 31) and below (low calories, filled triangle, n = 31) the cohort median calorie intake (2,091 kcal / day) at baseline. for the macronutrient density analyses, we did not observe significant associations between specific densities from fat, carbohydrates, or protein and rates of change in si (p > 0.10) or di (p > 0.36) with or without adjustment for covariates. high fat density at baseline was significantly associated with rates of increase in bmi, percent of body fat and fat mass (p < 0.036 for each), and the rate of decrease in 30-min change in insulin (p = 0.031) after adjusting for covariates. high fat density at baseline was marginally associated with the rate of increase in fasting glucose levels (p = 0.07). in this long - term, prospective, observational study of hispanic women with a history of gdm, we made a novel observation that higher daily calorie intake at baseline under free - living conditions predicted a long - term decline in insulin sensitivity and -cell compensation. the predictive value was independent of baseline characteristics and changes in adiposity, calorie intake, levels of pa and additional pregnancies during follow - up. on average, the women in this cohort consumed a diet at baseline containing a slightly higher number of calories than recommended ; the total calorie intake did not increase over time. on this background, adiposity, insulin sensitivity, thus, our findings indicate that a prolonged high calorie intake may contribute to rising insulin resistance and falling -cell function, independent of obesity. a few short - term clinical trials have evaluated the impact of calorie intake on insulin sensitivity (9,29) and -cell function (11,12). a 25% restriction in calories for 6 months caused a significant decrease in fasting insulin levels in nonobese subjects (29). a caloric restriction to 800 kcal / day for 3 months reduced hepatic glucose production, and increased insulin sensitivity and insulin secretion in seven obese subjects with type 2 diabetes (9). a 3-month trial (11) showed that 74% of subjects increased both insulin sensitivity and -cell function after a diet intervention of 1,200 kcal / day. a recent study (12) showed that very low calorie intake (600 kcal / day) could reverse declines in -cell function and hepatic insulin sensitivity in 11 type 2 diabetes patients. in the current study, we showed that long - term high calorie intake was significantly associated with deteriorating insulin sensitivity and -cell compensation, independent of changes in obesity, which we previously found to be associated with declines in insulin sensitivity and -cell function in this cohort (15). the association with caloric intake was a little stronger among women in whom diabetes developed during follow - up than among women in whom diabetes did not develop. further adjustment for the rate of change in insulin sensitivity explained 46% of the association between baseline calorie intake and the rate of change in -cell compensation, but did not completely eliminate the association. it is also possible that high calorie intake contributes to lipotoxicity (30) and inflammation (15), which further contribute to -cell decline. high - fat diets have been shown to be associated with increasing adiposity, and impaired glucose tolerance and insulin action (25,3134). we found that higher baseline fat density was related with a faster increase in adiposity and fasting glucose levels. our results support the concept that high fat consumption contributes to obesity and rising fasting glucose levels. the fact that we did not observe significant associations between dietary fat density and the decline in insulin sensitivity and -cell function could be due to the relatively narrow range of consumption in our cohort, which limits the ability to detect associations. it is also possible that it is the total calorie intake that drives the decline in insulin sensitivity and -cell function, since foods are not consumed in isolation in a free - living condition. declining insulin sensitivity and -cell function lead to an increase in glucose ; however, glucose levels rise slowly in the process of declining -cell function prior to the onset of diabetes (1). this explains why the deleterious effect of high calorie intake was less manifested on changes in fasting and 2-h glucose levels than on changes in insulin sensitivity and -cell function in this study. the strength of this study is the long time of follow - up with detailed ogtt, ivgtt, diet, and pa assessments at multiple times. the dietary questionnaire was validated and calibrated for the hispanic population (17) and was administered at the same time as ivgtts. unlike previous studies that investigated the impact of short - term calorie restrictions, we provided more realistic information about long - term dietary intake in a free - living environment. first, the relatively small sample size only allowed us to detect relatively large effect sizes. these women are at a high risk for the development of diabetes, and they were generally inactive. the variation in calorie intake and metabolic profiles in this cohort may be smaller than in the general population, which precluded us from detecting some important associations. third, although we considered weight, bmi, total body fat, and waist - to - hip ratio, we do not have a direct measure of abdominal obesity. last, causal inferences may not be drawn since this is an observational study, and it is possible that there are other unmeasured factors that may explain some of the associations. in summary, we found that high baseline calorie intake in a free - living environment predicted declining insulin sensitivity and -cell compensation over time in hispanic women who are at high risk for type 2 diabetes. the associations were independent of baseline characteristics, rates of change in calorie intake, adiposity, levels of pa, and additional pregnancies during follow - up. we recommend controlling calorie intake to preserve insulin sensitivity and -cell function, and to prevent type 2 diabetes. | objectiveto assess associations between dietary intake and rates of change in insulin resistance and -cell function in hispanic women with prior gestational diabetes mellitus (gdm).research design and methodssixty - two nondiabetic hispanic women with pregnancies complicated by gdm completed oral and intravenous glucose tolerance tests and bioelectrical impedance measurements of body fat every 1215 months postpartum for up to 12 years. self - reported dietary intake was collected at all visits by structured food frequency questionnaires developed for hispanics. mixed - effects models were used to assess the relationship between dietary intake and rates of change in metabolic outcomes during follow-up.resultsthe median length of follow - up from the first postpartum evaluation was 8.0 years (interquartile range 4.510.8 years). at baseline, women were 32 5.7 years old and had a median calorie intake of 2,091 kcal / day. over the course of follow - up, dietary intake did not change significantly. higher baseline calorie intake was associated with a faster decline in insulin sensitivity, measured by the insulin sensitivity index (si) (p = 0.029), and -cell compensation, measured by the disposition index (di) (p = 0.027), over time. these associations remained after adjustment for baseline characteristics ; changes in bmi, calorie intake, levels of physical activity ; and additional pregnancies during the follow - up period. the median rates were 0.06 vs. 0.02 units / year for si and 810 vs. 692 units / year for di for women with baseline calorie intake above versus below the cohort median.conclusionshigh calorie intake is associated with a faster decline in insulin sensitivity and -cell compensation in hispanic women who are at high risk for type 2 diabetes, independent of adiposity. |
cardiac resynchronization therapy (crt) is a major treatment for selected patients with heart failure (hf). crt has been demonstrated to improve hf symptoms, exercise capacity, and quality of life, and to reduce hf hospitalization rates and mortality. neutrophil - to - lymphocyte (n / l) ratio is a new prognostic marker in patients with cad undergoing coronary angiography, percutaneous coronary intervention, and coronary artery bypass grafting. recently, higher n / l ratio has been associated with increased risk for mortality in patients with acute decompensated hf. however, no data exist about the association between n / l ratio and response to crt. seventy consecutive patients (mean age 5813 years ; 40 men) undergoing crt were included in the study. patients were selected according to following criteria : (1) chronic heart failure (new york heart association functional class iii or iv), (2) wide qrs interval (120 ms), and (3) left ventricular ejection fraction (lv ef) 35%. patients with hematological disease, cancer, ongoing systemic inflammatory conditions, and autoimmune disease were excluded from the study. the remaining 4 patients had right bundle branch block. clinical evaluation included the assessment of nyha functional class. written informed consent was obtained from all patients. lv leads were inserted by a transvenous approach through the coronary sinus into a cardiac vein of the free wall. patients received a biventricular pacemaker (insync iii, medtronic inc, minneapolis, minnesota) or a biventricular cardioverter - defibrillator (insync icd, medtronic inc, minneapolis, minnesota). patients were imaged in the left lateral decubitus position with a commercially available system (vivid 7, general electric - vingmed ultrasound, horten, norway). images were obtained with a 2.5-mhz broadband transducer at a depth of 16 cm in the parasternal and apical views (standard long - axis, 2- and 4-chamber images). standard 2-dimensional and color doppler data triggered to the qrs complex were saved in cine - loop format. lv volumes were calculated using the teicholz method and lvef was calculated from the conventional apical 2- and 4-chamber images using the biplane simpson s technique. all echocardiographic measurements after crt implantation were made with the device in active pacing mode. echocardiographic response to crt was defined by a 15% reduction in left ventricular end - systolic volume at 6-month follow - up. the samples were centrifuged for 10 min and blood counts were measured by using cell - dyn 3700 (abbott, il, usa) at baseline and 6 months later. serum c - reactive protein (crp) levels were measured by a fluorescent polarization immunoassay (abbott diagnostics, abbott park, illinois). the mann - whitney u test was used to assess differences in baseline clinical, echocardiographic, and hematological parameters between responder and non - responder patients. a comparison of the clinical, hematological and echocardiographic variables before and after crt was performed by paired sample t - test or wilcoxon signed - rank test. variables associated with crt response in univariate analysis were entered into a forward stepwise logistic regression model. seventy consecutive patients (mean age 5813 years ; 40 men) undergoing crt were included in the study. patients were selected according to following criteria : (1) chronic heart failure (new york heart association functional class iii or iv), (2) wide qrs interval (120 ms), and (3) left ventricular ejection fraction (lv ef) 35%. patients with hematological disease, cancer, ongoing systemic inflammatory conditions, and autoimmune disease were excluded from the study. the remaining 4 patients had right bundle branch block. clinical evaluation included the assessment of nyha functional class. written informed consent was obtained from all patients. lv leads were inserted by a transvenous approach through the coronary sinus into a cardiac vein of the free wall. patients received a biventricular pacemaker (insync iii, medtronic inc, minneapolis, minnesota) or a biventricular cardioverter - defibrillator (insync icd, medtronic inc, minneapolis, minnesota). patients were imaged in the left lateral decubitus position with a commercially available system (vivid 7, general electric - vingmed ultrasound, horten, norway). images were obtained with a 2.5-mhz broadband transducer at a depth of 16 cm in the parasternal and apical views (standard long - axis, 2- and 4-chamber images). standard 2-dimensional and color doppler data triggered to the qrs complex were saved in cine - loop format. lv volumes were calculated using the teicholz method and lvef was calculated from the conventional apical 2- and 4-chamber images using the biplane simpson s technique. all echocardiographic measurements after crt implantation were made with the device in active pacing mode. echocardiographic response to crt was defined by a 15% reduction in left ventricular end - systolic volume at 6-month follow - up. the samples were centrifuged for 10 min and blood counts were measured by using cell - dyn 3700 (abbott, il, usa) at baseline and 6 months later. serum c - reactive protein (crp) levels were measured by a fluorescent polarization immunoassay (abbott diagnostics, abbott park, illinois). the mann - whitney u test was used to assess differences in baseline clinical, echocardiographic, and hematological parameters between responder and non - responder patients. a comparison of the clinical, hematological and echocardiographic variables before and after crt was performed by paired sample t - test or wilcoxon signed - rank test. variables associated with crt response in univariate analysis were entered into a forward stepwise logistic regression model. medication included angiotensin - converting enzyme inhibitors in 91%, beta - blockers in 89%, and diuretics in 90%. the baseline clinical, hematological and echocardiographic parameters for responders and non - responders showed no statistically significant differences (table 2). after 6 months, lvef had significantly increased from 217% to 3411% in responders (p=0.001). there was no significant increase in lvef in non - responders at 6-month follow - up (216% vs. 246%, p=0.06). mean nyha functional class in responders and non - responders were 3.10.6 and 3.20.5, respectively (p=0.62). at 6 months, mean nyha functional class there was no significant change in mean nyha functional class in non - responders (3.20.5 vs. 30.2, p=0.26). n / l ratio was decreased significantly, from 2.41 to 2.00.7 in responder patients (p=0.03). however, n / l ratio was increased from 31.7 to 3.61.5 in non - responder patients (p=0.37) (table 3). crp was decreased significantly, from 0.540.36 to 0.390.28 in responder patients (p=0.001). crp increased significantly, from 0.740.42 to 1.050.52 in non - responder patients (p=0.006) in multivariate analysis, significant associates of echocardiographic response to crt were evaluated adjusting for age, etiology of cardiomyopathy, baseline lvef, nyha functional class, crp, and baseline n / l ratio. baseline n / l ratio was the only predictor of response to crt (or 1.506, 95% ci, 1.0112.243, p=0.035). cardiac resynchronization therapy is considered an important treatment option of patients with wide qrs and advanced chf who are receiving optimal medical treatment. however, prediction of response to crt remains problematic and an important proportion of patients do not respond to crt, although they are selected according to current patient selection criteria [810 ]. additional echocardiographic, electrocardiographic, and blood markers have been investigated in various studies to find patients most likely to respond crt [1114 ]. to the best of our knowledge, our study is the first to investigate the prognostic significance of n / l ratio in hf patients who underwent crt. lymphocytopenia has been independently associated with increased mortality in patients with acute and chronic hf. downregulation of the proliferation and differentiation of lymphocytes, neurohumoral activation, and lymphocyte apoptosis have been suggested as potential mechanisms for lymphocytopenia. in our study, lymphocyte count was lower in the non - responder patient group. although the difference in lymphocyte count between responder and non - responder patients was not significant, low lymphocyte count in non - responder patients may reflect a more advanced disease stage. in addition, lymphocyte and neutrophil counts were not significantly changed in responder and non - responder patient groups. however, lymphocyte count was increased and n / l ratio was significantly decreased in responder patients. crp is a pentameric protein associated with inflammation, and elevated crp levels have been observed in hf patients. also, higher crp levels were associated with advanced hf and independently with mortality and morbidity. baseline crp levels were not statistically different, but crp levels were significantly reduced in responder patients in contrast to non - responder patients. the increased lymphocyte count, decreased n / l ratio, and decreased crp in responder patients may reflect decreased systemic inflammation with crt response, which in turn may help in development of reverse remodelling. in addition, the importance of baseline cardiac dimensions in prognosis and response to crt has been reported previously [2022 ]. the mean left ventricular end - diastolic diameter (lvedd) in responder patients was larger than in non - responder patients. although the difference was not statistically significant, increased mean lvedd in non - responder patients may also relate to a more progressive disease and extensive scar tissue. our study was limited in that it was a single - center, nonrandomized design and the study sample was small ; a larger study population might increase the significance of the presented data. our data suggest that determination of n / l ratio at baseline could help identify patients with response to crt. | backgroundneutrophil - to - lymphocyte (n / l) ratio has been associated with adverse outcomes in patients with acute coronary syndromes and increased risk for long - term mortality in patients with acute decompensated heart failure. we aimed to investigate the prognostic value of neutrophil - to - lymphocyte ratio on response to cardiac resynchronization therapy (crt).material / methodsseventy consecutive patients (mean age 5813 years ; 40 men) undergoing crt were included in the study. hematological and echocardiographic parameters were measured before and 6 months after crt. echocardiographic response to crt was defined as a 15% reduction in left ventricular end - systolic volume at 6-month follow-up.resultsafter 6 months of crt, 49 (70%) patients were responders. after 6 months, left ventricular ejection fraction (lvef) had significantly increased, from 217% to 3411% in responder patients (p=0.001). n / l ratio decreased significantly, from 2.41 to 2.10.7 in responders (p=0.04). in multivariate analysis, significant associates of echocardiographic response to crt was evaluated adjusting for age, etiology of cardiomyopathy, baseline lvef, new york heart association functional class, c - reactive protein, and baseline n / l ratio. baseline n / l ratio was the only predictor of response to crt (or 1.506, 95% ci, 1.0112.243, p=0.035).conclusionsn / l ratio at baseline could help to identify patients with response to crt. |
there are claims in the field of voluntary action that the sense of agency and hence of volition relies on a post - hoc (i.e., non - causal) evaluation of performed actions rather than on predictive mental processes (see reviews by wegner and wheatley, 1999 ; wegner, 2002 ; haggard, 2005, 2008, 2009 ; knoblich and sebanz, 2005 ; hallett, 2007). accordingly and in contrast with the priority principle (namely that thought comes before action ; wegner, 2002) the stand of a postdictive chain of mental events posits that action is promoted before thought (i.e., decision / intention to act) which is but an a posteriori interpretation of that action hence corrupted by the outcome of that action. (thought predicts / causes the ensued action) and postdictive with non - causal (action causes thought). most of the empirical approaches to the predictive postdictive debate capitalized on the comparison between the introspected timing of the decision / intention to act and the timing of that action 's precursory neural activity (libet, 1993 ; lau., 2007 ; this literature consistently showed that the introspected decision timing trails (sometimes by seconds ; soon., 2008) the timing of premotor activity. (e.g., nachev., 2008), about subject 's capability of estimating the moment of his intention to act and about the techniques used for such assessments (pockett, 2002 ; danquah., 2008). here we introduce a new method of testing the stand of a postdictive appraisal of one 's intentionality. to be specific, we equate here intentionality with subjects capacity of assessing the physical causes of their actions inasmuch as the notion of intentionality is meaningless in the absence of such assessment. the method capitalizes on subject 's retrospective judgments on the feasibility of a just executed synchronization motor task and on whether this task had been executed in a speeded/reactive or delayed/intentional mode. such distinction is not more conceptually debatable (pashler, 1998 ; nachev., 2008 ; haggard, 2009) than the notion of volition / free will itself, ultimately an introspective belief. here, the understanding is that a speeded/reactive action is spontaneous or irrepressible, hence non - intentional, while delayed/intentional requires pondering of stimulus parameters (such as speed) and internal factors (such as one 's reaction time, rt) and involves top - down processes (frank. a reactive action can be objectively operationalized as an action whose latency with respect to stimulation is in the range of subject 's simple rt and is based on bottom - up processes. this does not mean however that subjects necessarily use such a criterion for segregating their spontaneous vs. delayed responses. subjects were requested to synchronize a key - press with the termination of a synchronization interval (si ; randomly shorter or longer than their rt) and thereafter to judge (in separate sessions) whether that interval had been long enough to allow synchronization (q1), whether their motor response had been reactive or delayed (q2), or whether si had been long or short (q3). a valid answer to q1 requires subjects to compare their estimation of the current si with their introspected rt, irt (si irt). on the assumption that irt is a constant (say the introspected mean rt acquired through life experience or within the very first trials of a rt experiment), subjects responses should correlate with si irrespectively of their fluctuating synchronization response time (rst), i.e., the outcome of their action. however, the postdictive stand requires that their response to q1 should be based on their trial - by - trial rt appraisal, a capacity known to exist (james, 1890 ; gorea., 2010). to answer q2 subjects should compare their current rst and irt (rst irt) so that their responses should correlate with their fluctuating rst but not (or less) with si. q2 is all the more meaningful given that for the same si subjects showed a bimodal rst distribution with means separated by more than 100 ms (see results). to answer q3 subjects only need to appraise the mean si and the current si (si si). basing their responses to qs 1 and 3 on si would substantiate the view that subjects have an objective appraisal of the external world (i.e., si). in contrast, the stand of a postdictive / subjective appraisal of the world and hence of our intention to act upon it (i.e., react or delay our motor responses) predicts that subjects responses to qs 13 will correlate with their rst and/or with the rst for this to be the case, subjects must be able to appraise (consciously or not) and use their rst on a trial - by - trial base. the present results show that subjects responses to the three questions asked mostly correlate with si. we take this as evidence against the postdictive hypothesis. in the case of q2, subjects reliance on si shows that they prefer to rely on the external world even though such reliance is inappropriate (an over - objectivization). this suggests that subjects do not have conscious access to or, alternatively, do not remember what their decision (to react or delay their response) had been. the purpose of preliminary experiments was to assess subjects simple rt in a standard rt paradigm using the stimuli of the two main experiments. these rts were subsequently compared with subjects points of subjective synchronization (pss) derived as described below. main experiment 1 was run with a large but sparse sample of si that in conjunction with subjects answers to q1 were used to choose a narrower but denser range of si to be run in the main experiment 2. in the latter subjects the stimulus was a 1 thick virtual annulus (the gauge) with a 3 external edge radius (figure 1a) that started to fill up (randomly at one out of eight locations marked by a radial bar) either up white (100 cd / m) or in black (0.05 cd / m) on a 50 cd / m gray background. the filling gage was displayed on a 19 e96f+sb viewsonic screen (1024 768 pixels, 100 hz refresh rate) 40 cm away from observers eyes. the filling - up was always counterclockwise (figure 1b) and occurred randomly within one out of seven (0, 100, 200, 250, 300, 400, 500 ms ; preliminary 2 and experiment 1), or out of 21 sis (10 ms apart within a range of 200 ms more or less centered about each observer 's rt ; experiment 2 ; see procedure). stimulus presentation and response recording were controlled using the psychtoolbox (brainard, 1997 ; pelli, 1997) under matlab. spatial (a) and temporal (b) display of the stimulus. note the different ranges of the si used and the different questions asked in experiments 1 and 2. preliminary 1 : participants were asked to react (press a key) as soon as possible to the appearance of the annular gage that was filled up from the start (si = 0). preliminary 2 : as above but this time with seven sis (hence speeds) between 0 and 500 ms (see stimuli) randomized across trials. there were 80 trials per si and per subject distributed in two sessions. comparing the mean rts obtained in preliminary 1 and 2 as no such effect was found, mean rts were subsequently compared with subjects pss derived from their binary classification responses in experiments 1 and 2. in both these experiments participants were asked to synchronize their key - presses with the moment the filling of the gage was completed. in experiment 1 after each motor synchronization response participants provided a binary response to q1 : q1 : has the current si been long enough for you to synchronize with ? responses as a function of si is referred to as participant 's pss derived relatively to the si, psssi. for the reasons given below, this psssi was used to determine for each subject a narrower and denser si range to be run in experiment 2. in this second experiment, subjects responded to the following two additional questions : q2 : have you reacted or delayed your response to achieve synchronization ? q3 : has the current si been short or long ? to any of the questions above subjects responded by pressing the left (yes : long enough, reacted, short) or right arrow key on the keyboard. observers were specifically and repeatedly instructed not to base their response on the delay between the gauge filling up completion and their rst. clearly, very short (si rt) and very long (si rt) sis would (and did) entail 100% reliable responses to q1 as such intervals will most obviously be beyond and within subject 's synchronization capacities independently of his rst, or even in the absence of any motor response. this would also be the case for qs 2, 3 (see introduction and below). instead, an si range narrowly limited about the reaction / proaction transition point as given by psssi (and, as it will be shown, close to participant 's rt), will entail much less reliable answers to any of the three questions asked. this opens the possibility that participants will appeal to their introspectively estimated rst as an alternative source of information (even though less reliable than a. an additional advantage of using a relatively narrow si range about each participant 's psssi is that it dilutes the correlation between si and rst, a necessary condition for disentangling (at least partly) participant 's reliance on one or the other. reliance on rst should be even more pronounced given that subjects can compare the moment of the completion of the gage (a visual signal) and their own rst (motor and proprioceptive signals). under such conditions, observers are capable of judging their synchronization response (i.e., whether it had been early or delayed) with an accuracy of less than 50 ms, more than twice better than their perceptual estimation of the si they have to synchronize with (gorea., 2010). given the considerations above, experiment 2 was a re - run of experiment 1 but with a 200 ms si range centered on each participant 's psssi. each of the three questions was asked and answered in blocked sessions. there were 40 trials per si and per session (i.e., 40 7 = 280 trials / session) repeated four times (i.e., a total of 160 trials per si, participant and question asked). they were five master students (four male) with normal or corrected to normal vision aged in - between 2325 years. only one of them had had extensive training with the specific paradigm and was acquainted with the purpose of the experiment. the stimulus was a 1 thick virtual annulus (the gauge) with a 3 external edge radius (figure 1a) that started to fill up (randomly at one out of eight locations marked by a radial bar) either up white (100 cd / m) or in black (0.05 cd / m) on a 50 cd / m gray background. polarity was swapped on each trial to prevent adaptation effects. the filling gage was displayed on a 19 e96f+sb viewsonic screen (1024 768 pixels, 100 hz refresh rate) 40 cm away from observers eyes. the filling - up was always counterclockwise (figure 1b) and occurred randomly within one out of seven (0, 100, 200, 250, 300, 400, 500 ms ; preliminary 2 and experiment 1), or out of 21 sis (10 ms apart within a range of 200 ms more or less centered about each observer 's rt ; experiment 2 ; see procedure). stimulus presentation and response recording were controlled using the psychtoolbox (brainard, 1997 ; pelli, 1997) under matlab. spatial (a) and temporal (b) display of the stimulus. note the different ranges of the si used and the different questions asked in experiments 1 and 2. preliminary 1 : participants were asked to react (press a key) as soon as possible to the appearance of the annular gage that was filled up from the start (si = 0). preliminary 2 : as above but this time with seven sis (hence speeds) between 0 and 500 ms (see stimuli) randomized across trials. there were 80 trials per si and per subject distributed in two sessions. comparing the mean rts obtained in preliminary 1 and 2 as no such effect was found, mean rts were subsequently compared with subjects pss derived from their binary classification responses in experiments 1 and 2. in both these experiments participants were asked to synchronize their key - presses with the moment the filling of the gage was completed. in experiment 1 after each motor synchronization response participants provided a binary response to q1 : q1 : has the current si been long enough for you to synchronize with ? responses as a function of si is referred to as participant 's pss derived relatively to the si, psssi. for the reasons given below, this psssi was used to determine for each subject a narrower and denser si range to be run in experiment 2. in this second experiment, subjects responded to the following two additional questions : q2 : have you reacted or delayed your response to achieve synchronization ? to any of the questions above subjects responded by pressing the left (yes : long enough, reacted, short) or right arrow key on the keyboard. observers were specifically and repeatedly instructed not to base their response on the delay between the gauge filling up completion and their rst. clearly, very short (si rt) and very long (si rt) sis would (and did) entail 100% reliable responses to q1 as such intervals will most obviously be beyond and within subject 's synchronization capacities independently of his rst, or even in the absence of any motor response. this would also be the case for qs 2, 3 (see introduction and below). instead, an si range narrowly limited about the reaction / proaction transition point as given by psssi (and, as it will be shown, close to participant 's rt), will entail much less reliable answers to any of the three questions asked. this opens the possibility that participants will appeal to their introspectively estimated rst as an alternative source of information (even though less reliable than a. an additional advantage of using a relatively narrow si range about each participant 's psssi is that it dilutes the correlation between si and rst, a necessary condition for disentangling (at least partly) participant 's reliance on one or the other. reliance on rst should be even more pronounced given that subjects can compare the moment of the completion of the gage (a visual signal) and their own rst (motor and proprioceptive signals). under such conditions, observers are capable of judging their synchronization response (i.e., whether it had been early or delayed) with an accuracy of less than 50 ms, more than twice better than their perceptual estimation of the si they have to synchronize with (gorea., 2010). given the considerations above, experiment 2 was a re - run of experiment 1 but with a 200 ms si range centered on each participant 's psssi. there were 40 trials per si and per session (i.e., 40 7 = 280 trials / session) repeated four times (i.e., a total of 160 trials per si, participant and question asked). they were five master students (four male) with normal or corrected to normal vision aged in - between 2325 years. only one of them had had extensive training with the specific paradigm and was acquainted with the purpose of the experiment. the median and mean rts assessed with the filled gage (227.4 15.9 ms and 230.6 13.1 ms ; preliminary 1) and with the randomized filling - up durations (230.4 8 ms and 242.6 12.1 ; preliminary 2) were not statistically different. figure 2a presents mean rsts for each participant (different symbols ; keep in mind that rt and rst refer to response time measured in speeded and in a synchronization response mode, respectively) as a function of si in experiment 1. this representation is not quite legitimate as at least three of the seven rst means per subject were derived from bimodal rst distributions (as assessed by akaike information criterion, aic ; akaike, 1974), with all subjects showing bimodal rst distributions for si = 0 and for si = 500 ms. these bimodal rst distributions for the two extreme sis are exemplified in figures 2a1,a2 for a typical subject, sl). the average difference between the means of the two distributions is 129 ms for si = 0 and of 109 ms for si = 500 ms. averaging rsts over such bimodal distributions accounts for subjects too early synchronization responses (i.e., rsts below the main dashed diagonal) for the longest two sis (400 and 500 ms ; for such sis, subjects show perfect synchronization under blocked si conditions ; gorea., 2010). while the source of this bimodality is of critical interest in understanding the synchronization process (an issue addressed in an ongoing study) it is worth noting, however, that the means of the fastest rst distributions averaged over the five subjects for si = 0 (249 29.8 ms) are close to and not significantly different from their mean rts assessed in the two preliminary (rt) experiments (230.6 13 and 242.6 12.1 ms). this suggests that in mixed si blocks including sis too short to synchronize with, subjects behavior alternates in about equal proportions between reactive and delayed response modes. synchronization response times (rst) as a function of si in experiments 1 (a) and 2 (b) of all five participants (different symbols) together with representative bimodal rst distributions of participant sl for si = 0 (a1) and si = 500 ms (a2) as well as a representative cumulative gaussian fitted to this subject 's yes the dashed main diagonals in (a) and (b) show perfectly synchronized rsts. the double arrowed right - angle lines in a3 point to this subject 's rst at 50% yes responses (i.e., the mean of the fitted gaussian), i.e., his point of subjective synchronization referred to si (psssi). figure 2a3 displays the cumulative gaussian (,) fitted to subject 's sl yes responses to q1 in experiment 1 (neider mead simplex algorithm). the means of such functions fitted to each participant 's yes responses are their psssi and have been used as midpoints of the 200 ms si range tested in experiment 2. a first notable observation from figure 2 is that the psssi averaged over the five participants in experiment 1 (246.4 34.2 ms) are similar to these participants mean rts assessed in the preliminary experiments and to their mean rsts derived from their fastest rst distributions for si = 0 (249.0 29.8 ms). this suggests that subjects have an accurate implicit knowledge of their mean rt and use it when answering q1. the restricted si range about each participant 's psssi used in experiment 2 (in - between 117183 to 317383 ms) ensured that they operated in an uncertainty react / delay range and that their rsts were minimally correlated with si (so as to maximize the disentanglement between subjects putative reliance, when answering any of the three questions, on si from their reliance on rst and, as a consequence, on the si - rst difference). that this was achieved is demonstrated by the shallow slopes [0.060.3 ; r (0.110.48) ] of the mean rst vs. si functions displayed for each participant in figure 2b (same symbols as in figure 2a). it remains however that for this restricted 200 ms si range, the rst distributions covered an average range of 214 27 ms across the five subjects, much smaller than the rt range obtained in the preliminary rt experiments over an equivalent si range (126 9 ms). this indicates that subjects tempted to synchronize their key - press with the completion of the si rather than reacting to the onset of the gage. on a number of occasions, subjects may have nonetheless responded to the onset of the gage (i.e., reacted) rather than to its completion. responses of the same representative participant as in figures 2a1a3 for each of the three questions asked (circles, squares and triangles for q1, q2, and q3) together with the corresponding cumulative gaussian fits as a function of si (3a), rst (3b), and si - rst (3c). whatever the question asked, the steepest sigmoids (smallest) are obtained when the yes responses are related to si (si = 36.9 1.7 ms), slightly shallower when related to si - rst (si - rst = 43.1 3.0 ms) and flat for all practical reasons when related to rst (rst = 235.8 102.2 ms). these observations are true for all participants (with two marginal exceptions out of 15 comparisons for q1, subjects md and cr) as shown in table 1. while the possibility that subjects may have partially based their judgments on the si - rst difference can not be entirely excluded, the present results show that this difference was not their main source of information. responses to qs1 - 3 (circles, squares, and triangles, respectively) of a representative subject (sl) as a function of si (a), rst (b), and si - rst (c) together with the best cumulative gaussian fits (sigmoids of corresponding colors). standard deviations of the cumulative gaussians fitted to subjects yes responses to each of the three questions asked in experiment 2 as a function of the synchronization interval (si), response time (rst), and si - rst (si - rst ; odd cols) together with the corresponding point - biserial correlations (rpb ; even cols). at a first look, the accuracy of participants judgments appears to depend on the question asked (q1 = 170.9 134.1 ; q2 = 68.3 23.8 ; q3 = 76.7 38.1 ms) but this is mainly due to the very inaccurate responses to q1 when related to rst (rst, q1 a two - way anova (factors : question - type and relevant index, i.e., si, rst, and si - rst) confirms these observations, namely a significant index effect [f(2,8) = 13.8, p = 0.021 ] but no question - type effect (f = 5.3, p = 0.076). a partial comparison reveals a significantly steeper si - related than (si - rst)-related slope [f(1,4) = 3.44, p = 0.02 ]. table 1 also displays the point - biserial correlations (rpb ; lev, 1949) between the binary (yes / no) and the continuous (si, rst, and si - rst) variables. as it should, the steepness of the fitted functions and rpb show a high negative correlation (r = 0.62, p 0.001). in short, the data clearly show that whatever the question asked participants base their responses on the actual duration of si with practically no postdictive interference of their motor synchronization response. additional support for this conclusion transpires from a closer look at subjects psss and rts. as already noted, rts are practically identical when directly measured in the preliminary rt experiments with the gage instantaneously filled (si = 0) or filling - up over 0 to 500 ms intervals or when derived from the fastest rst distribution to the instantaneously filled up gage in the synchronization experiment 1 (black circle, square, and triangle in column rt of figure 4). to answer the introspective qs 1 and 2, the fact that the psssi (column pss_si in figure 4) derived from their answers to these questions (q1, solid red and open blue circles for experiments 1 and 2, respectively ; q2, open blue square, experiment 2) are about equal to their rts, while the corresponding pssrst (column pss_rst ; same colors and symbols) differ markedly from the rts as well as across experiments (circle vs. square) and subjects (error bars) support the notion that subjects introspection was based on the sis rather than on their own rst. as the mean of the sis used in experiment 2 was by construction equal to the psssi derived in experiment 1 (red solid circle), the psss derived from subjects responses to q3 (that bore on the si itself) should coincide with the mean si. this is the case for the psssi but not for the pssrst implying once again that subjects based their responses to q3, as they should have, on the estimated si and not on their own rst. hence, subjects base their judgments on the objective duration of the interval they have to synchronize with whether in response to an introspective (q1, q2) or objective (q3) question. finally, the possibility that subjects calibrated their responses (whatever the question asked) relatively to their average rst is not supported by the data as these averages (av_rst column in figure 4) are significantly above subjects rt and psssi. reaction times (rt ; black symbols), points of subjective synchronization derived from the cumulative gaussian fits of the yes responses as a function of the si (pss_si) and as a function of subjects synchronization response time (pss_rst) and the means of these rst (av_rst) as measured or derived from the different experiments. rts measured in response to the instantaneously filled (si = 0) gage and to a gage filling over 0 to 500 ms intervals are shown as the black circle and square, respectively. rt derived from the fastest rst distribution to the instantaneously filled gage in the synchronization experiment 1 is shown a the black triangle. pss and av_rst derived from responses to questions 1, 2, and 3 are shown as circles, squares, and triangles with their colors referring to the experiment where they have been assessed (red, experiment 1 ; blue, experiment 2). the solid red circle is the psssi from experiment 1 used as the midpoint of the si range tested in experiment 2. each symbol is the average over the five participants with the vertical bars showing 1se. the present experiments demonstrate that despite observers having explicit feedback on their motor synchronization response with the completion of a random time interval, their introspective judgment of their synchronization capacity relative to the length of this interval (q1) or of them having been in a reactive or delayed motor response mode (q2) are poorely, if at all, related to the timing of their actual synchronization response but strongly related to the physical duration of this interval. in fact, the accuracy of such introspective judgments is no different from that assessed when subjects are asked to judge the absolute duration of these same intervals (q3). the statistical analysis shows that the accuracy of subjects judgments is higher when these judgments are referred to the time interval itself rather than to the difference between this interval and the timing of their synchronization response. the data also show that subjects pss are not significantly different from their mean rt and less variable (over experiments and subjects) when derived from subjects answers to any of the three questions asked as a function of the actual duration of the si than as a function of their synchronization rst. while it has been shown that judgements of a stimulus having entailed an action (such as the location of a saccade target and of the corresponding saccade landing) are not biased by putative motor errors (collins., 2009 ; munuera., 2009 ; ostendorf., 2010), the present study is the first to show that introspection on one 's actions in relation to the events having triggered them is based on these events rather than on the outcome of the actions they have entailed. notwithstanding, such introspection appears to be calibrated with respect to subjects introspected mean reaction time. the observation that responses to q2, that bears on subjects past decision to react or to delay action, do not correlate with their actual rst implies that, at least in the present experimental conditions, subjects can not access or, alternatively, do not remember what this decision had been. this observation raises serious doubts about the experimental paradigm used in many studies where subjects were asked to specify when their decision to act had been taken (e.g., libet, 1993 ; see pockett, 2002 ; danquah., 2008). reactive and delayed movements are known to reflect (e.g., frith., 1991 ; jahanshahi., 1995 ; jenkins., 2000 ; brass and haggard, 2007 ; haggard, 2008, 2009) and actually be triggered by the activity of distinct neural networks hypothesized to subtend reflexive and deliberate actions, respectively (desmurget., 2009). while the distinction between such responsive modes is fuzzy (nachev. 2011), it is widely accepted that they involve bottom - up vs. top - down processes (e.g., frank., 2009 ; ridderinkhof., the anatomo - physiological distinction between the two response modes does not appear to be sustained by subjects presently assessed introspective judgments of their actions having been reactive or delayed (q2). this is so even though, in contrast with previous laboratory manipulations of volition (of the kind have free will now ! see haggard, 2009), subjects switch from a reactive to a delayed response mode was entirely under their own control. the conundrum appears to be solved, though not conceptually, by desmurget.s (2009) astonishing findings that stimulation of distinct cortices may trigger the intention and desire to move a limb or the lips together with the feeling of having done so in the absence of any corresponding electromyographic activity (inferior parietal regions) and reciprocally, an overt movement without the feeling of having executed it (premotor area). in as much as the consciousnesses of acting and the action itself appear to be dissociated (or at least dissociable), it is not more remarkable to admit that reactive and delayed actions may not be consciously dissociable either. such dissociations are actually amply documented in amputees and anosognostic patients (for the first type of dissociation) and in patients with utilization behavior or with delusions of control (for the second type of dissociation ; frith., 2000). temptingly, one would take such intention / action dissociations as evidence in favor of the notion that free will is but an illusion (wegner, 2002). the present results provide paradoxical denotations in this respect. on the one hand, subjects incapacity of retrospectively classifying their reactive vs. delayed motor response (q2) based on the actual latency of this response, the fact that subjects judge their capacity of synchronizing a motor response with a random time interval based on their estimation of this interval rather than on their introspected response time (q1) implies that their judgment is causal (or predictive) in nature (i.e., not based on the outcome of their action or postdictive), a prerequisite of the free will stand. hence, not astonishingly, the present experiments and results reveal an intrinsic duality of the free will concept debated since the antiquity (e.g., bobzien, 1998 ; o'keefe, 2005). the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | by means of a new visuo - motor synchronization paradigm we test the frequently made proposition that one 's feeling of having voluntarily made a decision to act is in fact postdictively established contingent on the outcome of his action rather than on its aim. subjects had to (1) synchronize a key - press with the end of a random synchronization interval (si) shorter or longer than their reaction time (rt) and (2) judge thereafter whether (q1) si had been long enough to allow synchronization, (q2) their motor response had been reactive (i.e., close to their rt) or delayed, or (q3) whether si was short or long. si was denoted by the filling - up time of an annular gauge. in principle, the synchronization key - press should be reactive for si rt and delayed in proportion with si for si > rt. instead, response time distributions were bimodal for the shortest (0 ms) and longest (500 ms) sis and widely spread for intermediate sis. to all three questions asked, subjects responses strongly correlated with si itself (r = 0.620.76) and barely with their actual response times (r = 0.030.42). hence subjects introspective judgments on their trial - by - trial potential capability to synchronize their motor response (q1) and on their reactive vs. delayed response mode reflected the objective cause of their action rather than being corrupted by its outcome (namely their actual response time). that subjects could not reliably decide whether their motor response was reactive or delayed implies that they did not have retrospective access to (or did not remember) their motor decisions which amounts to say that they could not decide on the intentionality of their actions. |
a total of 231 cases of dlbcl treated with r - chop (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) or r - chop like (with or without radiotherapy or surgery) chemotherapy were selected for the study. cases were retrieved from the archival files from the department of pathology, severance hospital, from 2005 to 2011. all cases were independently reviewed by two pathologists (s.o.y. and s.h.k.) based on current world health organization criteria, and discordant cases were consulted to other expert hematopathologists. in hotair expression analysis, 164 cases were selected from the above 231 cases and investigated after quality assessment of extracted rna. all study protocols were performed according to the ethical guidelines of the world medical association declaration of helsinki ethical principles for medical research involving human subjects. formalin fixed paraffin embedded (ffpe) tissue sections were prepared and stained with hematoxylin and eosin, and then the tumor areas were confirmed and marked under the microscope. the marked areas mainly contained packed tumor cells, and the stromal component was less than 10% of the marked area. the unstained slides of ffpe tissues were prepared after dissecting ffpe tissue blocks at 10-mm thickness using a microtome, and the marked area was scraped using a scalpel blade. total rna was isolated using an rneasy ffpe kit (qiagen, hilden, germany) according to the supplier s instructions. extracts of rna were verified by measuring the ratios ofa260/a280 and a260/a230 with a nd-1000 nanodrop spectrophotometer (nanodrop, wilmington, de, usa). the expression patterns of hotair were assayed by relative quantification using expression of the house - keeping gene glyceraldehyde-3-phosphate dehydrogenase (gapdh). primers for hotair and gapdh were as follows : hotair (forward, 5'-agccagaggagggaagagag-3 ' ; reverse, 5'-tcccgttccctagattttcc-3 ') and gapdh (forward, 5'-caaattccatggcaccgtca-3 ' ; reverse, 5'-atcgccccacttgattttgg-3 '). primers of hotair were designed to detect all three transcript variants (transcript variant 1, 3, and 2). in brief, a 20 l mixture containing 1.0 l of cdna, power sybr green pcr master mix (applied bio - systems, carlsbad, ca, usa), 1.0 l of 10 pmol/l forward primer, 1.0 l of 10 pmol/l reverse primer, and 7.0 l of tertiary distilled water was prepared. amplification was performed using a step one plus real - time pcr instrument (applied biosystems) under the following conditions : denaturation at 94c for 10 minutes, followed by 35 cycles of 94c for 15 seconds, 55c for 30 seconds, and 72c for 30 seconds (fluorescence signal acquisition was performed at this phase). immediately after amplification, all samples were analyzed in triplicate to confirm reproducibility. using the step one plus real - time pcr system software ver. 2.1 (applied biosystems), the threshold cycle (ct, beginning of the polymerase chain reaction exponential phase) value of amplified hotair was normalized versus that of amplified gapdh (2). after quality assessment of extracted rna and expression analysis of the housekeeping gene gapdh, 164 cases were finally selected for the analysis of hotair expression. normal palatine tonsil tissue was obtained from 10 cancer - free individuals and used as age - matched cancer - free controls. the cutoff value for high expression of hotair (hotair) was determined at the uppermost value among those of normal control tonsil tissues. the hematoxylin and eosin slides were reviewed and two representative core tissues of the tumor area were selected in each case. core tissues (3 mm in diameter) were taken from the individual donor blocks and arranged in new recipient tissue microarray paraffin blocks using a trephine apparatus. immunohistochemistry of ezh2 (1:100, invitrogen, carlsbad, ca, usa), suz12 (1:50, abcam, cambridge, uk), eed (1:1,000, abcam), h3k27me3 (1:100, clone c36b11, cell signaling technology, beverly, ma, usa) was performed using the ventana benchmark xt autostainer (ventana medical systems, tucson, az, usa). immunohistochemistry for c - myc (1:50, clone y69, abcam), bcl2 (1:50, novocastra, newcastle, uk), cd10 (1:100, novocastra), bcl6 (rtu, novocastra), and mum1 (1:200, cell marque, rocklin, ca, usa) was performed using the leica bond - iii autostainer (leica biosystems, newcastle upon tyne, uk). for in situ hybridization for epstein - barr virus (ebv), the inform eber probe (ventana medical systems) was used with the ventana benchmark xt autostainer (ventana medical systems) and ish iview blue detection kit (ventana medical systems). for ezh2, suz12, eed, and h3k27me3, staining intensity (0, no staining to weak ; 1, moderate to strong) and proportion of positive tumor cell nuclei (0, 60 (p 60 (p<.001), eastern cooperative oncology group performance status 2 (p<.001), elevated lactate dehydrogenase (p<.001), extranodal sites 2 (p=.001), ann - arbor stage iii iv (p=.003), and international prognostic index (ipi) risk 3 (p<.001) showed significant association with reduced overall survival rate. expression of c - myc and co - expression of c - myc / bcl2 were also related to inferior overall survival rate (p=.032 and p=.006, respectively). among the prc pathway markers (ezh2, suz12, eed, and h3k27me3), suz12 and h3k27me3 were related to inferior overall survival rate (p=.028 and p=.010, respectively) (fig. hotair, however, was significantly related to superior overall survival rate than hotair (p=.004) (fig., h3k27me3 revealed an independent effect on poor overall survival (hazard ratio, 2.0 ; p=.023). ipi risk 3 was still determined as an independent prognostic factor (hazard ratio, 3.5 ; p<.001), while hotair showed a tendency to be related to improved survival with marginal significance (hazard ratio, 0.5 ; p=.086). in our previous study, high level of global h3k27me3 was found to be a negative prognostic indicator in patients with dlbcl. this subsequent study aimed to explore the role of hotair, possibly functioning via induction of h3k27me3. we found the positive correlation between prc2 proteins, global h3k27me3 levels, and c - myc in dlbcl as expected. in the present study, the positive correlation among prc2 proteins (ezh2, suz12, and eed), global h3k27me3 levels, and c - myc was also confirmed in dlbcl as expected. in addition these findings could support that the lncrna hotair may be involved in inducing h3k27me3 through recruiting polycomb repressive complex, the methyltransferase ezh2 and core accessory proteins, suz12 and eed. based on many other studies, interaction between c - myc and prc2 (ezh2, suz12, and eed) is well known in the tumorigenesis of various cancer types including lymphomas. protein c - myc interacts with ezh2 as well as suz12/eed, and they induce the histone modification of h3k27me3 on the promoter of target genes, which then represses gene expression. many other studies have shown that ezh2 and c - myc activate each other [13 - 15 ]. from the present findings, it could be also suggested that c - myc may be the possible mechanism for inducing h3k27me3 via prc2-related pathways in dlbcls. in other studies of solid tumors originating from various organs, high expression of hotair showed a close association with poor prognosis. in the present study, however, high levels of hotair expression showed an association with good prognosis in dlbcl, although the impact was not significant in the multivariate analysis. there are no comparable reports to support the present findings because this is the first study for the expression status of hotair in hematologic malignancies. although interaction between c - myc and hotair has not been established in hematologic malignancies, a close relationship between them may be plausible when considering the close association of prc2 and hotair, and prc2 and c - myc. however, expression of c - myc showed a negative correlation with hotair in the present study. one of the possible reason is autoregulation of c - myc reported in one study ; c - myc represses itself via forming autoregulatory loops with ezh therefore, hotair might be involved in that process via ezh2. whether hotair expression is directly linked to the suppression of c - myc or h3k27me3 should be investigated in further functional studies. in the hotair - related suppression of c - myc, prc2-associated histone modification (h3k27me3) might be induced in the promoter region of c - myc gene. for confirmation of this the favorable outcome of dlbcls with high hotair expression might be associated with the negative correlation with c - myc and/or bcl2. recent evidence has shown that co - expression of c - myc and bcl2 proteins is associated with poor prognosis in dlbcl patients regardless of gene signature. this was also observed in the present study in the univariate survival analysis. when the factor of hotair expression was added in the multivariate analysis ; however, the prognostic effect of coexpression of c - myc and bcl2 became weak, and only the factor of high h3k27me3 level was important as well as the ipi risk score. h3k27me3, the chromatin modification status induced via changes of hotair, c - myc, or other various known and unknown mechanisms, seems to be the most important factor in determining the fate of disease aggressiveness of dlbcl. though little is known about the key role of hotair in the malignant lymphoma, hotair might be involved in regulation of various genes in the lymphomagenesis. further study should follow to determine the explicit mechanism among the sophisticated modulatory networks of c - myc, prc2, h3k27me3, and hotair. recent evidence has shown that inhibition of ezh2 methyltransferase activity provides a potential target therapy for ezh2-deregulated lymphomas. in addition, the pharmacological inhibition of ezh2 activity results in a decrease of global h3k27me3 levels and a reactivation of silenced prc2 gene targets, and it inhibits growth of dlbcl cells. from these overall findings as well as the present findings, hotair may also be used in a target therapy by modulating the c - myc ezh2/prc2 loop in a subset of dlbcls with a high level of h3k27me3. in conclusion, we found frequent expression of prc2 proteins and h3k27me3 and positive correlation between these proteins and c - myc in dlbcls. high expression of h3k27me3 was determined as an independent predictor of poor prognosis, however, hotair was associated with favorable overall survival, which can be partly explained by negative correlation with c - myc. lncrna hotair expression could be one of the possible mechanisms to be involved in aggressive behavior of dlbcl via induction of h3k27me3 and ezh2-related prc2 activation. | backgrounda long non - coding rna hox transcript antisense intergenic rna (hotair) is involved in epigenetic regulation through chromatin remodeling by recruiting polycomb repressive complex 2 (prc2) proteins (ezh2, suz12, and eed) that induce histone h3 trimethylation at lysine 27 (h3k27me3). deregulation of c - myc and interaction between c - myc and ezh2 are well known in lymphomagenesis ; however, little is known about the expression status of hotair in diffuse large b - cell lymphomas (dlbcls).methodsthe expression status of prc2 (ezh2, suz12, and eed), h3k27me3, c - myc, and bcl2 was analyzed using immunohistochemistry (n = 231), and hotair was investigated by a quantification real - time polymerase chain reaction method (n = 164) in dlbcls.resultsthe present study confirmed the positive correlation among prc2 proteins, h3k27me3, and c - myc in dlbcls. expression level of hotair was also positively correlated to ezh2 (p <.05, respectively). between c - myc and hotair, and between c- myc / bcl2 co - expression and hotair, however, negative correlation was observed in dlbcls (p <.05, respectively). high level of h3k27me3 was determined as an independent prognostic marker in poor overall survival (hazard ratio, 2.0 ; p =.023) of dlbcl patients. high expression of hotair, however, was associated with favorable overall survival (p =.004) in the univariate analysis, but the impact was not significant in the multivariate analysis. the favorable outcome of dlbcl with hotair high expression levels may be related to the negative correlation with c- myc expression or c - myc / bcl2 co-expression.conclusionshotair expression could be one of possible mechanisms for inducing h3k27me3 via ezh2-related prc2 activation, and induced h3k27me3 may be strongly related to aggressive dlbcls which show poor patient outcome. |
leukemia is a malignant tumor of the hematopoietic system, which often has a poor prognosis. several factors, such as radiation and chemical carcinogens exposure, have been reported to be risk factors for leukemia, and internal factors, such as gene variation, have also been regarded as important factors. in our previous study, xrcc1 polymorphism was found to confer leukemia susceptibility. however, despite findings in the literature, the etiology of leukemia is still poorly understood. for years, the roles of parasitic infection in the genesis of tumors have attracted much attention. for example, the parasites clonorchis sinensis, opisthorchis viverrini, and schistosoma haematobium are associated with a number of cancers, such as nasopharyngeal carcinoma, leukemia, and hepatocellular carcinoma. toxoplasma gondii (t. gondii) is a wide - spread parasite reported to infect about one - third of the world population, mainly in low- or middle - income countries. human infection results from accidental ingestion of oocysts from unsanitary food or water that contains t. gondii tissue cysts. toxoplasmosis in immunocompetent individuals is generally asymptomatic, but it often leads to serious pathological effects in immunocompromised patients (e.g., people with hiv / aids or transplant patients). usually, infection with t. gondii is regarded as an established risk factor for poor obstetric history and is one of the major causes of congenitally acquired infections. moreover, t. gondii infection has also been implicated in the development of several disorders, such as liver cirrhosis, epilepsy, and even schizophrenia. recently, reports have indicated a possible association of t. gondii infection with cancer risk. for leukemia, therefore, we aimed to conduct a meta - analysis addressing this issue to obtain a reliable result. a systematic literature search was conducted in the biological databases medline, sciencedirect, and chinese national knowledge infrastructure (cnki), without language restriction. the following keywords were used for searching : toxoplasma, toxoplasmosis, leukemia, hematology, malignancy, neoplasm, and cancer. all relevant studies found in the search were retrieved and their bibliographies were checked for other relevant publications. the potentially eligible articles were read in full, but only those that met the inclusion criteria were selected. the criteria for the literature selection were : first, leukemia as a disease and toxoplasmosis as an exposure ; second, presence of a control group, with details of techniques and relevant targets used to diagnose t. gondii infection ; and third, data on sample size, odds ratios (ors), and their 95% confidence intervals (cis) or information from which these could be inferred. articles that met the following criteria were excluded : first, the designs were obviously different from other selected papers ; second, lack of controls and other essential information ; third, reviews and repeated articles. two of the authors carefully read the full texts and selected the papers according to the criteria mentioned above. a discussion was conducted. if we did not reach a consensus, another author of the present study was consulted to resolve the dispute and a final decision was made by majority vote. the newcastle - ottawa scale (nos) was used to evaluate the quality of the included studies, which generated scales ranging from 0 to 9 stars. studies with less than or equal to 3 stars were indicated to be low - quality and were excluded. the odds ratio (or) of leukemia risk associated with presence of t. gondii was estimated for each study. the or and its 95% confidence interval (ci) in each study was plotted against the number of participants for detection of any possible sample size biases. if the result of the q test was p>0.1, ors were pooled according to the fixed - effects model (mantel - haenszel) ; otherwise, the random - effects model (dersimonian and laird) was used. sensitivity analysis was assessed by changing the effects models and using one - way sensitivity analysis to assess stability of the results. publication bias was evaluated by creating funnel plots, in which the standard error of the log (or) of each study was plotted against its log (or). an asymmetric plot indicates a possible publication bias. to minimize the potential subjective effects on the results, egger s linear regression test was used to assess the symmetry. the fail - safe number for p=0.05 (nfs0.05) was also used for assessment of possible publication bias. statistical analysis was performed using excel 2003 (microsoft) and stata 11.0 software (stata corporation, tx). a systematic literature search was conducted in the biological databases medline, sciencedirect, and chinese national knowledge infrastructure (cnki), without language restriction. the following keywords were used for searching : toxoplasma, toxoplasmosis, leukemia, hematology, malignancy, neoplasm, and cancer. all relevant studies found in the search were retrieved and their bibliographies were checked for other relevant publications. the potentially eligible articles were read in full, but only those that met the inclusion criteria were selected. the criteria for the literature selection were : first, leukemia as a disease and toxoplasmosis as an exposure ; second, presence of a control group, with details of techniques and relevant targets used to diagnose t. gondii infection ; and third, data on sample size, odds ratios (ors), and their 95% confidence intervals (cis) or information from which these could be inferred. articles that met the following criteria were excluded : first, the designs were obviously different from other selected papers ; second, lack of controls and other essential information ; third, reviews and repeated articles. two of the authors carefully read the full texts and selected the papers according to the criteria mentioned above. a discussion was conducted. if we did not reach a consensus, another author of the present study was consulted to resolve the dispute and a final decision was made by majority vote. the newcastle - ottawa scale (nos) was used to evaluate the quality of the included studies, which generated scales ranging from 0 to 9 stars. studies with less than or equal to 3 stars were indicated to be low - quality and were excluded. the odds ratio (or) of leukemia risk associated with presence of t. gondii was estimated for each study. the or and its 95% confidence interval (ci) in each study was plotted against the number of participants for detection of any possible sample size biases. if the result of the q test was p>0.1, ors were pooled according to the fixed - effects model (mantel - haenszel) ; otherwise, the random - effects model (dersimonian and laird) was used. sensitivity analysis was assessed by changing the effects models and using one - way sensitivity analysis to assess stability of the results. publication bias was evaluated by creating funnel plots, in which the standard error of the log (or) of each study was plotted against its log (or). an asymmetric plot indicates a possible publication bias. to minimize the potential subjective effects on the results, egger s linear regression test was used to assess the symmetry. the fail - safe number for p=0.05 (nfs0.05) was also used for assessment of possible publication bias. statistical analysis was performed using excel 2003 (microsoft) and stata 11.0 software (stata corporation, tx). a total of 235 publications were identified, of which 221 irrelevant papers were excluded after a review of their titles and abstracts. as shown in figure 1, 14 publications were preliminarily eligible, of which 2 reviews and 1 study whose selection of cases obviously led to selection bias were excluded. then, after a careful review of the texts, 4 studies without controls [2225 ] and 1 study with insufficient data were further excluded. finally, 6 studies [2732 ] were selected for analysis. of the selected publications, no papers in english or other languages were included because relevant papers did not meet the inclusion criteria. the quality assessment scales of these included studies were all over 3 (data not shown) ; thus, they were all selected for analysis. as shown in table 2, we first analyzed the heterogeneity of the pooled data. thus, the fixed - effects model was used for data pooling. for the overall data, a significant association was observed (or=3.05 ; 95%ci=1.835.08 ; p=0.147 for heterogeneity), indicating that the infection rate of t. gondii in the leukemia cases was significantly higher than that in the controls (figure 2). considering the potential effect of confounding factors on the results, we conducted subgroup analysis according to source of controls and target of detection. the data showed that the results of the subgroups regarding these 2 factors were in line with the overall data, suggesting that both the source of controls and the detection targets exerted little influence on the overall data. to test whether the results were stable, we changed the effects model for pooling the data. as expected, the significance of the random - effects model (or=3.39 ; 95%ci=1.647.03) was in accordance with the fixed - effects model. then, we repeated the analysis by sequentially omitting each study, and found that the results were not significantly changed (data not shown). the results suggest that the results of the present meta - analysis were stable. for detection of possible publication bias, the funnel plot was further estimated by egger s linear regression test, in which 95%ci (2.512, 4.964) of the point of intersection between the regression curve and y axis included the original point, with t value of 0.91 and p value of more than 0.05, indicating the symmetry of the funnel plot (figure 3). the result was 53, which is more than 8 times the number of included studies. these data suggest that publication bias did not exert an evident influence on the results. a total of 235 publications were identified, of which 221 irrelevant papers were excluded after a review of their titles and abstracts. as shown in figure 1, 14 publications were preliminarily eligible, of which 2 reviews and 1 study whose selection of cases obviously led to selection bias were excluded. then, after a careful review of the texts, 4 studies without controls [2225 ] and 1 study with insufficient data were further excluded. finally, 6 studies [2732 ] were selected for analysis. of the selected publications, no papers in english or other languages were included because relevant papers did not meet the inclusion criteria. the quality assessment scales of these included studies were all over 3 (data not shown) ; thus, they were all selected for analysis. as shown in table 2, we first analyzed the heterogeneity of the pooled data. thus, the fixed - effects model was used for data pooling. for the overall data, a significant association was observed (or=3.05 ; 95%ci=1.835.08 ; p=0.147 for heterogeneity), indicating that the infection rate of t. gondii in the leukemia cases was significantly higher than that in the controls (figure 2). considering the potential effect of confounding factors on the results, we conducted subgroup analysis according to source of controls and target of detection. the data showed that the results of the subgroups regarding these 2 factors were in line with the overall data, suggesting that both the source of controls and the detection targets exerted little influence on the overall data. to test whether the results were stable, we changed the effects model for pooling the data. as expected, the significance of the random - effects model (or=3.39 ; 95%ci=1.647.03) was in accordance with the fixed - effects model. then, we repeated the analysis by sequentially omitting each study, and found that the results were not significantly changed (data not shown). the results suggest that the results of the present meta - analysis were stable. for detection of possible publication bias, the funnel plot was further estimated by egger s linear regression test, in which 95%ci (2.512, 4.964) of the point of intersection between the regression curve and y axis included the original point, with t value of 0.91 and p value of more than 0.05, indicating the symmetry of the funnel plot (figure 3). the result was 53, which is more than 8 times the number of included studies. these data suggest that publication bias did not exert an evident influence on the results. whether infection with t. gondii is a risk factor for leukemia has been uncertain and only a few reports have addressed this topic, with inconsistent results. in the present meta - analysis, we found that t. gondii infection appears to be associated with increased leukemia susceptibility. for instance, parasitization by liver flukes, such as opisthorchis viverrini and clonorchis sinensis, has been suggested to confer cholangiocarcinoma susceptibility. schistosoma haematobium infection has been reported to play a role in genesis and development of bladder cancer. thus, parasite infection as a risk factor for malignancy may be of interest and value for research in this field. to the best of our knowledge, the present meta - analysis is the first to address the possible relationship between t. gondii infection and leukemia risk, and it provides new insights into research on cancer etiology. the mechanisms by which t. gondii initiates tumorigenesis are unclear. reports showed that t. gondii can export mirnas into its host cell, which might regulate the hosts gene expression, and thus cause cancer onset. by modifying host mirna expression, the genes involved in apoptosis or anti - apoptosis were both targeted by the differentially - expressed mirnas, the change in balance of power between the mirnas targeting host apoptosis genes and those modulating host anti - apoptosis genes leads to the fate of the host apoptosis process. the above evidence might be helpful in elucidating the roles of t. gondii in the genesis of leukemia. future studies are needed to provide more precise evidence. considering that hospital - based controls were patients with other benign disorders that might have an association with t. gondii infection, we conducted subgroup analysis stratified by source of controls. nevertheless, the results of the 2 subgroups were consistent with the overall data, indicating that source of controls probably did not affect the results. moreover, toxoplasma dna was used as a target for detection in some studies, while in other studies toxoplasma ig g was used. similarly, subgroup analysis regarding detection indicates little influence of this factor on the overall results. several limitations must be considered in the present meta - analysis when interpreting the results. first, only studies written in chinese and turkish were included because papers in english did not meet the inclusion criteria. this might be because t. gondii infection mainly affects people in low- or middle - income countries, such as china and turkey. since the biological characteristics and clinical features are different between acute leukemia and chronic leukemia, and between lymphocytic leukemia and myeloid leukemia, it will be necessary to increase the sample sizes and divide the cases according to the clinical type in future primary studies. third, the controls in most included studies were not well - matched to the cases, and this might have affected the accuracy of the estimates. furthermore, as shown in our previous paper, genetic factors might exert an influence on the susceptibility to leukemia. whether t. gondii infection interacts with genetic factors and contributes to leukemia risk this subject could not be assessed in the present meta - analysis because this interesting point has not been evaluated in the primary literature. since t. gondii might influence host gene expression, ethnic variation may play a role in the pathogenesis of leukemia. therefore, studies in different geographic areas and involving different ethnicities are needed to obtain a more reliable estimate. the results of the present meta - analysis indicate that t. gondii infection might have an association with increased leukemia risk. these interesting results may be helpful in research on leukemia etiology. studies in different regions and ethnicities, with large sample sizes, together with basic laboratory studies, are warranted to confirm this association and explore the potential molecular mechanisms. | backgroundpossible associations of parasite infection with cancer risk have recently attracted much attention. published studies concerning the association between toxoplasma gondii (t. gondii) infection and leukemia risk have generated inconsistent results. in the present study, we aimed to address this topic by conducting a quantitative meta-analysis.material/methodsrelevant publications were searched in electronic databases and eligible studies were rigorously screened and selected. essential information was extracted and the data were pooled. subgroup analysis on source of controls and detection target was also performed.resultsa total of 6 studies that met the inclusion criteria were selected. the overall data show that t. gondii infection might have an association with increased leukemia risk (or=3.05 ; 95%ci=1.835.08). similar results were shown in the subgroups regarding source of controls and detection target.conclusionsour results suggest that t. gondii infection might be a risk factor for leukemia, providing new insight into the etiology of leukemia. future studies with large sample sizes in different geographic areas are needed to confirm this conclusion. |
infectious keratitis is one of the important causes of corneal blindness which is a major public health problem worldwide. the incidence of fungal keratitis varies around the world and is more prevalent in areas with hot humid climates. however, under some circumstances such as corticosteroid therapy and trauma to the eyeball, these fungi might invade the eye and cause fungal ocular infections. among these fungi, fusarium species are the most frequent cause of fungal keratitis and account for up to one - third of these infections. they are fast - growing hyalohyphomycetes that have been isolated from soil, plants, and water. most incidences of fusarium keratitis are caused by an eye injury with vegetative matter, such as trauma to the eye with a palm branch. fusarium keratitis infections through contact lens wear have been reported, but they are less prevalent. fusarium solani species complex (fssc) can cause severe types of fungal keratitis because of its high level of virulence and its resistance to antifungal medications. keratitis caused by fssc may lead to serious complications such as endophthalmitis, descemetocele, perforation, and blindness. considering its difficult diagnosis, fungal keratitis is one of the most serious and hazardous forms of corneal infections. different classes of antifungals including polyenes, triazoles, and echinocandins have been used previously in the treatment of fungal keratitis. in india, second - generation triazoles such as posaconazole and voriconazole seem to be effective in the treatment of ocular or corneal fungal infections. few studies are available about the efficacy of these drugs on fungal keratitis and the route of prescription. reported results of intrastromal voriconazole injection in the treatment of two patients with recalcitrant fusarium keratitis. they concluded that intrastromal injection of voriconazole together with topical voriconazole effectively reduced the infiltration size and controlled the infection in patients with fusarium keratitis. in this study, we aimed to evaluate the therapeutic effect of intrastromal voriconazole on fusarium keratitis and compare it with topical voriconazole and topical natamycin. this study was performed at the department of ophthalmology at khalili eye hospital (shiraz university of medical sciences, shiraz, iran) and was approved by ethics committee of shiraz university of medical sciences. the isolate was subcultured on potato dextrose agar (merck, germany) plates at 28c to induce sporulation. the conidia were harvested by washing the surface of the colonies with 0.1% tween 80 in the sterile physiological saline and filtering the suspension through 2 layers of sterile gauze to remove hyphal residue. the spore suspensions were then transferred to eppendorf tubes and centrifuged at 10000 g to pellet the conidia. the supernatant was carefully removed, and the conidia were resuspended with sterile normal saline to yield the final inoculum of 8.6 10 colony forming units (cfu) per milliliter. the number of conidia was measured by hemocytometer and their viabilities were determined and confirmed by quantitative plating of serial dilutions of stock inoculum. twenty - four new zealand white rabbits weighing 2.5 to 3 kg were selected from the animal laboratory of shiraz university of medical sciences. they were systemically anesthetized with 50 mg / kg of intramuscular ketamine hydrochloride and 5 mg / kg of xylazine before all interventions. the stromas of their right corneas were inoculated with 0.1 ml suspension of fssc (10 cfu / ml under sterile condition in the animal operating room). one week after injection they were checked for fungal keratitis and the size of corneal ulcer and corneal clouding were measured. the rabbits were randomly divided into four groups : the first group received topical voriconazole 1% once each hour (q1h) (except from 1 a.m. to 7 a.m.) for one week, the second one received a single injection of intrastromal voriconazole 50 microgram/0.1 ml (figure 1), the third group received topical gel of natamycin q1h (except from 1 a.m. to 7 a.m.) for one week, and the last group did not receive any antifungal treatment and was considered as the control group. all groups received chloramphenicol drops four times a day for 2 weeks for prophylaxis of bacterial superinfection. the eyes in four groups were examined at day 7 (baseline) and at day 14 (7 days after beginning of treatment, before enucleation) using an operative biomicroscope. the severity of keratomycosis in the rabbits was scored with the scoring system described by wu.. each of the following criteria was graded : area of corneal opacity, density of opacity, and surface regularity (table 1). a normal cornea was given a score of 0 in each category and a summation score of 0. the scores from these categories were tallied for each cornea to show a possible total score ranging from 0 to 12. a total score of 5 or less was considered mild, a total score of 6 to 9 was categorized as moderate, and a total score of more than 9 was considered severe. on day 14 the eyes were enucleated and sclerocorneal buttons were sent for microbiological and histological examinations. in each of the examined groups, the corneal buttons were weighed, homogenized, and inoculated onto the sabouraud dextrose agar plates. following 27 days of incubation at 28c, the specimens were immediately inoculated onto the sabouraud dextrose agar plates and incubated at 28c for 7 days. then, the buttons were halved and underwent routine tissue processing and tissue block preparation. the sections were examined under light microscope (olympus bx41). presence or absence of ulceration, fungal hyphae, type and severity of inflammation, extent of stromal scarring and vascularization, and characteristics of infiltrating cells the data were presented as mean sd. the statistical analyses of differences between different groups were performed using the tukey hsd and mann - whitney tests. the wilcoxon test was used to compare the results for each group between days 7 and 14. fusarium corneal ulcer was developed in all eyes one week after intrastromal injection of fungal suspension. tables 2 and 3 show the mean clinical scores obtained from each group on days 7 and 14. kruskal - wallis one - way analysis of variance showed that statistically there was no significant difference between these four groups at baseline. mann - whitney u test showed that, on day 14, the difference between the control group and intrastromal voriconazole group was significant statistically. the difference between control group and natamycin group was also significant, but there was statistically no significant difference between the control and the topical voriconazole group or between the intrastromal voriconazole and natamycin groups (table 4). these data indicate that rabbits in the intrastromal voriconazole and natamycin groups developed infection with less severity than the control and topical voriconazole groups. the mean sd weight of the samples sent to the microbiological laboratory was 0.1494 gr 0.0085. the values about the colony forming units (cfus) that were obtained from the cultured samples are given in table 5. tukey 's hsd test showed a statistically significant difference between the control and intrastromal voriconazole groups (table 6). there was a significant difference between the control and natamycin groups, but the differences between the control and topical voriconazole and between intrastromal voriconazole and natamycin groups were not significant statistically. these data indicate that fusarium proliferation was significantly lower in intrastromal voriconazole and natamycin groups. in pathological evaluation of the control group similar findings including moderate chronic inflammation, moderate eosinophils, and anterior stromal vascularization were found. the pathological comparison of the experimental groups with the control group is shown in table 7. plasma cells, lymphocytes, neutrophils, eosinophils, and mast cells were the inflammatory cells that were investigated. in pathological evaluation, fewer chronic inflammations were reported in 2 of the 3 buttons from the topical voriconazole group and 3 of the 3 buttons from the intrastromal voriconazole and natamycin groups in comparison with the control group. in 1 of the 3 buttons from the topical voriconazole group, 2 of the 3 buttons from the intrastromal voriconazole group, and 3 of the 3 ones from the natamycin group, the diagnosis of these infections is very difficult and currently the therapy for fungal diseases is not as forceful and effective as antibacterials. lin. reported that almost 70% of patients with deep lesions of fusarium keratitis do not respond to medical therapy alone. fssc is very virulent and can destroy an eye completely within a few weeks because the infection is usually severe and perforation, deep extension, and malignant glaucoma may supervene.. found no difference in three - month best spectacle corrected visual acuity or scar size between natamycin- and voriconazole - treated patients in fusarium keratitis. however voriconazole - treated patients were more likely to perforate than natamycin - treated cases.. stated that natamycin treatment was associated with significantly better clinical and microbiological outcomes than voriconazole treatment for smear - positive filamentous fungal keratitis, especially in patients with fusarium infection. in this study we found that topical natamycin - treated fusarium ulcers had fewer clinical scores, cfu, and less severe pathological findings than topical voriconazole - treated patients. other researchers reported that all of fusarium species were sensitive to natamycin, but that did not translate to good clinical outcome in patients with fusarium keratitis irrespective of early or late presentation. this probably indicates the poor penetration of natamycin especially in the presence of advanced fungal keratitis affecting deeper layers of the cornea. several studies have evaluated the effect of more potent drugs and delivery to the site of action in the posterior stroma using intrastromal or intracameral injections [18, 19 ]. described the outcome in 3 patients with recalcitrant fusarium keratitis and reported that intrastromal injection of voriconazole together with topical voriconazole is effective in reducing the infiltration size and control of the infection. sharma. offered intrastromal injection of voriconazole as a modality of treatment for managing cases of recalcitrant fungal keratitis. in this study we found that, in rabbits with fusarium keratitis, intrastromal injection of voriconazole seems to be as effective as topical natamycin. it had lower clinical score, cfus and fewer chronic inflammations, eosinophils, and anterior stromal vascularization than topical voriconazole. since the cost of intrastromal injection of voriconazole is less than the frequent use of topical natamycin, it appears to be more economical to inject intrastromal voriconazole in fusarium corneal ulcers. on the other hand, the use of one - time intrastromal injection of voriconazole is more comfortable than applying topical natamycin every hour. we believe that intrastromal voriconazole can be useful in the treatment of fusarium keratitis especially in ulcers that do not respond to other treatment modalities. we propose further studies on different concentrations of intrastromal voriconazole to investigate their influence on fusarium keratitis to find the most effective concentration. | purpose. evaluating the therapeutic effect of topical and intrastromal voriconazole and topical natamycin on fusarium keratitis. methods. 24 rabbits were selected. the stroma of their corneas was inoculated with suspension of fusarium solani species complex. seven days after injection they were divided into 4 groups randomly : the first group was treated with topical voriconazole (tv) 1% for one week, the second one with one - time intrastromal injection of voriconazole (isv) 50 microgram/0.1 ml, and the third group with topical gel of natamycin (tn) for one week, and the last one did not receive any antifungal treatment. finally the eyes were enucleated and sclerocorneal buttons were sent for histological and microbiological examinations. results. after treatment the isv group and tn group showed significantly lower clinical score and colony forming units than the control group (p = 0.040 and p = 0.026, resp.), but there was statistically no significant difference between control and tv groups (p = 0.249) or between isv and tn groups (p = 0.665). in pathological evaluation, fewer chronic inflammations were reported in 2 of the 3 buttons from tv group and 3 of the 3 buttons from isv and tn groups in comparison with the control group. conclusion. intrastromal injection of voriconazole seems to be effective in treatment of fusarium keratitis as much as topical natamycin and these are more effective than topical voriconazole. |
the atherosclerosis risk in communities (aric) study is an ongoing community - based prospective cohort study of 15,792 middle - aged adults from four u.s. communities : washington county, md ; suburban minneapolis, mn ; jackson, ms ; and forsyth county, nc. the first study visit occurred between 1987 and 1989 with three follow - up visits that occurred approximately every 3 years (13,14). visit 2 (19901992) was attended by 14,348 participants and is the baseline for the present analysis. we excluded participants who self - identified as other than white or black race (n = 48) or who were missing data on hba1c or other covariates of interest (n = 970), leaving a final sample size of 13,288 participants in this analysis. institutional review boards at each clinical site approved the study protocol, and written informed consent was obtained from all participants. frozen whole - blood samples collected at aric visit 2 were thawed and assayed for the measurement of hba1c using high - performance liquid chromatography (tosoh a1c 2.2 plus glycohemoglobin analyzer method in 2003 to 2004 and the tosoh g7 method in 2007 to 2008 ; tosoh corporation) (15). both instruments were standardized to the diabetes control and complications trial assay (16). aric study investigators conduct continuous surveillance for all hospitalizations and deaths among participants via annual phone calls to participants or proxies, and detailed information on deaths is obtained from family members, coroner reports, or health department death certificates. methods for the ascertainment of death and its causes in aric have been published previously (13). we classified deaths according to underlying cause, on the basis of coding from the icd-9 and -10. we divided causes of death into the following major diagnosis categories defined by the icd codes : 1) cancers (icd-10 codes of c00-d48, icd-9 codes of 140239) ; 2) cardiovascular system (icd-10 codes of i00i99, icd-9 codes of 390459) ; 3) respiratory system (icd-10 codes of j00j99, icd-9 codes of 460519) ; 4) digestive system and liver (icd-10 codes of k00k93, icd-9 codes of 520579) ; and 5) genitourinary system and kidney (icd-10 codes of n00n99, icd-9 codes of 580629). we also identified incident liver disease hospitalizations from hospital discharge records with an icd-9 code for liver disease using the following icd-9 codes (listed anywhere in the hospital discharge record) : 570.0573.9. all covariates were assessed during visit 2 (19901992), except education and physical activity, which were assessed during visit 1 (19871989). covariates evaluated as potential confounders included : age (continuous), race / field center (washington county, md whites ; minneapolis, mn whites ; forsyth county, nc whites, forsyth county, nc blacks ; and jackson, ms blacks), sex (male / female), education (less than high school, high school or equivalent, or more than high school), bmi (continuous), waist - to - hip ratio (continuous), cigarette smoking (current, former, or never), alcohol intake (current, former, or never), physical activity [assessed with the use of baecke index (17) ], family history of diabetes, presence of hypertension (defined as systolic blood pressure 140 mmhg, diastolic blood pressure 90 mmhg, or use of antihypertensive medication during the previous 2 weeks), total cholesterol (continuous), hdl cholesterol (continuous), prevalent coronary heart disease, prevalent stroke, fasting glucose (continuous), hemoglobin concentration (continuous), red - cell mean corpuscular volume (mcv ; continuous), total leukocyte count (continuous), and plasma fibrinogen levels (continuous). we divided the baseline study population into groups according to clinical categories of glycated hemoglobin (14.7 g / dl). adjusted associations of low hba1c (0.1). however, in sensitivity analyses among persons with anemia (10.2% of the study population), we observed stronger associations of low hba1c with all - cause mortality (1.60, 95% ci : 1.072.39), cancer mortality (1.94, 95% ci : 1.033.65), and cardiovascular death (1.51, 95% ci : 0.743.09). among participants without anemia, the results were somewhat attenuated but remained significant for all - cause mortality (1.22, 95% ci : 1.031.46) and cancer death (1.35, 95% ci : 1.051.74). adjusted hrs (95% ci) for cause - specific mortality by hba1c category compared with persons with hba1c 5.0 to < 5.7%, those persons with high hba1c (6.5%) also had an increased risk of all - cause mortality and deaths from cancer, cardiovascular disease, diseases of the respiratory system, digestive system and liver disease, diseases of the genitourinary system, and kidney disease (table 3). similar results were observed among persons with and without anemia, after adjustment for fasting serum glucose, pulmonary function tests, white - cell differential, platelet count, mean corpuscular hemoglobin, and when the analysis was restricted to persons with 3 years of follow - up (data not shown). fig. 1 presents the adjusted hrs and 95% cis from the restricted cubic spline model for the association of baseline hba1c with risk of hospitalization for liver disease. we observed a pronounced j - shaped association ; both very low and high hba1c values were associated with an increased risk of liver hospitalization in this population. adjusted hrs (95% ci) for liver disease hospitalization by baseline hba1c value. the hr is expressed per absolute increase in one percentage point in the hba1c value at baseline. the model is centered at the median (5.5%) with knots at the 20th, 40th, 60th, and 80th percentiles. the hrs were adjusted for age, sex, race, field center, total and hdl cholesterol, bmi, waist - to - hip ratio, hypertension, family history of diabetes, education level, alcohol use, physical activity, smoking status, hemoglobin, mcv, fibrinogen, and leukocyte count. compared with persons with hba1c in the normal range, we found that low hba1c values (< 5.0%) were associated with an increased risk of all - cause mortality and death from various causes, including cancer. this finding is consistent with previous studies that have documented a j- or u - shaped association between hba1c and all - cause mortality (3,7,8,10,11). our results are contrary to a recent analysis of the european prospective investigation of cancer - norfolk study that reported no increase in risk of mortality at low normal hba1c values in a relatively homogenous population of caucasians (20). the debate regarding the hba1c mortality association also extends to the mortality curves for fasting and 2-h glucose, which are reported to be j - shaped in some studies (2124). no single cause of death appeared to drive the association between low hba1c and risk of death in our study. prior studies have suggested that liver disease may be an important contributor to low hba1c values (10,12). consistent with this hypothesis, we observed that low hba1c values were associated with an increased risk of hospitalization due to liver disease in the aric study population. the exact mechanisms underlying the association of low hba1c values with increased risk of death are not known. we found that low hba1c values were significantly associated with higher mean red - cell volume and low hemoglobin. other studies have demonstrated associations of red - cell indices with long - term mortality in the general population (25,26). the etiology of these relationships is largely unclear but may reflect that some disease processes affect red - cell turnover and thus hba1c. whereas high hba1c values are almost entirely determined by circulating glucose levels (27), it is probable that nonglycemic factors such as red - cell turnover may be of disproportionate importance in the low range of hba1c. our observations suggest low hba1c values may be a general marker of ill health, analogous to the well - documented u - shaped association between cholesterol and mortality (2830). there is strong evidence that some disease processes reduce circulation of cholesterol - bearing lipoproteins (28). low hba1c values may characterize a mix of healthy people and a group in which low hba1c is a marker or signal of underlying health issues, such as liver disease or early stages of cancer (3,10). this hypothesis is consistent with the negative confounding observed in our study ; once traditional risk factors for death were added to our models, the association of low hba1c with all - cause mortality and cause - specific mortality became stronger. we were limited to information available from the death certificate for the classification of the underlying cause of death, which may have resulted in misclassification, particularly for the noncancer and noncardiovascular outcomes (3133). we also did not have data available on iron indices, reticulocyte count, or red - cell distribution width. nonetheless, the aric population is a large, diverse cohort with virtually complete follow - up for vital status and comprehensive information on important confounders and major risk factors for mortality. to our knowledge, this study is one of the first comprehensive examinations of the association of low hba1c with cause - specific mortality in a general population. in conclusion, our results add to the evidence that low hba1c values may be a marker of elevated mortality risk in the general population. with new recommendations for the use of hba1c for diagnosis of diabetes, hba1c testing will undoubtedly increase. to the extent that hba1c is adopted for diabetes screening, clinicians will frequently observe hba1c values in the normal range, including low normal values. additional work should focus on understanding the nonglycemic determinants of hba1c and the mechanisms that explain why low hba1c values in some individuals may reflect an elevated risk state. | objectiveto identify predictors of low hemoglobin a1c (hba1c) (< 5.0%) and to investigate the association of low hba1c with cause - specific mortality and risk of liver disease hospitalization.research design and methodsprospective cohort study of 13,288 participants in the atherosclerosis risk in communities study. logistic regression was used to identify cross - sectional correlates of low hba1c, and cox proportional hazards models were used to estimate the association of low hba1c with cause - specific mortality.resultscompared with participants with hba1c in the normal range (5.0 to < 5.7%), participants with low hba1c were younger, less likely to smoke, had lower bmi, lower white cell count and fibrinogen levels, and lower prevalence of hypercholesterolemia and history of coronary heart disease. however, this group was more likely to have anemia and had a higher mean corpuscular volume. in adjusted cox models with hba1c of 5.0 to < 5.7% as the reference group, hba1c < 5.0% was associated with a significantly increased risk of all - cause mortality (hazard ratio [hr ] : 1.32, 95% ci : 1.131.55) and of cancer death (1.47, 95% ci : 1.161.84). we also noted nonsignificant trends toward increased risk of death from cardiovascular causes (1.27, 95% ci : 0.931.75) and respiratory causes (1.42, 95% ci : 0.782.56). there was a j - shaped association between hba1c and risk of liver disease hospitalization.conclusionsno single cause of death appeared to drive the association between low hba1c and total mortality. these results add to evidence that low hba1c values may be a generalized marker of mortality risk in the general population. |
emphysema is a lung disease 1 that results in narrowing of the small airways and breakdown of the lung tissues. it is due to long - term exposure of irritants like inhaling of cigarette smoke, and other toxic particles that cause lung inflammation. emphysema is a phenotype of copd 2 and is amongst the leading cause of deaths in most of the countries 3. treatments like methylxanthines 7, long - term oxygen therapy, corticosteroids, and bronchodilators are used as a conventional therapy. but long - term usage of these treatments increases the risk of pneumonia, arrhythmia, and fractures 3,8. previous studies have shown that cell - based therapies have a potential for lung repair and rehabilitation of lungs after injury 9. cell - based therapies play an important role and reduce both alveolar damage and inflammation of the airways 10. in our previous studies, we have shown improvement in patients with cerebral palsy 11 patients with cortical visual impairment 12 and patients with other conditions after human embryonic stem cell (hesc) therapy 13. here, we present a case of 52-year - old male with emphysema having paraseptal emphysematous changes seen in bilateral upper lobes. the cell lines were cultured and maintained as per our in - house patented technology in a gmp, glp, and gtp certified laboratory (patent - wo 2007/141657a pct/1b 2007 published 13 december 2007). the evidence for the use of hescs at nutech mediworld has been submitted in written and accepted at house of lords, regenerative medicine, science and technology committee 14. the patient provided with a written consent form before the start of the treatment. a complete examination of the patient was done and video recordings were made before and after the treatment. the treatment consisted of three phases in which 0.25 ml hescs were administered through intramuscular route twice daily and 1 ml of hescs were administered through intravenous route twice daily for 7 days. a 52-year - old male was admitted at the nutech mediworld on 6 august, 2012 with chief complaints of shortness of breath, pain radiating to the neck, increased cough and wheezing especially at night and after getting up from sleep for the past 9 months. the patient and his wife noticed that he had increased shortness of breath after mild physical exertion (like walking). a visit to a local physician revealed that he had emphysema copd with not much hope of cure, and only symptomatic treatment the patient was a heavy smoker since last 30 years and drank alcohol frequently. the family history of patient revealed that the patient s father died of carcinoma of the lungs and his mother died of stomach cancer. the first contrast - enhanced computed tomography (cest) performed on 06 august, 12 showed paraseptal and panacinar emphysematous changes in bilateral lung fields with flattening of the diaphragm. on examination of the central nervous system (cns) and other parameters, the patient was conscious, alert, well oriented to time and with fair nutritional status. after 101 days of the hesc therapy, the patient showed an improvement in symptoms like absence of cough and phlegm. a cest of chest performed after the therapy on 25 september, 2012 showed paraseptal emphysematous changes in bilateral upper lobes and rest of parenchyma appeared normal. he was also able to completely quit smoking during his stay at our facility. on his last follow - up on (2014, november), he did not report cough, phlegm, or wheezing, was able to work full time and to walk long distances. in the present study, an improvement in cough, phlegm, and overall stamina was observed in a patient with emphysema after hesc therapy. cest performed after the therapy showed normal parenchyma of the lungs. no adverse events (aes) previous studies have shown that there is a potential in cell lines to consummate multipotent lung progenitors in association with combined growth factors and novel markers (lifr and nrp1) 15. the mesenchymal stem cells (mscs) release chemokines, cytokines, and growth factors to the injury site and act as migratory cues. these cues influence selectins and result in activation of integrins on the stem cell surface. this permits the stem cells to interact, adhere, and transmigrate through endothelium 16. a study by lee and colleagues stated that mscs can mitigate the inflammatory and lung injury in isolated perfused human lungs. mscs released anti - inflammatory mediators to the injury site and repair the damaged cells 17. the study showed a base for therapeutic use of hescs in a variety of lung diseases. so, we might possibly explain that hescs used in our study also rely on the same mechanism of action. we assume that our hescs also penetrate across the parenchyma and migrate to the affected region. the use of stem cells is restricted due to the fear of aes as teratoma formation and immune rejection. in our patient, we have not observed any ae. we have already established the safety of hesc therapy in patients with terminal / incurable conditions 18. no study till date assessed the therapeutic potential of hescs in the treatment of patients with emphysema. this is the first study to report the use of hescs as therapy in lung repair. our patient showed remarkable clinical improvement and an improved quality of life. though he quit smoking, but improvement observed in the patient can not be justified for his improvement that was observed in a very small span of time. he had been a heavy smoker for last 30 years and his stay at our facility was about 3.5 months only. it has also been reported previously that after smoking cessation, the inflammation, apoptosis, and oxidative stress can remain and sustain to contribute to copd 19. there is an ethical concern regarding the use of hescs like using fertilized embryo, misuse of cells and use in experimental purposes. the fertilized ovum for the present hescs was donated willingly by a woman undergoing ivf and she was cognizant that it would be used for experimental and research purpose. moreover, we have used this therapy on patients with incurable conditions and nothing can be more unethical than denying a ray of hope to the patients who have tried all treatments and still shown no improvement. however, we were not able to perform bronchoalveolar lavage sample examination of the patient before and after the treatment. future well - designed studies can further clarify the role of hesc therapy in patients with emphysematous lung disease. therefore, it is summarized that hescs showed good therapeutic potential in the treatment of patients with emphysematous copd. clinical trials reveal that cell - based therapies have a potential for lung repair and rehabilitation of lungs after injury. however, more clinical trials and follow - up studies are needed to prove the long - term efficacy and safety of hescs in the treatment of patients with emphysema copd. | key clinical messageemphysema results in narrowing of the small airways due to inhaling of cigarette smoke and other noxious particles. oxygen therapy, corticosteroids, and bronchodilators increase the risk of pneumonia, arrhythmia, and fractures in long term. therapy with human embryonic stem cells resulted in improved symptoms of a patient with emphysema. |
when you publish, you get to claim scientific priority for a discovery. in return, you share what you know in sufficient detail so that others can reproduce and build on that discovery. what is surprising is that many papers fail to report experimental procedures in sufficient detail for this to happen. i do not think it is intentional in most cases ; rather, i think that we tend to use other published papers as examples of what to report and, facing word limits at many journals, often the methods are the first place we cut corners. we do this even with the knowledge that compromising detail in this section of the paper might be detrimental to readers. when papers do nt provide experimental procedures in sufficient detail another group may have to start from scratch, wasting valuable time and resources. this is unfortunate in any case, but especially so some might even say unethical when the procedures involve experimental animals during my phd in peter walter s lab, part of my first major project was to figure out how to separate and purify the proteins in signal recognition particle (srp). it took about a year of testing different kinds of columns and loading and elution conditions, but i finally got it right, and i published the experimental procedure in my first research paper in 1985 (siegel and walter, 1985). a couple of years after i started my postdoc, a technician in peter s lab called to tell me she was going to purify some srp proteins, and asked me whether my published experimental procedure was complete and reproducible. i told her i thought it was, but i was terrified that i had left out some crucial step, and that the purification in her hands would fail which would mean that the paper i had published was flawed because, without the purification, the experiments using the purified proteins would be impossible to reproduce. when she called a few days later to announce her success, i was relieved. in a research paper that you write, the methods usually comprise some experimental procedures that you have personally fine - tuned or even designed, and others that were established by others or that you might consider standard. those that are new and yours alone to describe in detail are probably the ones reported most precisely in your research articles. then there are scores of other experimental procedures that have been handed down from colleagues or in lab notebooks and which you consider standard. rather than describing these in detail in your article, you might reference a previous paper that you know used the method, even if you re not sure the method is actually detailed in that paper and if it is nt, you expect it will reference the paper in which it was first described. and then there are the experiments performed at a core facility at your institute, which you trust have been done according to some standard procedure and you may or may not obtain and report those precise details in your paper. you look to other papers to see what details they have reported, and you include what they include, leave out what they leave out. on the flip side, almost all of us have tried to repeat the experimental procedures described in a published research paper, only to find ourselves frustrated by the lack of detail and the need to look up references to other papers that also do nt provide the procedure we re after. so, we start on a wild goose chase, reading the methods sections of several generations of referenced papers eventually to find the goose, or, alternatively, contacting the author to find out what they really did and hope that they know. when papers do nt provide experimental procedures in sufficient detail to know precisely how the experiments were performed at a level of detail sufficient to reproduce them, another group may have to start from scratch, wasting valuable time and resources. this is unfortunate in any case, but especially so some might even say unethical when the procedures involve experimental animals. how, then, can we improve the situation ? when i teach manuscript writing, i urge my students to provide methods like a that s great advice, but as a procedure for manuscript preparation it too is faulty, because it lacks the detail to really guide our students. what we really need are detailed guidelines checklists for describing every type of experiment. perhaps the most well known of these are the consort guidelines for clinical trial reporting (http://www.consort-statement.org/consort-statement/), which enable both clear reporting of the clinical trial itself and also systematic reviews of related trials that may be informative in a way the individual trials were not. more recently, in 2010, a study group comprising animal researchers, journal editors, statisticians and research funders convened to develop guidelines for the reporting of research involving experimental animals [see accompanying editorial in this issue (percie du sert, 2011) and http://www.nc3rs.org.uk/arrive ]. dmm is pleased to endorse these guidelines and to recommend them to our authors and reviewers. their recommendations for writing the main text of the paper, from title through discussion, are for the most part very sensible, but i especially value the guidelines for describing methods. i urge our authors to use them as a checklist when preparing manuscripts for submission, and to read the accompanying editorial from the programme director of the center that provides these guidelines (percie du sert, 2011). we welcome the development of guidelines for research reporting, and will continue to endorse and to publish guidelines that we find relevant to our authors. as one example, in 2010, we published a special article called standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice by ayala. for the mouse metabolic phenotyping center consortium (ayala., 2010). we encourage you to participate in guideline development in your research area and to publish those guidelines in dmm, where they will be immediately freely available upon publication for everyone to read and to use. | summaryprogress in biomedical research depends in part on being able to build on the findings of other researchers and thereby on being able to apply others methods to your own research. however, most of us have struggled to understand how to repeat or adapt another researcher s study because of minimal or missing details in the methods section of a published paper. in expensive and complex experiments involving animal models, clear descriptions of the methods are particularly important. in this and the accompanying editorial in this issue, we discuss how crucial the methods section is to the integrity and utility of a biomedical research paper, and encourage researchers working with animal models to follow the recently released arrive guidelines when preparing their studies for publication. |
a healthy 25-year - old caucasian female presented 2 months postpartum complaining of a mass above her right eye. she first noticed it when she was 6 months pregnant and it had progressively enlarged. examination revealed a freely mobile 1 1 cm firm, solitary nodule above the superomedial aspect of the right orbital rim. a computed tomography (ct) scan examination of the orbits showed a rounded, soft - tissue mass in the subcutaneous soft tissue at the right supraorbital notch [fig. 1 ]. there was no associated osseous defect and no intraorbital, sinus or other extension identified. a rounded subcutaneous soft - tissue mass at the right supraorbital notch. there is no associated osseous defect or extension identified the nodule was completely excised and the surgical site closed primarily. they were arranged in interlacing fascicles with occasional storiform pattern in a myxoid stroma [fig. 2 ]. there were numerous fine capillaries with scattered collections of extravasated lymphocytes and red blood cells present. bland plump spindled cells in a loose myxoid background with extravasated red blood cells and scattered chronic inflammation, (h and e, 200) the etiology remains unknown although trauma and inflammatory reactions have been blamed even though limited evidence substantiates this. periorbital nf can be very concerning since its rapid onset and growth may simulate a malignant process. although periorbital lesions have similar histological features when compared to nf identified at other anatomic sites, the lesions tend to be smaller presumably due to the lack of abundant subcutaneous fat and earlier patient presentation. recurrence of the excised lesion has been documented, but it is so uncommon that it has been suggested that recurrence of a lesion initially diagnosed as nf should lead to a careful reevaluation of the original diagnosis. ophthalmologists are unlikely to consider nf in their differential of lesions in the periorbital region. clinically, the differential diagnosis may include fibroma, solitary fibrous tumor, dermatofibroma, fibrosarcoma, neuroma, neurofibroma, lipoma, granuloma, dermoid, and epidermal inclusion cyst. the clinical and imaging features of nf are not pathognomonic and this can lead to potential diagnostic confusion. histopathologic examination and immunohistochemistry are usually required for the precise diagnosis. despite the absence of cellular atypia, nf can be confused with a spindle cell sarcoma because of its infiltrative quality, rich cellularity, and variable mitotic figures. these include fibrosarcoma, neurofibrosarcoma, myofibroblastic sarcoma, and osteosarcoma. as a spindle cell tumor, nf stains positive for vimentin and variably for sma. similar to our case, nf in the head and neck region more commonly stains diffusely for sma than lesions of the trunk and extremities, suggesting stronger expression by myofibroblasts. a weak presence of estrogen receptors was reported in a series with three out of four cases of back and upper extremity nf, demonstrating focal cytoplasmic positivity for estrogen receptors. a case of intravascular fasciitis, a nf variant, was discovered during pregnancy and the authors hypothesized that the hormone - related changes during pregnancy may have contributed to the development of the lesion. they reasoned that estrogen is known to stimulate fibroblasts and smooth muscle cell types and has been implicated in other fibroproliferative disorders such as carpal tunnel syndrome. since our patient developed nf during pregnancy without a history of trauma, we also reasoned that hormone - related changes may have contributed to the development of the lesion by inducing smooth muscle and fibroblast proliferation. the lesion stained negative for estrogen and progesterone receptors, but this can sometimes be due to poor fixation and embedding procedures. although rare, nf should be considered in the differential diagnosis of periorbital soft - tissue masses arising during pregnancy. | nodular fasciitis (nf) is a benign proliferation of fibroblasts and myofibroblasts that rarely occurs in the periorbital region. we report what we believe to be the first case of periorbital nf associated with pregnancy. a case of intravascular fasciitis, a nf variant, has been reported during pregnancy, but it was not located in the periorbital region. a weak presence of estrogen receptors has been reported in nf. this may make it more susceptible to the hormone - related changes during pregnancy and contribute to the development of the lesion by stimulating fibroblasts and smooth muscle cell types. although rare, nf should be considered in the differential diagnosis of periorbital soft - tissue masses arising during pregnancy. |
recently, yttria partially stabilized tetragonal zirconia (y - tzp) has come into wide clinical use mainly due to its high fracture strength.1,2 although zirconia ceramic restorations may be luted using conventional luting cements,2 bonding with resin to the ceramic would be advantageous for many clinical applications.3 wegner and kern3 demonstrated that a durable bond to zirconia was achieved by applying resin luting cements containing 10-methacryloyloxydecyl dihydrogenphosphate (10-mdp) to an air - abraded zirconia surface. resin - ceramic bonding might be compromised in clinical situations when compared with clean laboratory situations.1 after the try - in of all - ceramic restoration, the ceramic surface might be contaminated by saliva, blood, or silicone fit - indicators.4 among them, saliva contamination is reportedly the main cause of decreased resin bond strength.1,5 some previous studies demonstrated that saliva contamination significantly affected the strength and durability of resin bonds to zirconia and that air - abrasion was the most useful cleaning method.1,4,5,6 recently, a commercial cleaning solution (ivoclean, ivoclar vivadent, schaan, liechtenstein) has been introduced to the dental market. the manufacturer claims that a simple application of the solution, followed by water rinsing and air - drying, effectively cleans the saliva - contaminated bonding surfaces of various dental restorations including zirconia ceramic. however, little research has been carried out with respect to the cleaning efficacy of such cleaning solutions on saliva - contaminated zirconia in terms of resinzirconia bonding. in this in vitro study, we tested the cleaning efficacy of four (one commercial and three experimental) cleaning solutions in enhancing resin - zirconia bonding following simulation of try - in with saliva exposure and compared it to that of air - abrasion. the hypothesis tested was that the cleaning solutions are less effective than air - abrasion in removing saliva contaminants from zirconia surfaces with respect to zirconia bonding with a 10-mdp - containing resin cement. saliva was collected from one non - smoking male who had refrained from eating and drinking 1.5 hours before the collection procedure, in accordance with the institutional review board of kyungpook national university hospital (bmri 74005 - 452) and with the informed consent of the donor.1,6 all experiments were performed with fresh saliva.1 ivoclean (ic, lot # : r78201), which contains zirconium oxide, water, polyethylene glycol, sodium hydroxide, pigments, and additives, was tested. sodium dodecyl sulfate (sds), hydrogen peroxide (hp, h2o2), and sodium hypochlorite (shc, naocl) were purchased from bio - rad (richmond, ca, usa ; lot # : l1610301) or duksan pure chemicals (seoul, korea ; lot # : lc8eb41 and 032516, respectively) and diluted with distilled water into 1.0 wt% solutions. zirconia (lava, 3 m espe, seefeld, germany) disk specimens (20 mm diameter and 1.5 mm thickness) were fabricated according to the manufacturer 's instructions. initially, one surface of all specimens was polished with 600 grit silicon carbide (sic) paper, air - abraded with 50 m al2o3 at 0.25 mpa for 15 seconds at a distance of 10 mm,4 ultrasonically cleaned in isopropyl alcohol for 3 minutes, rinsed with water, and finally air - dried.2,7 the specimens were classified into seven study groups. except for the control group (group nc, no saliva contamination), all specimens were immersed in saliva for 1 minute and rinsed with water - spray for 15 seconds and air - dried for another 15 seconds.4 in group ws, no further cleaning was performed. in group aa, specimens were air - abraded, ultrasonicated, rinsed, and air - dried as described above. in group ic, a microbrush was used to apply ic, which was allowed to react for 20 seconds, followed by rinsing with water - spraying for 15 seconds and air - drying for another 15 seconds, according to the manufacturer 's recommendation. in groups sds, hp, and shc, the corresponding solutions were applied, respectively, rinsed, and air - dried in the same way as for group ic. the study design for surface analysis and shear bond strength testing is illustrated in fig. 1. to assess the cleanability of the surfaces for each study group,8 water contact angle (ca) measurements were performed. since roughness may also alter the ca values, surface roughness measurements were performed prior to ca analysis.9,10 the surface roughness ra of each specimen was measured using a profilometer (surftest sv-400, mitutoyo corp., kawasaki, japan) at a stylus speed of 0.1 mm / second, a cutoff of 0.8 mm, and a range of 600 m.2 the ra of each specimen was determined as the average of five readings (n=5/group). the ca measurements were performed using a ca goniometer (oca 15 plus, data physics instrument gmbh, filderstadt, germany) in a temperature - controlled room at 23 1 with relative humidity at 50 5%.11 using the dynamic sessile drop method, the advancing ca of water was measured after settling 6 l droplets on the material surface, the receding one then being measured after sucking 2 l from the droplet into the syringe (n=5/group).9,12,13 the ca hysteresis (h) was calculated using the equation8 : h = cosr - cosa, in which r and a are the receding and advancing water cas, respectively.14 the degree of correlation between the cosa and the h was determined by the pearson correlation coefficient. to determine the effectiveness of the cleaning methods, specimens of the seven test groups were examined with x - ray photoelectron spectroscopy (xps).1,6,7 all measurements were performed using an xps system (phi quantera sxm, ulvac - phi inc., tokyo, japan) with an x - ray source providing al k x - rays and kinetic energy of 1486.6 ev.1 the emission angle of the photoelectrons was kept constant at 45. a 180 hemispherical analyzer with 32 channel detectors was used for detection of the photoelectrons.7 a wide scan survey spectrum (0 - 1100 ev) was obtained to examine the surface composition of the specimens under ultra high vacuum at 10 pa.7 high resolution scans of the carbon (c1s), oxygen (o1s), zirconium (zr3d), nitrogen (n1s), and aluminum (al2p) peaks were obtained.1,5 ratios of c / o, c / zr, o / zr, n / zr, and al / zr were calculated. for shear bond strength testing, a total of 28 zirconia disks were prepared and embedded in round silicone rubber molds using an acrylic resin. the uncovered (to be bonded) surfaces were treated according the study design (fig. 1) and isolated using a bonding jig (ultradent products inc., south jordan, ut, usa).9,15 freshly - mixed panavia f 2.0 (kuraray noritake dental inc., okayama, japan ; lot # : 00586d (a paste), 00114d (b paste, light shade)) was applied to the surface by packing the material into cylindrical - shaped plastic matrices with an internal diameter of 2.38 mm and then irradiated for 20 seconds using a halogen curing light (elipar trilight, 3 m espe ; output intensity=750 mw / cm).9 in this manner, three bonded resin cylinders were made on one zirconia disk specimen and a total of 12 resin cylinders (i.e., four disk specimens) prepared for each group. prior to debonding, all bonded specimens were stored in water at 37 for 24 hours and then thermocycled 5000 times between 5 and 55 water baths with a dwell time of 30 seconds and a transfer time of 5 seconds between each bath.16 the specimens were perpendicularly engaged at their bonded resin cylinder bases with a round - notched custom shear blade in a universal testing machine (model 3343, instron inc., canton, ma, usa) at a crosshead speed of 1.0 mm / minute until bonding failure occurred.9,15 bond strengths (mpa) were calculated from the peak load of failure (n) divided by the bonded surface area. following debonding, all fractured interfaces were examined under an optical microscope (smz800, nikon corp., tokyo, japan) at 10 magnification to determine the failure mode : a, adhesive failure at the zirconia - resin interface ; c, cohesive failure within resin ; and m, a combination of these failure modes (mixed failure). in addition, a scanning electron microscope (sem, jsm-6700f, jeol, japan) operating at 5 kv was used to observe the debonded zirconia surfaces. the shapiro - wilks normality test and levene 's variance homogeneity test were applied to the surface roughness and bond strength data. the surface roughness data, which met both the normality and variance homogeneity assumptions, were analyzed using one - way anova. as the bond strength data were normally distributed but showed inhomogeneity of variances between groups, they underwent a log10 transformation to meet homogeneity of variance prior to analysis (leven 's test, p=.135).14 shear bond strength comparisons between the seven test groups were conducted using one - way anova followed by the bonferroni post hoc test. the analyses were done under an assumption of independence among the three resin cylinders bonded to each zirconia disk specimen (st 1).17 in addition, a simple random effect in mixed model anova was conducted to allow correlation between the resin cylinders (st 2).17 statistical analyses were carried out using spss 17.0 for windows (spss inc., differences were considered statistically significant at p.05 (marginally significant at p.1, highly significant at p.01, and extremely significant at p<.001).18 in addition, a post hoc power analysis was carried out to examine the power of the bond strength data using gpower 3.1.7 software. one - way anova revealed no significant differences among the seven groups tested (p=.683), indicating that the additional air - abrasion cleaning (group aa) did not significantly increase the ra value of the primary air - abrasion (group nc). 2 shows the results of the pearson correlation analysis between the cosine values of the advancing cas (cosa) and the contact angle hysteresis (h). groups nc, aa, ic, and shc showed higher cosa values, whereas groups ws, sds, and hp yielded lower values. a significant strong negative correlation was found between the parameters (r=-.866, p=.012).. the peak intensity ratios of c / o and c / zr were the highest in group ws (2.1 and 11.6, respectively). the ratios were reduced after air - abrasion (group aa), being comparable to those of group nc. the four cleaning solution groups showed c / o and c / zr ratios that were similar to group aa - except for group sds, which exhibited notably higher ratios. the n element was not detected only in groups nc and aa. in group ws and hp, the phosphorus (p) element was also detected (0.5 and 0.9 at%, respectively). in group sds, each p value from post hoc comparison bonferroni 's test is presented in table 4. group shc showed the highest shear bond strength value (10.9 1.7 mpa). groups nc, aa, ic exhibited statistically similar bond strength values to that of group shc. in contrast, groups sds, ws, and hp showed significantly lower bond strengths than the aforementioned four groups (i.e., groups shc, nc, aa, and ic). for groups shc, nc, aa, and ic, mixed failures outnumbered adhesive failures. for groups sds and hp, the higher frequency of adhesive failures observed when compared to mixed failures. for group ws, all failures were adhesive. fig. 4 shows representative sem images of zirconia surfaces after debonding. groups nc, aa, ic, and shc show the typical air - abraded zirconia surfaces. for groups ws, sds, and hp, blister - like bubble formations on the airabraded surfaces were observed. according to the manufacturer, ic contains sodium hydroxide and is meant for extraoral use only. the three substances used to prepare the cleaning solutions also have potentially adverse intraoral effects when present in high concentrations and with prolonged exposure. clinically relevant concentrations of sds are 0.015 - 1.5%, and toothpastes usually contain 1 - 3% of sds as the detergent.19 home mouth rinses and dentifrices contain low concentrations (1% or less) of hp.20 although 5.25% shc is a common tissue solvent, 1% shc solution has effective tissuedissolving capability.21 considering the potential use of such cleaning solutions for intraoral repair procedures with restorative composite resin, three experimental zirconiacleaning solutions with relatively low concentration (1.0 wt%) were prepared and tested (fig. non - covalent adsorption of salivary proteins, simulated by saliva immersion in this study, occurs on zirconia surface after try - in.1 only water - spray rinsing of the specimens after saliva contamination significantly lowered the bond strength value, compared to the control (table 3). the additional air - abrasion after contamination effectively removed saliva contamination without significantly increasing the ra value (table 2) and restored the bond strength significantly to the same level as that in the control, in accordance with some previous studies.1,4,5,6 in this study, moreover, some of the cleaning solutions (ic or shc) were found to be also effective in removing saliva contamination and in enhancing the resin bond strengths. thus, the null hypothesis that the cleaning solutions are less effective than air - abrasion in terms of resin bond strength was rejected. surface hydrophobicity / hydrophilicity can be determined by ca measurement.22 it is known that surface roughness also alters the ca values of the surface.9 in this study, the influence of surface roughness on the ca values can be excluded because the ra values among the seven test groups were not significantly different (table 1).9,10 although zirconia is rather hydrophobic and has a low surface free energy,23 air - abrasion creates high surface energy and promotes microretention.24 thus, air - abraded zirconia surfaces without saliva contamination may be considered relatively hydrophilic. according to the ca measurements (fig. 2), groups nc, aa, ic, and shc, which produced higher bond strength values (table 3 and table 4), showed lower advancing ca values (39.7 - 52.2) and therefore indicated more hydrophilic surfaces. on the contrary, groups ws, sds, and hp, which exhibited lower bond strength values, yielded lower ca values (62.9 - 65.2) and indicated more hydrophobic surfaces. thus, more hydrophilic zirconia surfaces indicate more effective cleaning ; whereas more hydrophobic surfaces indicate less - effective cleaning.8 in addition, since chemical heterogeneity can also cause ca hysteresis,9 greater surface inhomogeneity due to less - effective cleaning of saliva - contaminated zirconia surfaces may induce greater ca hysteresis.8 thus, a significantly strong negative correlation between the cosa and the ca hysteresis (fig. 2) indicates that ca hysteresis is also relevant in assessing the cleanness of rough surfaces.8 nonetheless, ca measurements alone are not sufficient to characterize surface chemical changes on zirconia before and after cleaning.22 therefore, xps was also used to identify the chemical elements on the surfaces. the depth and spatial resolutions for xps are 1 - 25 nm and 8 - 150 m, respectively.25 since the subtended zirconia surface signal was detectable (fig. 3), the thickness of the contamination layer was less than 10 nm.7 in dental practice, shc solution has been widely used as an endodontic irrigant due to its effective antimicrobial and tissue - dissolving capabilities.26 it is also known that residual shc may interfere with resin polymerization due to oxygen generation.27 among the experimental cleaning solutions, however, 1% shc solution was the most effective in removing the saliva contaminants from the zirconia surface (table 3). xps analysis also showed a lower o / zr ratio for the zirconia surface cleaned with shc than that cleaned with hp. sem observation of the debonded surface revealed little bubble formation at the interface (fig. these findings may indicate that shc effectively cleaned the surface ; water - spray rinsing then removed most of the residual shc on the zirconia surface. the results for group aa confirms again that air - abrasion is a useful cleaning method of saliva - contaminated zirconia.1,4,5,6 in clinical practice, however, the more complex surface geometry of zirconia - based restorations may make it difficult to remove contamination using air - abrasion.1 in such cases, a microbrush would be more convenient for applying cleaning solutions such as shc to the inner surfaces of the restorations. according to the manufacturer (ivoclar vivadent scientific documentation, 2011), the alkaline suspension of zirconium oxide particles in ic removes salivary phosphate contaminants by adsorption. although 0.5% p element was detected in group ws, no p element was detected in group ic (fig. it appears that the application of ic effectively removed various contaminants (including salivary phosphate) from the surface and provided a clean surface for improved resin bonding (table 3). sds is an anionic surfactant commonly used in the removal of proteins.28 however, group sds showed a significantly lower bond strength than groups nc and aa (tables 3 and 4). as stated above, groups ws, sds, and hp showed similar higher water cas (i.e., rather hydrophobic surfaces) (fig. 2).29 nonetheless, the ca hysteresis of group sds was greater than that of group hp and similar to that of group ws. similarly, xps analysis showed a higher c / zr ratio in group sds than in group hp (5.6 vs. 3.5). these findings indicate less complete removal of carbon by 1% sds solution when compared with 1% hp. moreover, small bubbles remained on the sds - treated and water - washed zirconia surface (fig. thus, the solution seems ineffective in cleaning the saliva - contaminated zirconia surface. for group ws, the n element was detected on the zirconia surface (table 2). after cleaning with the solutions, the n / zr ratio was reduced but n remained on the zirconia surfaces. nitrogen was not detected only in groups nc and aa, in which the surfaces were air - abraded. groups ic and shc showed lower cosa values and higher ca hystereses than group aa (fig. 2). these findings might imply the superior cleaning potential of saliva - contaminated zirconia surface by air - abrasion than by the application of ic or 1% shc, although no significant differences in shear bond strength were found among the three cleaning methods (table 3). further research is still needed to clarify whether the cleaning solutions are superior to air - abrasion in terms of long - term clinical bond strength. hp is one of the principal reactive products of oxygen metabolism and often used as a bleach or cleaning agent. although ca measurements and xps analysis indicated a relatively effective cleaning efficacy of 1% hp, group hp exhibited the lowest bond strength value among the seven test groups. this may be due to the more extensive bubble formations on the zirconia surface after cleaning with hp (fig. microtensile bond strength testing, each tooth can be considered a statistical unit because the variation within the tooth may be larger than the inter - tooth variation.30.31 in this study, a zirconia disk, which is expected to show less heterogeneity as opposed to tooth material, was used,31 three resin cylinders being bonded on one zirconia surface.22 in post hoc power analysis, a power of about 0.80 is regarded as acceptable for most purposes.32 for the bond strength data in this study, the power values were 1.00. in our linear mixed model, responses from a subject are thought to be the sum (linear) of fixed and random effects, the latter contributing only to the covariance structure of the data. although the fixed effect was the primary interest in our study, it was necessary to adjust for the covariance. thus, the statistical analyses for the shear bond strength data were done in two ways after transformation : under the assumption that the data are independent (st 1) and that the data on one zirconia are correlated (st 2).17 when the preset threshold value of alpha was set to 0.05, the statistical results in the pairwise comparisons were not affected by the methods of statistical analysis (i.e., sts 1 or 2). nevertheless, the p values in some of the pairwise comparisons differed between the two methods. in particular, the bond strength of group sds was " highly " significantly lower than those of groups aa and ic according to st 1. the value of group hp was " marginally " significantly lower than that of group ws according to st 1, but not significantly different from each other, according to st 2. although a zirconia ceramic is expected to show less heterogeneity as opposed to tooth material, it may thus be necessary to adjust for the covariance structure of the data. during the try - in of zirconia restorations, the surfaces to be bonded may be additionally contaminated by blood or silicone indicators, which might also compromise resin bonding.1,4,6 these contaminants were not included in the present study and further experiments are needed to determine whether the cleaning solutions tested are also effective for such contamination. the cleaning efficacy of 1% shc solution on other prosthetic restoration surfaces should also be studied. the findings of this study confirm that saliva contamination significantly reduces resin shear bond strength to zirconia and that air - abrasion is a useful cleaning method. however, a simple application of ic or 1% shc effectively removed the saliva contaminants and provided a clean surface. the resin bond strength results were supported by water ca measurements and chemical identification of the zirconia surface with xps. however, long - term clinical studies are still required to clarify the efficacy of the cleaning solutions in improving resin bonding of saliva - contaminated zirconia. | purposethis study aimed to investigate the efficacy of cleaning solutions on saliva - contaminated zirconia in comparison to air - abrasion in terms of resin bonding.materials and methodsfor saliva - contaminated airabraded zirconia, seven cleaning methods)-no contamination (nc), water - spray rinsing (ws), additional airabrasion (aa), and cleaning with four solutions (ivoclean [ic ] ; 1.0 wt% sodium dodecyl sulfate [sds ], 1.0 wt% hydrogen peroxide [hp ], and 1.0 wt% sodium hypochlorite [shc])-were tested. the zirconia surfaces for each group were characterized using various analytical techniques. three bonded resin (panavia f 2.0) cylinders (bonding area : 4.5 mm2) were made on one zirconia disk specimen using the ultradent jig method [four disks (12 cylinders)/group ; a total of 28 disks ]. after 5,000 thermocycling, all specimens were subjected to a shear bond strength test with a crosshead speed of 1.0 mm / minute. the fractured surfaces were observed using an optical and scanning electron microscope (sem).resultscontact angle measurements showed that groups nc, aa, ic, and shc had hydrophilic surfaces. the x - ray photoelectron spectroscopy (xps) analysis showed similar elemental distributions between group aa and groups ic and shc. groups ic and shc showed statistically similar bond strengths to groups nc and aa (p>.05), but not groups sds and hp (p<.05). for groups ws, sds, and hp, blister - like bubble formations were observed on the surfaces under sem.conclusionwithin the limitations of this in vitro study, some of the cleaning solutions (ic or shc) were effective in removing saliva contamination and enhancing the resin bond strength. |
chondrosarcoma (cs) is a malignant tumor characterized by the formation of cartilage and usually arises in long and flat bone. head and neck cs is an uncommon entity accounting for approximately 0.1% of all head and neck malignancies. several variants of cs have been proposed including clear cell, dedifferentiated, myxoid and mesenchymal. mesenchymal chondrosarcoma (mc) is a rare variant of cs that accounts for up to 3 - 9% of all cs and has high predilection for the head and neck region. mc is one of the most unusual rare malignant cartilaginous malignancy of bone and soft tissues with distinct histopathological appearance and biological behavior and was described by lichteinstein in 1959. approximately 33% of these tumors occur in an extraskeletal location or are associated with a large, extraskeletal, soft tissue mass, commonly in the meninges. similar to conventional chondrosarcomas, lesions occur in the ribs, pelvis, and jaw bones more frequently than other bone neoplasms. mc occurs between ages 30 - 60 years with a mean age of 47.5 years. patients typically present with vague symptoms of pain and swelling that may have been present for a relatively long duration. histologically, it is characterized by a biphasic pattern consisting of areas of hyaline cartilage mixed with small cell malignancy. a 60-year - old male patient reported with the chief complaint of swelling in the lower right side of the face since 1 month. history revealed that the swelling was small 1 month back which rapidly grew to attain the present size. extra - oral examination revealed a diffused swelling on the right side of the face measuring approximately 6 7 cms in size, extending from the temporal region to inferior border of the mandible superior - inferiorly and from the nose to the ear anteroposteriorly. the skin over the swelling was slightly stretched with no secondary changes [figure 1 ]. on palpation the swelling was non - tender, varied in consistency from soft in the center to hard at the periphery. the right sub - mandibular lymph nodes were palpable, non - tender, hard and fixed to underlying structure. clinical photograph of the patient showing a diffused swelling on the lower right side of the face intraoral examination showed an oval swelling present on the right side of the jaw in the retromolar area measuring approximately 4 3 cms in size. intraoral photograph showing an oval swelling present on the right side of the jaw in the retromolar area radiological examination revealed a diffused radiolucency involving the lower body and angle of the mandible extending from right second premolar to angle of the mandible [figure 3 ]. orthopantomogram revealed a diffused radiolucency involving the lower body and angle of the mandible in relation to 46, 47 considering the size and aggressive nature of the lesion, surgical resection with wide margins of the lesion was carried out. the gross soft tissue specimen received for histopathological examination measured approximately 12 5 mms in size, was soft to firm in consistency, irregular in shape and whitish brown in color. on histopathological examination, the hematoxylin and eosin (h and e) stained section showed sheets of tumor cells interspersed by cartilage area showing calcification [figures 4 and 5 ]. higher magnification showed pleomorphic cells with large basophilic ovoid nuclei and small darkly stained nuclei with little cytoplasm [figures 6 and 7 ]. scattered, small, poorly circumscribed foci of cartilaginous tissue were also seen. correlating the clinical, radiological and the histopathological features a diagnosis of mc the patient showed no signs and symptoms since 2 years and is being followed up 6 monthly periodically for possible recurrence. (h&e stain, 200) photomicrograph showing tumor cells and cartilaginous area with calcification. (h&e stain, 100) photomicrograph showing pleomorphic cells with large basophilic ovoid nuclei and few cells with small darkly stained nuclei and little cytoplasm. (h&e, 200) photomicrograph showing pleomorphic chondrocytes (h & e, 400) mc was first described by lichtenstein l and bernstein d in 1959 as a biphasic tumor, comprising of spindle cell mesenchyme interspersed with areas of chondroid differentiation. it is a specific variant of cs and represents approximately 1% of all css. mc of the head and neck occurs most often in the third to the sixth decades of life that is very much consistent with the present case. however, they are formed from cartilage in tissues not normally harboring cartilage or, secondly, from the cartilage cap of exostosis or enchondromas. it originates from bone, meninges or less commonly, soft tissues. according to thomas rs., (2010), 3 to 25% of all skeletal mc occurs in the maxillofacial region the premolar - molar region being the most common site. other most commonly reported symptoms are nasal obstruction, epistaxis, tooth mobility, headache, bleeding, ulceration, facial asymmetry, paresthesia and trismus. radiographically, these lesions appear as osteolytic, radiolucent shadows with ill - defined, ragged borders. undifferentiated areas appear as sheets of primitive mesenchymal spindle / round cells similar to small cell anaplastic sarcoma. however, islands of relatively well differentiated cartilaginous tumor help in making a specific diagnosis. neoplastic cartilage may be replaced by bone in a manner similar to normal endochondral ossification. histologically, the lesion must be differentiated from similar other lesions like hemangiopericytoma, ewing 's sarcoma, pnet, leukemia / lymphoma, rhabdomyosarcoma, and malignant melanoma the most effective therapeutic modality is wide surgical excision. wide local excision with a tumor free margin of 2 - 3 cm is recommended. also, the postoperative radiotherapy and chemotherapy offer a good prognosis and eradicate chances of micrometastases. the prognosis for mc is poor because tumors have a tendency for late recurrence either locally or as metastasis. mc is a rare mesenchymal tumor that occurs frequently in both hard and soft tissue locations. these tumors show local aggressive behaviour as well as a high metastatic and recurrence potential. due to these features the prognosis of mc is poor. considering the propensity of these tumors to metastasize and the poor prognosis of patients with mc, early identification may allow earlier, more aggressive interventions. | mesenchymal chondrosarcomas (mcs) are rare malignant connective tissue neoplasms representing approximately 1% of all chondrosarcomas (css) that can arise from both soft and hard tissues. they are distinct tumors arising in unicentric or multicentric locations. the tumor is most unusual as it has been described as a particularly aggressive neoplasm with a high tendency for late recurrence and delayed metastasis. it is a biphasic tumor with areas comprising of spindle cell mesenchyme having areas of chondroid differentiation. here we report a case of 60-year - old male with mesenchymal cs of the mandible. |
plasmodium falciparum infection remains a major cause of death in most of developing countries, particularly in sub - saharan africa. it generates different clinical manifestations with either few or no symptoms or severe signs such as impaired neurological function termed cerebral malaria (cm) [3, 4 ]. these variable courses of the disease have been related to different factors such as the levels of parasites transmitted, the age of the infected subjects, the genetic background of the population studied [3, 4 ], and, above all, the immune status of affected individuals. thus, unprotected children living in endemic zones or unprotected adults coming from nonendemic zones have a high susceptibility to severe malaria. it is postulated that partial protective immunity can be induced after iterative infection through triggering the action of the immune response, particularly the humoral response [6, 7 ]. recent studies have highlighted the influence of the autoantibody response [8, 9 ]. therefore, appropriate analysis of the serum self - igg repertoire could contribute to a better understanding of the immuneregulation processes involved during the course of the disease. in healthy subjects, despite of interindividual differences, the human serum self - igg response is thought to be well conserved and restricted to the recognition of a few self - antigens in autologous tissues. in contrast, durable distortion of these immune profiles has been found in our laboratory among patients with multiple sclerosis (ms) or other autoimmune diseases with predominant neurological signs such as neuropsychiatric systemic lupus erythematosus [12, 13 ]. when we induced experimental autoimmune encephalomyelitis, dynamic changes in immune profiles related to pathogenic or protective events were also identified [14, 15 ]. despite the predominant neurological symptoms in clinical and experimental situations, discriminant self - igg reactivity involves mostly ubiquitous antigens rather than specific targets in nervous system tissue. although these footprints have allowed the identification of new useful biomarkers [12, 13 ], their pathophysiological significance remains to be defined. in the present study, we aimed to evaluate the impact of the environment and self - reactive natural and acquired antibody repertoires on humoral immune profiles. the findings of numerous epidemiological and clinical studies suggest that the risk of allergic and autoimmune diseases is related to the hygiene hypothesis. parasitic infections, especially malaria, may influence the development, or the course of autoimmune disease such as ms. in contrast some self - reactive antibody responses might also influence the course of malaria leading to protective or pathogenic events. to further evaluate the relationships between environmental factors, autoimmune profiles, and the clinical status of malaria, the natural and acquired self - igg antibody responses were analyzed in subjects of different age brackets living in endemic zones of parasitic transmission. immune profiles were compared between malaria patients with (cerebral malaria) and without (including uncomplicated disease, asymptomatic plasmodium falciparum carriers) neurological symptoms. our data revealed the presence of antigenic bands specifically targeted by plasma igg collected in patients of a well - defined clinical status and age range. blood samples were collected from subjects exposed to malaria parasite, termed malaria - exposed individuals ([mei ] n = 102, mean age sd : 21.07 20.2), and from healthy subjects living in european nonendemic areas, termed nonendemic controls ([nec ] n = 17, mean age sd : 39 4.5). one subgroup consisted of patients with neurological symptoms, usually termed cerebral malaria ([cm ] n = 28, mean age sd : 16.2 21.4). the other subgroup was termed mei without neurological symptoms (n = 74, mean age sd : 21.1 21.3) and was comprised of parasite carriers with classical symptoms of malaria but without other complications and asymptomatic carriers (without any detectable symptoms). meis with symptoms (neurological and classical) were recruited from the emergency department of the university hospital of cocody - abidjan (cte d'ivoire). using available data bases, thus, mei with neurological symptoms included patients with a blantyre coma scale 2 (concerning the children), a modified glasgow coma scale 9 (concerning the adult), the occurrence of at least one convulsive episode during the 24 h before admission to the emergency department, and plasmodium falciparum - positive thin blood smears. blood samples of asymptomatic parasites carriers were obtained from the national blood transfusion centre of abidjan, from the mother - infant department of the district hospital, and from some volunteer students. all the procedures were reviewed and approved by the national health office ethics committee of cte d'ivoire. for the nec group, blood samples were obtained from healthy volunteers following approval by the centre de ressources biologiques - centre d'investigation clinique of chru - lille. isolated plasmas were then stored at 40c in our laboratory (ea 2686, universit lille - nord de france). for the mei group, the samples were taken on the day of admission (in the emergency department before any therapy concerning those who were ill, the mother - infant department, the national centre of blood transfusion, or in the school for those who were asymptomatic parasite carriers). plasma was isolated after centrifugation at 500 g for 15 minutes and stored at 40c at the laboratoire d'immunologie of chu cocody - abidjan before being sent the ea 2686 research group (lille - france). parasitemia was assessed at the department of parasitologie - mycologie, facult de mdecine d'abidjan - cocody - cte d'ivoire as follows. giemsa - stained thick and thin blood films were prepared for detection and identification of plasmodium falciparum and for parasite counts. anti - falciparum total antibodies detection and quantification were performed at the laboratoire de parasitologie mycologie - centre de biologie, de pathologie - chru de lille - france. briefly, sera were diluted 1/100, 1/200, 1/400, 1/800, 1/1600, and 1/3200 in phosphate - buffered saline (ref 77511 biomerieux). diluted serum was incubated with acetone - fixed plasmodium falciparum at 37c for 30 mn. the secondary antibody used was fluorescein - conjugated goat anti - human igg, igm, iga, heavy and light chains - h, and l-(ref 74511 diagnostic pasteur) in bleu evans solution (ref 75491 biomerieux). any fluorescence in fluopep (ref 75521 biomerieux) obtained at a dilution 1/100 was considered negative. briefly, cerebral tissue was extracted by autopsy from the frontal lobe in broadman 's area 10, from healthy subjects with no history of neurological disease (department of neuropathology, centre hospitalier de l'universit de lille, and institut national de la sant, de la recherche mdicale, unit 422, lille, france). autopsies were performed within the framework of a tissue collection program that had been approved by the local ethics committee. the brain samples were homogenized in a tris buffer containing 5% sds at a final concentration of 10 mg / ml and heated at 95c for 10 min. 100 l of this lysate was loaded onto a 1020% gradient polyacrylamide gel (navix tris - glycine gels - invitrogen), beside a well of 15 l of molecular mass marker (amersham pharmacia biotech). just before 1-dimension electrophoresis (1d), the homogenates were reduced for 10 min with 10 mm dtt (sigma - aldrich) at 100c. then electrophoresis was run in laemmli buffer using the following conditions : run time 2 h at constant 125 v and 3040 ma / gel (start) ; 812 ma / gel (end). proteins were transferred onto ecl nitrocellulose membranes (amersham pharmacia biotech) at 0.8 ma / cm and later saturated with 5% fat - free dried milk. western blotting was conducted with total sera, diluted 1/100 in tnt (100 mm tris ph 8.3-, 0.3 m nacl containing 0.5% tween) and 5% fat - free dried milk. reaction igg antibodies were revealed with an anti - human fc hrp - conjugated antibody (1/10000 ; sigma - aldrich) after overnight incubation at 4c. image analysis was performed on a molecular imager gs-800 calibrated densitometer (bio - rad) to localize and compare the igg immune profile patterns. band detection, alignment, and matching of the antibody reactivities were performed using phoretix 1d and 1d pro software version 10 (totallab - nonlinear dynamics) and validated by two operators. we performed comparative analyses using detection - band parameters that allowed us to consider each band intensity that was > 13% higher than the global background intensity of the gel to be significant. to calibrate and more accurately define the alignment of antibody reactivities, molecular weight marker protein standards (stdmw - benchmark pre - stained protein ladder, invitrogen) as well as internal reference standards were used the level to which each molecular standard migrates is called relative front of migration (rf). a function curve defined by rf values on the x axis and standard molecular weight on the y axis allowed the molecular weight of each antigenic band to be determined by extrapolation because the reactive band align on its rf. data were expressed in binary mode (0 = absence of an antigenic band and 1 = presence of an antigenic band) for submission of igg antibody patterns for analysis using either the chi - square test or fisher 's exact test. this approach allowed us to select the most relevant antigens that qualitatively supported different types of immune recognition. we then used linear discriminant analysis (lda) to balance the discriminating antigens between the populations of individuals, as described previously [22, 23 ]. using a stepwise logistic regression model and supported by the lda method, we isolated a subgroup of brain antigens according to their strength of discrimination between the different populations involved in the study. using 2 parameters (for the presence1or absence0of each selected antigen) and the coefficient previously defined by the lda, a score was calculated for each subject as a representative value of the individual immune profile, using the following formula : (1)score = ag1coef1[0(absent) or 1 (present) ] + ag2coef2[0(absent) or 1 (present)]+ag3coef3, where ag represents antigen and coef represents coefficient. a threshold value was determined using a receiver operating characteristic curve, and the sensitivity and specificity of this approach were evaluated. when the number of patients was too small to apply lda, chi - square and fisher 's exact tests were performed to retain the most discriminant band. to compare the number of bands between the different groups of subjects the frequency of each discriminant self - reactive band has been calculated determining its percentage of its presence within the group of subjects. using principal component analysis (pca) and nonparametric test of kruskal - wallis, we evaluated the relationship between the discriminant band and the clinical status. self - igg reactivity against human brain was compared between patterns obtained with plasma samples from the mei group, with or without neurological symptoms (subjects from 1 to 86 years old), and those obtained with samples from the nec group (figure 1). self - igg responses appeared to be quantitatively (number of bands) and qualitatively (presence or absence of bands) heterogeneous within or between the different populations studied. quantifiable data were given after the mapping and alignment of all patterns which revealed 4 to 19 bands according to the strips. when all of the strips were considered, there were more antigenic bands in the mei (n = 102, mean sd : 14.9 3.9) than in nec group (n = 17, mean 4.7 1.9 ; p 30 years) to detect any antigenic bands that discriminate between malaria with or without neurological symptoms. chi - square, fisher, and lda analyses were performed to discriminate between mei with or without neurological symptoms for each of the defined age groups. for ages over 30 years, lda combined with fisher and chi - square tests did not identify any discriminatory antigenic bands, in mei with or without neurological symptoms. however, for ages under 30 years, lda and fisher tests identified the two most discriminant antigenic bands in mei with or without neurological symptoms (with p 30 years). age influences the severity of the infection since it is known that malaria in children living in endemic zones is often severe. they are the first victims of cm because of their unprotected immune status [5, 43, 44 ]. in our study, two types of singular self - igg reactivity were noted (common or discriminatory antigenic bands) when we examined the three youngest age groups of mei with or without neurological signs (15 years, 615 years, and 1630 years). the lowest number of mei with cm was for the subgroup aged over 30 years and does not allow us to discuss the results obtained for this age group. however, all self immune prints from mei, with or without neurological symptoms, were superimposed in this age bracket. these data on self - reactive prints suggest that during parasitic infection of adults, there is a point where stop - evolving events appear. during aging, internal changes in self - proteins may occur, and the general network of natural autoantibodies called immunculus will be also modified. furthermore, some internal events (metabolic deviations related to diseases) and external events (environmental factors) could lead to immunculus distortions. common antigenic bands (p49) appeared in mei age groups from 1 to 30 years irrespective of clinical status. this nondiscriminatory reactivity might be linked to the previously described immunculus characterized by constant or minimal individual variations of the immune repertoire. these natural autoantibodies derived from human germline immunoglobulin genes without somatic mutations are often poly reactive and expressed a low affinity for antigens [39, 40, 47 ]. natural autoantibodies in different members of a malaria population may also react against the conserved public self - epitopes [9, 4850 ]. in return, the microbial, viral, and parasitic infections can all modify the antibody repertoire leading to the emergence of potent pathogenic auto - antibodies. natural and acquired auto - antibodies are often directed against ubiquitous antigenic targets with different seric titers, that is, antinuclear, antimuscle, or antiphospholipid, antibodies. nevertheless, they occur at higher frequencies among individuals exposed to viral, bacterial, and parasitic diseases, including malaria [9, 40 ]. in our work, some specific antigenic bands, presented as a peculiar intragroup self - reactivity pattern, are expressed according to each age group in different proportions in mei with or without cm. in children aged 1 to 15 years, discriminant antigenic bands were identified (p130-p30 in subjects aged from 1 to 5 years, and p180-p43 in subjects aged from 6 to 15 years). however, they are not good bio - markers for cm, as they were low in frequency within each age group of mei with cm. in contrast, in mei subjects aged from 16 to 30 years, significant discriminatory bands were found. subjects of this group displayed singular self - immune reactivity characterized by only two discriminatory self - reactive antigenic bands this appears stable in all subjects since they were immunized against almost all prevalent parasitic strains encountered in the area [4, 52, 53 ]. when we considered only the presence of cm irrespective of age, three antigenic bands were identified (p145, p140, and p94). a double band localized at p147 kda has been previously detected and characterized as isoforms of nonerythroid spectrin. regarding the degree of resolution of our methodological approach of we can not exclude that p140, p145, and p147 could be the similar antigenic targets. it discriminated, not only mei and nec, but also it discriminated both mei with or without neurological symptoms and cm patients according to age. as previously postulated, this new discriminant antigenic target could be a fraction of the p147 band, cleaved during apoptosis following the activation of a neutral calcium - activated protease (calpain). such dynamic changes occurring before and after 16 years might be related to the immune response against different strains of plasmodium falciparum that underlie recurrent infections. indeed, in malaria - endemic zones, acquired protective immunity (called premunition) induced by parasitic diversity is strengthened with increasing age [5557 ]. consequently, in children, iterative infection episodes may induce antibodies to specific parasitic strains which can cross - react against self - epitopes. furthermore, these recurrent parasitic attacks occurring in childhood may induce inflammatory and deleterious tissue events. this may generate neoantigenicity by epitope spreading processes, or by revealing self cryptic / hidden antigens [14, 15, 59 ], thereby, favoring expression of new antibodies involved in self - immune reactivity and potent pathophysiological events. the present work is focused on the age - related evolution of the self - immune patterns. therefore, the characterization of the nature and structure of identified antigenic bands of interest (such as p94) will be assessed in future studies requiring mass - spectrometry analysis. their presence in malaria infection may be of prognostic interest as in other infections where some auto - antibodies reveal a new repertoire that heralds susceptibility to future autoimmune diseases. self - reactivity to nonerythroid alpha spectrin and beta tubulin iii have been associated with cm in gabonese children and indian populations, respectively. however, to our knowledge, no specific brain auto - antigens related to age of infection with malaria have been reported until now. it is, therefore, of interest to characterize such self - antigens because cm is one of the principal causes of neurodisability in sub - saharan africa. although a few studies suggest that there is full neurological recovery in cm [35, 63, 64 ], it has become clear that many children have sustained severe brain injury over the past 15 years. thus, 25% of cm cases result in long - term neurological and cognitive deficiency or epilepsy [65, 66 ]. our data show that parasite pressure seems to cause quantitative and qualitative changes of systemic self - igg immune - reactive profiles in mei. however, the singularities of such an immune response related to age allow the identification of some specific antigenic bands as potent useful biomarkers. their further characterization is of particular prognostic interest in cm since such evolutionary changes involving the repertoires of self - natural antibodies and acquired antibodies may lead to pathogenic or protective events. | background. absence of acquired protective immunity in endemic areas children leads to higher susceptibility to severe malaria. to investigate the involvement of regulatory process related to self - reactivity, we evaluated potent changes in auto - antibody reactivity profiles in children and older subjects living in malaria - endemic zones comparatively to none - exposed healthy controls. methods. analysis of igg self - reactive footprints was performed using western blotting against healthy brain antigens. plasmas of 102 malaria exposed individuals (meis) from endemic zone, with or without cerebral malaria (cm) were compared to plasmas from non - endemic controls (necs). using linear discriminant and principal component analysis, immune footprints were compared by counting the number, the presence or absence of reactive bands. we identified the most discriminant bands with respect to age and clinical status. results. a higher number of bands were recognized by igg auto - antibodies in mei than in nec. characteristic changes in systemic self - igg - reactive repertoire were found with antigenic bands that discriminate plasmodium falciparum infections with or without cm according to age. 8 antigenic bands distributed in mei compared with nec were identified while 6 other antigenic bands were distributed within mei according to the age and clinical status. such distortion might be due to evolutionary processes leading to pathogenic / protective events. |
in order to examine resources of nslbp, a definition of nslbp is first given. nslbp refers to pain symptoms anywhere in the lower back between the twelfth rib and the top of the legs. it is defined as pain or discomfort, localized below the costal margin and above the inferior gluteal folds, with or without leg pain [2, page 171 ]. no recognizable, specific pathology such as infection, tumor, osteoporosis, fracture, radicular syndrome, or cauda equina syndrome is attributable to the pain sensations. about four out of five persons experience low back pain at least once in their lifetime with a one - year prevalence of 15% to 45% in industrialized countries. since the natural history of nslbp is favorable, most individuals recover within six weeks [2, 5 ]. however, not all individuals recover spontaneously, and if nslbp persists for longer than 12 weeks, acute nslbp becomes chronic nslbp. an epidemiological study with data out of 16 european countries estimates that 19% of the european population suffered from chronic pain in 2003. the largest category with 47% out of this 19%is based upon back pain. a recent inception study presents even higher numbers : more than 40% of 973 individuals developed chronic nslbp after presenting themselves to primary care with acute nslbp. first are the chronological dimensions with acute nslbp lasting less than four weeks, subacute nslbp lingering for between four to twelve weeks, and chronic nslbp persisting for longer than twelve weeks. krner - herwig postulates that acute pain, for example, a knife wound or a sprain, is related to a distinct trigger, whose concrete function is to warn about an injury. although this definition of acute pain is not completely transferable to nslbp, since the pain triggers might remain unclear, the distinctions with chronic pain are noteworthy. the pain sensation of chronic nslbp is no longer related to a possibly unclear peripheral trigger ; pain is centralized. two kinds of neuronal plasticity, functional and structural, are relevant to this phenomenon. functional plasticity occurs rather quickly as a physiological adaptation measure. structural plasticity relates to medium and long - term anatomical and biochemical modifications due to the altered requirements in the pain processing mechanisms. for this reason, factors, mechanisms, and treatment options of chronic nslbp will be explained below. although pain symptoms are often implicitly attributed to medical reasons such as serious pathologies in the lumbar spine, other causes besides medical reasons also have to be considered as the origin. therefore, nslbp is often explained with the biopsychosocial model of pain [11, 12 ]. in his 1977 article, engel postulated that the appearance of illness resulted from the interaction of diverse causal factors biological, psychological, and social factors and that psychosocial variables were crucial with respect to the susceptibility, severity, and course of illness. engel also pointed out that the patient - clinician relationship influenced medical outcomes as well as scientific results with regard to the hawthorne effect [13, 14 ]. (the hawthorne effect relates to studies conducted in the twenties of the last century and refers to a noticeable change in the behavior of study participants without any experimental condition. the adaptations in the behaviors of the participants were explained by the arguments that participants knew they were observed and part of the study.) it was waddell who integrated the latest findings of the biopsychosocial model of nslbp into the who - icf model (figure 1), the international classification of functioning, disability, and health, and took a further step toward establishing the model as a result. a parallel research line toward an understanding of pain is melzack and wall 's gate control theory of pain. two central aspects of the theory are crucial : the description of the transmission and modulation of nociceptive signals and the recognition of pain as a psychophysiological phenomenon. much like engel in 1959 [17, page 901 ], who mentioned the affective aspect of pain : when we scrutinize more carefully the identifying quality of pain we note that it includes an affective tone. it is usually unpleasant, but it may also be pleasant, if only in a relative sense. this effective quality brings pain into a very central position in terms of psychic development and function, melzack and wall [16, page 978 ] stressed the psychological aspect by declaring that the (gate control theory) model suggests that psychological factors such as past experience, attention, and emotion influence pain response and perception by acting on the gate control system. ever since then, a number of investigations have examined details of the gate control theory, as well as neurophysiological and neuroanatomical pathways of pain. because its descriptions would clearly stretch this works ' focus, interesting readers are referred to butler and moseley pain management for a detailed historical summary, or to krner - herwig and colleagues, quite a few pain models have been published integrating interrelationships between complex factors which enhance chronic nslbp. however, they often focus on specific pathways, such as psychological, biological, or behavioral risk factors of chronic nslbp. by introducing the modified salford model, a broad overview of possible pathways to chronicity and of the momentary evidence - based knowledge the salford model has been adapted in order to better demonstrate the impact of psychological factors (dark lateral squares) on the physiological dimensions (bright middle square). a single arrow does not stand for a single transition ; the model rather shows several, interrelating self - enhancing circles. the model demonstrates now several circuli virtuosi, self - enhancing circles which can cause or increase chronicity. pain often provokes guarded movements or muscle spasms [22, 23 ], which are reversible. guarded movements lead to momentary reduced activity and decreased circles of actions, such as work or social activities. as a consequence, local physical deconditioning occurs by a decreased intra- and intermuscular coordination, as well as neuromuscular perception. until here if, however, the circle mechanisms endure for too long and can not be interrupted, the circle continues to increase the problem, and the reactions start to be pathological [23, 24 ]. when turning toward the dark lateral square on the right, the self - enhancing circle of (mis)attributions and fear - avoidance behavior is pictured.. some might think not to worry or that the pain will pass in itself, while others start to worry about the nature of the pain sensation and search for signs of severe pathologies or false behavior. beliefs and misattributions lead to a so - called fear - avoidance behavior in order to restore the homeostasis. all movements which are subjectively prognosed to increase pain will be avoided, further guarded movements occur, and the basic circle keeps turning, as does the attribution circle. the second dark lateral square on the left includes affective components of nslbp. prolonged guarded movements or muscle spasms individuals start to become annoyed about the pain sensation and the resulting disability which, again, can enhance muscle spasms or guarded movements as well. a normal and effective reaction to this anger is to ease the pain or to change the situation. if, however, all techniques and measures do not ease, and situations can not be attained, feelings of helplessness or loss of control occur, which consequently can lead to psychological distress or even to depression. moreover, symptoms of depression and stress are found to mediate the effect of pain on disability [30, 31 ]. the emotional state of depressed individuals is altered, as they usually have a low mood that is accompanied by low self - esteem. furthermore, they have often lost interest or pleasure in things that they used to enjoy. depressive individuals this declares that a similar state of emotion during learning eases the individuals ' recall abilities and experiences. for example, learning something when happy will be harder to recall in a depressive state. first, when looking at the basic circle, withdrawal from work or social activities can also induce helplessness or psychological distress and start or intensify the affective circle in this way. second, iatrogenic influences need to be mentioned. sometimes, treatment by medical staff is not successful. despite the willingness to help and heal, treatments can fail ; thus symptoms, beliefs, or behavior become worse and can result in a failed treatment. unsurprizingly, this can lead to anger or frustration toward the medical staff, the hospital, or even the pain symptoms. failed treatment can provoke learned helplessness, and support subjective (mis)attributions toward the symptoms. iatrogenic influences can also directly enhance (mis)attributions as well as fear - avoidance beliefs. third, family or learnt behavior can have an impact on (mis)attributions, fear - avoidance beliefs, and behaviors, as well as learned helplessness. finally, socioeconomic and occupational factors, like high job stress levels, can cause withdrawal from work or social activities and influence anger and frustration as well as learned helplessness. which is not shown in the figure is the fact that patients do not feel understood as a consequence and lose confidence, which leads to a bad doctor - patient relationship that also decreases treatment success. taken together, the salford model gives an overview on how chronic nslbp can develop and be maintained. first, the basic circle in the bright square is physiological and only develops into a pathogenic pathway when it repeatedly occurs and can not be interrupted. second, psychological as well as exogenic processes can provoke a pathogenic development of nslbp. the complexity of the interrelations is shown to some part and one might assume how fragile the balance can sometimes be. further endogenous factors, such as personal traits like introversion or neuroticism, self - efficacy, or resilience, as well as social factors like support from relatives or superiors at work and material aspects always influence possible confounding pathways. with regard to the influencing factors of the development of (chronic) nslbp, a clinical aim within the last 15 years has been to improve the understanding of risk factors which could serve in a prognostic way and help to identify individuals at risk developing prolonged nslbp or transforming nslbp to chronic lbp (e.g., [5053 ]). as a result, flags containing different risk factors and obstacles to recovery have been developed [5456 ]. the highest risk factor for experiencing nslbp is a previous occurrence of nslbp [57, 58 ], which is not surprizing considering the prevalence rate of nslbp [4, 58 ]. as for body weight, a recent population - based study with more than 60,000 participants estimated a significant odds ratio (or) per 5 kg / m increase in body mass index for men (or = 1.07, 95% confidence interval : 1.031.12) and for women (or = 1.17, 95% ci : 1.141.21) after controlling for age. being married, well - educated, and employed was negatively associated with costs due to nslbp. a french population - based study on nslbp and its risk factors illustrated a low educational level as crucial for the development of nslbp. however, when considering work postures or heavy workloads, the educational level lost its predictability. with regard to the underlying mechanisms, a low educational level and work postures have to be considered as confounders. the least - educated men demonstrated the highest frequency of physically tiring postures (63%), while the lowest frequencies of physically tiring postures were confirmed for the highest and second highest education levels. being female [62, 63 ] and being older than 50 years are well also defined risk factors [57, 63 ]. while women demonstrate a higher probability of suffering of nslbp and therefore to incur costs, men generate higher costs. the authors of the population - based german back pain study concluded that women tend to utilize healthcare more quickly than men, but that once men utilize healthcare it leads to higher costs on average. roughly summarized, there is evidence for demographic risk factors for the onset of nslbp. however, evidence varies between studies and despite the investigations on prognostic factors, uncertainty remains regarding the strength of the associations and the extent of confounders. risk factors mentioned in the flag system are often associated with a delayed recovery and thus with the transition from acute to chronic nslbp [19, 54 ]. clinical red flags : biomedical factors. red flags are not considered as risk factors, rather as warning lights. since nslbp is defined as nonspecific, it is crucial to exclude possible medical problems. for that reason, red flags were first proposed by guidelines [65, 66 ] and applied in primary care to identify patients with an urgent need for a specialist opinion. they were defined as medical - biomedical signs and symptoms that indicate an organic pathology or a concurrent serious medical problem. examples are cauda equine syndrome signs, such as bladder or bowel incontinence, significant trauma, pain which gets worse when lying down, or unexpected weight loss with or without fever. they include subjective appraisals, unhelpful beliefs and expectations about pain, or negative expectations of recovery. these adverse cognitive appraisals can enhance the fear of movement, the avoidance of activities due to expectations of pain and possible reinjury, and feelings of being helpless, worried, and distressed [54, 67 ]. clinical orange flags : psychiatric symptoms. they include excessively high levels of distress, severe personality disorders, drug and alcohol addiction, or clinical depression [19, 56 ]. they include aspects of the employee and the workplace. with regard to the workplace, blue flags comprise physical job demands, low possibilities to modify work, or a stressful job environment. likewise, regarding employee aspects, low job satisfaction, as well as low social support at work, can enhance delayed recovery [55, 68 ]. two occupational constructs with strong evidence as risk factors for nslbp shall be introduced more profoundly : (low) job satisfaction and (low) social support at work. job satisfaction includes an individual 's thoughts and feelings about their work, with respect to the feeling of overall satisfaction or overall dissatisfaction. overall satisfaction can be understood not only as a single, global construct but also as an accumulation of various different facets like the work itself, social relationships at work, the supervisor, wages, or the travel distance to work. low job satisfaction, in comparison to high job dissatisfaction, was found to act as a risk factor for nslbp or its transition to chronic nslbp. more recent literature did not confirm the influence of job satisfaction on prolonged sickness absence due to nslbp [73, 74 ] or on the outcome of nslbp in primary care settings. however, satisfaction with one 's job was described as a resource protecting against the development of chronic nslbp and disability. as for social support at work basically, social support implies that a person feels cared for and appreciated and has access to help when needed [78, 79 ]. with respect to the complexity of social support, the literature differs between social support from colleagues or from superiors. receiving support from close confidants had a detrimental effect on nslbp, while support form supervisors or less confident colleagues correlated negatively with the nslbp duration [48, 80 ]. these findings indicate that by focusing on neutral issues, support from more distant individuals does not disturb the integrity of the person seeking help. (or, in other words, although a more distant person supported an employee in need, the distance between the persons kept the supporting person from intervening too much. the employee in need did not succumb to a dependency towards the supporter.) occupational black flags : system and contextual factors. black flags are defined as the occupational and systemic context in which a person functions. they include misunderstandings or disagreements between key players, such as employers, or insurances due to financial and compensation problems. further black flags include process delays with regard to treatment approval or financial security or social isolation due to dysfunction as a result of a lack of co - ordination among health care providers [55, 82 ]. therapeutic aims include (a) the reduction of the factors responsible for pain maintenance and (b) the improvement of individual pain management. with regard to the biopsychosocial aspect of nslbp, the most highly recommended way of tackling chronic nslbp is via a multidisciplinary treatment (mdt) program [6, 83 ]. ever since turk and colleagues integrated a cognitive - behavioral approach to pain has become the framework that most current pain management programs have been drawn from. they focus on pain management rather than cure ; contain a behavioral rather than disease perspective ; enclose various measures like medical, manual, exercise, or psychological treatments ; and include an interdisciplinary skill mix. the emphasis of the group therapy settings lies on active and self - helping approaches with regard to augmenting the patients ' responsibility [26, 85 ]. due to its high economic impact direct medical costs include physician consultations or medications, while direct, nonmedical costs incorporate transportation costs to attend medical appointments. supplementary, indirect costs include decreased or lost productivity due to disability or sickness absence. despite the difficulty in measuring indirect costs, it is well known that costs resulting from lost work productivity represent the majority of nslbp - associated costs. nslbp costs estimations for switzerland in 2005 were 2.6 billion for direct costs, representing 6.1% of the total healthcare expenditure. indirect costs were estimated between 2.2 billion and 4.1 billion, depending on the economical approach or the individual productivity loss. the economical approach focuses on the time span until the productivity losses were compensated by a successor and assume lower productivity losses, whilst the overall individual productivity losses are summarized for the entire absence of a missing individual. the overall economic burden of nslbp in 2005 was between 1.6 and 2.3% of the swiss gross national product. work absenteeism (wa), or sickness absence due to nslbp, which quite often results in indirect costs mentioned above, also needs a brief overview. (sickness absence is often used as a synonym to wa. however, the significance of the word might not be totally alike. sickness absence refers to days absence due to a sickness usually nonpermanent while wa emphasizes the permanent absence from work due to a limiting factor, such as nslbp.) approximately 20% of the employees with a current nslbp - episode experience long - term wa. nonspecific lbp is part of the problem causing wa, and it is well known that pain, disability, and wa are linked, but the relationship is complex and influenced by many factors (e.g.,). the influencing factors of wa share similar risk factor patterns to the influencing factors of nslbp. for example, recovery expectations and fear - avoidance beliefs also belong to the psychosocial risk factors of wa [73, 90 ]. a recent study by elfering and colleagues even proposed a relationship between the two factors. in addition, pain intensity, previous wa, and nslbp disability or pain behavior [93, 94 ] were described as biomedical risk factors. occupational risk factors comprised heavy physical workload and low job control [92, 94 ]. high job insecurity, which could have been related to insecure organizational downsizing strategies, also belonged to the occupational risk factors. finally, depression or negative life events further contributed to the development of wa. clearly, wa is the most important impact of nslbp. its social as well as financial consequences explain the political interest in nslbp and its possible resulting incapacity of work. despite the amount of work studying nslbp and its implications, a lot of questions remain unanswered about the mechanisms, confounding and risk factors, treatment measures, and the efficacy or cost effectiveness of those treatment measures for nslbp. with all of the impacts of nslbp presented above in mind, a change of perspective through a new and resource - oriented approach towards nslbp seems reasonable and warranted. more than twenty years ago, only a handful of studies had inquired about individuals who were considered nslbp asymptomatic [96100 ]. however, this specific line of inquiry was not of much interest in the 80s and 90s. only recently has the scientific community seemed to recall the fact that approximately 20% of all individuals never experience nslbp in their lifetime [101103 ]. the emphasis in this review is on a health- and resource - oriented, hence salutogenetic, perspective of nslbp. first, two quite approved salutogenetic approaches to nslbp, the salutogenetic model and resilience, will be presented in order to broadly introduce the theoretical frameworks of resources. second, the state of the art of health - enhancing resources will be introduced. included in the state of the art are personal resources, behaviors, and physical resources, and occupational resources. the following discussion finally discusses all of the presented resources on the basis of the published technical literature. the salutogenic model, based on antonovsky, focuses on resources enhancing recovery or keeping individuals healthy. (soc), which can be understood as a basic sense of trust or a stable feeling of confidence towards life. thus, a high soc proposes that internal as well as external stimuli are structured, predictable, and explainable (comprehensibility) ; that the individual has enough and eligible resources to cope with stressful situations (manageability) ; and that the external demands represent subjective challenges worth fighting for (meaningfulness). antonovsky further rejected the traditional dichotomous health versus illness model and postulated instead that the relationship between health and illness is dynamic and continuous. therefore, he suggested a dynamic health ease / dis - ease continuum [107, page 15 ]. if, for example, a person can not sufficiently cope with an external demand, a state of stress occurs, resulting in a change of the continuum towards the dis - ease end. however, by successfully coping with the stressor or resolving the state of stress, a change towards the ease and an increase in soc arise. previous studies have confirmed the relationship between soc and general health, as well as lbp [109111 ]. for example, soc overlaps with other, well - established concepts like optimism [112, 113 ], self - efficacy, or locus of control, and the question remains whether soc is a unique, clearly definable trait. it refers to one 's capacity to navigate psychological, sociocultural, and physical resources that sustain well - being despite facing adverse and stressful situations and to provide these resources in daily routine. for a broad overview two different aspects of resilience are distinguished : resilience as a personal trait and relational resilience which includes the person - environment constellation. if resilience is considered as a personal trait, the logic consequence investigating on resilience is to identify these resistant and resilient characteristics within individuals on certain outcomes. for example, antonovsky 's soc or kobasa 's hardiness is already established protective factors for negative impacts of stress. the latter, hardiness, describes three core personality characteristics to be in control, to show a high commitment, and to search for challenges. while these characteristics provide the motivation and courage needed to tackle a difficult situation, they also increase personal growth. alternatively, resilience can be described as a specific person - environmental constellation which varies over time. protective factors may include personality characteristics like intelligence, personal aims, or coping strategies, as well as environmental characteristics including social network, educational style, or school support. a crucial aspect is the individual 's ability to develop and to change over their life and hence to successfully adapt to environmental situations. resilience is best understood as a resulting process of individuals interacting with their environments in order to endorse well - being or protect themselves against the influence of risk factors. therefore, a logical consequence is to ask for the processes and protective factors that endorse resilience by promoting well - being and protecting against risk [123, 124 ]. currently, to the knowledge of the authors, no literature exists on resilience and nslbp. however, two recent studies describe resilience as a new paradigm for adaptation to chronic pain [125, 126 ]. though the main focus remains on individuals suffering from chronic pain. nevertheless, processes and protective factors which protect against the onset of nslbp have been examined and will be described in the next paragraph. the following literature on processes and protective factors against nslbp can be considered state of the art since no further literature was found in an extensive search for the period from january 1980 to july 2012 in the following electronic databases : web of science (isi web of knowledge), medline (via pubmed), the cochrane library, pubmed central, ovid, and manually searched in google scholar with links to other articles taken from bibliographies. selection criteria included personal, behavioral, physical, and occupational resources protecting against nslbp, nonpregnant subjects above 18 years of age explicitly not suffering of nslbp, and comparisons between samples with nslbp or chronic nslbp to healthy controls without acute nslbp. the exception with regard to completeness, though, refers to physical resources. the presented physical resources give an overview of the current literature, but there remains no doubt about additional, undetected published literature describing physical differences between individuals with (chronic) nslbp and healthy controls. however, before the resources protecting against nslbp will be introduced, the handful of studies that have been inquiring about individuals who were considered nslbp asymptomatic are briefly recollected and described. enquired about either physical and psychological working conditions [96, 99, 100 ], musculoskeletal status [96, 97 ], or life conditions of individuals with an nslbp - free lifetime prevalence. two, possibly three of these studies are based on the same data [97, 99 ]. nslbp - resilient participants (n = 36) who stated that they had never had nslbp or only occasionally very slight problems and had never been sick - listed with nslbp were recruited from a large manufactory. all statements were checked against the records of the social insurance office. whether the participants of the third study, a previous pilot study, were related to the two studies by hultman and colleagues [97, 99 ] is unclear, but this is a probability since 21 men within a similar age group were recruited from two enterprises. again, statements in this third study were checked against records of the social insurance office. in the fourth study, the swedish central bureau of statistics performed a cross - sectional study of nslbp - resilient individuals (n = 1839) from a random geographically standardized 1 : 1000 sample of the swedish population. they were asked by questionnaire if they had ever experienced any disease or illness related to nslbp and whether they had suffered from nslbp or sciatica. the prevalence of pain - free individuals was very high, with 72% in the youngest age group (3039 years), 62% in the intermediate group (4049 years), and 55% in the oldest group (5059 years). regarding methodological aspects, study three, the pilot study, assessed interview and physical examination data, and study two also assessed physical examination data, while studies one and four compared the questionnaire data of nslbp - resilient persons with nslbp sufferers [96, 99 ]. a total of 216 workers out of the 1773 questioned (12%) reported a prevalence rate decreased over time, and only 6% of workers reporting high stress levels were without back pain. however, the reliability of the postal questionnaire selecting persons with healthy backs remains unclear. more recent studies investigating nslbp - asymptomatic individuals. two longitudinal studies assessed population - based, representative subgroups in sweden and the united kingdom. the swedish study assessed a questionnaire at baseline with two followups of one and five years. this was similar to the british study, which assessed followups at 15 months and four years after the baseline. the swedish study group around reigo and colleagues defined the absence of back problems as not having previous or ongoing nslbp at any of the three evaluations and calculated adjustments based on the nonrespondent analysis at the baseline survey. they further examined two age groups, young adults (2534 years), and older adults (5459 years). overall, 37% of the young and 43% of the older subgroup remained nslbp - free. the british study asked their participants at each time point if they had suffered from any aches or pains which had lasted for one day or longer in the past month. overall, 17.4% of the british study subgroup experienced no nslbp. however, no study checked the pain - free reliability of the answers. a further study from carragee and cohen observed nslbp - asymptomatic soldiers who reported no nslbp at the time of interview or in the previous three years. the authors discovered that 84% of all nslbp - asymptomatic soldiers mentioned at least weak nslbp at least once. the surprizing fact was that five years after the initial data collection, 97% of all nslbp asymptomatic soldiers still considered themselves as nslbp - asymptomatic. thus, carragee and cohen concluded that the subjective statements of nslbp - asymptomatic individuals were not completely reliable and that an nslbp recall bias existed. however, soldiers were assessed in a monthly interval at the end of each weekend drill. when asked why they considered themselves nslbp asymptomatic, the answers indicated that soldiers considered nslbp after military drills not to be a medical problem rather a similar arguments were reported by rolli salath and colleagues. the 21 out of 42 nslbp - resilient participants of the study who reported muscle tension in the back after sporting activities, a bad night, or gardening clearly defined these symptoms clearly as no pain. overall, 42 nslbp - resilient workers between 50 and 65 years of age were pairwise compared to propensity score - matched population - based case controls with and without momentary nslbp. the aim of the study was to explore if the nslbp resilient individuals had a better health, demonstrated more positive health behaviors, and were better able to achieve routine activities than the compared case control groups. interestingly, the nslbp resilient individuals differed from the controls without momentary lbp by being more vital, having a lower workload, a healthier attitude towards health, and by drinking less alcohol. the authors concluded that three underlying traits seemed to be relevant about nslbp - resilient individuals : the personality, favourable work conditions, and subjective attitudes and attributions towards health. the following paragraphs present results of the extensive literature search on personal resources. cognitive appraisals and individual coping strategies are regarded as a distinct subgroup of personal resources, since relationships between personality, cognitive appraisals and coping strategies are well depicted. several personal resources, like the soc, life satisfaction, or extraversion, have been described. for individuals without nslbp, compared to individuals with chronic nslbp, a higher level of soc was mentioned in two studies. in the first study, the difference seemed to be influenced by stress manageability. however, in the second study, the environment of persons with chronic nslbp was perceived to be less comprehensible, manageable, and meaningful. the same study found significantly higher levels of life satisfaction, extraversion, and less emotionality in individuals without nslbp. in other studies, high life satisfaction prevented individuals with acute or subacute nslbp from sickness absence due to nslbp, while good mental health reduced the likelihood of persistent nslbp in individuals with acute or subacute nslbp after twelve - week followup. the ability to seek, understand, and use health information is considered health literacy. a recent study examined broad elements of health literacy among individuals with no or chronic nslbp. out of the eight health literacy domains, only one domain was different between the two groups. patient attitudes towards their health, which included two personal abilities : first, the individual 's ability to attend to the personal health needs ; second, the individual 's willingness to change or adapt their personal lifestyle to maintain their health state. the authors concluded that individuals with chronic nslbp seem to have greater difficulty engaging in general proactive health behaviors. in a different study, a large sample of nslbp - free individuals was prospectively examined over four years. in a univariate analysis, individual characteristics included low anxiety and low health anxiety, as well as low depression and only a few recent adverse life events, indicating that individuals without lbp seem to be emotionally stable and do not often have to cope with stressful life events. two different health behaviors have been found in the extensive literature search regarding personal behaviors. for this reason, the first paragraph will illustrate healthy behaviors, whereas the second paragraph highlights risky health behaviors. with regard to briggs ' conclusion, general proactive health behaviors, such as taking part in sports and being active, seem to influence nslbp in a positive way. by exploring salutogenetic factors of chronic lbp, participating in sports furthermore, the influence of subjective workload on the degree of nslbp was moderated by sports activity. for individuals doing sports more than twice per week, subjective work load no longer enhanced nslbp. similar results confirmed these findings : for example, individuals without nslbp were more often moderately physically active for one to two hours during their leisure time,, as well as more regularly and more enduring physically active than nslbp - sufferers. further, they were better able to do routine activities such as climbing stairs or regular walking. although one study found better health status to be associated with lower medical care, saraste and hultman could not confirm differences in activity and leisure time behaviors. however, a recent study illustrated that individuals without nslbp walked 0.7 hours longer per day, accomplished 3480 steps more per day, and had an altered physical activity pattern than individuals with chronic nslbp. while sleep duration does not play an important role in the differentiation, sleep quality does [101, 138 ]. subjective sleep quality can be divided into self - reported sleep onset latency and self - reported sleep efficacy. on the other hand, objective sleep quality is divided into sleep efficacy and waking after sleep onset, both of which can be measured using actigraphy. furthermore, the study identified significant associations between nslbp, physical health, and disability levels, as well as the subjective, but not objective, sleep quality in the group with chronic nslbp. risky health behaviors. referring to risky health behaviors, like smoking or drinking alcohol, to the knowledge of the authors only two studies found higher alcohol consumption for individuals with nslbp versus healthy individuals. nslbp - resilient individuals drank significantly less alcohol than individuals without momentary nslbp but did not differ from individuals with momentary nslbp. however, the results of the second study are based on a univariate analysis and could not be confirmed by the multivariate analysis. besides, individuals without nslbp appear to be less frequent smokers [98, 135, 136, 141 ] although the gender question is not yet exclusively answered. bjrck - van dijken and colleagues found evidence for more frequent nslbp - free female nonsmokers, while saraste and hultman described this phenomenon for 5059 year - old males only. moreover, smoking seems to affect the extensor muscle strength : non - smokers without nslbp seem to have stronger back muscles than non - smokers with nslbp, while no distinction could be found between the smokers. before turning to the physical resources, a short summary of the personal and behavioral resources will be given. individuals without nslbp appear to have more personal resources, like higher levels of soc and life satisfaction and less emotional instability, and were more able to attend to personal health needs. with regard to health behaviors, individuals without nslbp were more physically active and had a better subjective as well as objective sleep quality, whereas risky health behaviors, like smoking or drinking alcohol, were less common. although physical resources are the most well - investigated resource factors, they have not been included in the extensive literature search. however, it is common to compare two subgroups in medical literature in order to differentiate between individuals with and without nslbp symptoms. therefore, all of the findings described in the following section refer to such investigations. examinations have been performed on six, highly interrelated aspects : (a) higher order kinematics during complex movement tasks, such as displacement, velocity, or acceleration ; (b) proprioception of the spine ; (c) spinal movement patterns ; (d) postural control ; (e) body perception ; and (f) muscle strength. higher order kinematics in complex movements seem to distinguish well between nslbp - sufferers and healthy individuals. the neurophysiological foundation of higher order kinematics, however, is the proprioception of the spine (b). this is described as [] the sense of position and movement of one 's own limbs and body without using vision. there are two submodalities of proprioception : the sense of the stationary position of the limbs (limb - position sense) and the sense of limb movement (kinesthesia) [144, page 443 ]. investigating proprioceptive aspects, studies have asked participants to reproduce predetermined target body positions, such as standing or four - point kneeling [145, 146 ]. if participants did not manage to achieve the position, the repositioning error was calculated as the absolute difference between the target position and the participant - perceived target position. all three studies found significant, but not the same, differences in lumbar proprioception between individuals with and without nslbp. newcomer and colleagues described controversy repositioning errors for flexion and extension movements with a higher repositioning error for flexion and a lower error for extension in individuals with nslbp. in addition, descarreaux and colleagues found modifications in movement time, peak velocity, and acceleration in some, but not all, nslbp - study participants. however, they found a greater motion perception threshold in nslbp patients than in healthy individuals. a similar result was reported regarding the pre- and postlumbosacral position sense after paraspinal muscle vibration. individuals with nslbp had a less refined position sense due to altered paraspinal muscle spindle afferences and central processing of sensory input. spinal movement patterns and postural control. with reference to spinal movement patterns (c) and postural control (d), lumbar and hip movements were investigated before and in response to rapid bilateral arm flexion movements while participants were asked to control their trunk in motion. individuals with nslbp used the preparatory extension of the lumbar spine less frequently and provoked a greater spinal displacement, which was induced by shoulder flexion. a further aspect of postural control is body sway, which is defined as deviation of the body away from the center of the body 's gravitation line. multiple factors are attributed to causing body sway, for example, an inherent noise within the human neuromotor system, (or a) reflexive of an active anticipatory search process, or an output of a control process to maintain postural control [151, page 358 ]. two recent reviews illustrated the association with an increase in anteroposterior body sway in individuals with nslbp exhibiting a greater postural instability than healthy individuals [151, 152 ]. similar results were described in a study exploring the balance performance in unstable sitting : individuals with nslbp showed poorer balance performances and delayed lumbar muscle response times in the highest difficult balance task levels, compared to healthy individuals. in order to find possible mechanisms of postural control strategies, a study investigated the body sway of individuals with and without nslbp by manipulating the acute inspiratory muscles fatigue (imf). after imf, individuals without much alike the individuals with nslbp used a more rigid proprioceptive control strategy instead of the normal multisegmental control, as in a nonfatigue condition. regarding body awareness or body perception (e), some recent studies have been exploring the body image, body schema, or the tactile acuity by testing the two - point discrimination on the back. comparing the accuracy of trunk rotation judgment in individuals with bilateral or monolateral nslbp versus individuals without nslbp, a decrease in the accuracy healthy individuals achieved a 20%, respective to 33%, higher accuracy than individuals suffering from monolateral, respective to bilateral, nslbp. in addition, a decreased tactile acuity was found in the area of usual pain in individuals with chronic nslbp, indicating a distorted body image. tactile acuity is described as a clear signature of primary sensory cortex organization ; therefore decreased tactile acuity might refer to a change in primary sensory cortex organization. a recent review portrayed such functional as well as structural brain changes in chronic nslbp. a change in cortical representation resulted in changed cortical activity and responsiveness, with implications for the response pattern to noxious stimuli, psychological and cognitive effects, and altered body perceptions. as for hultman, who revealed flexible backs, flexible hamstrings muscles, and stronger extensor compared to flexor isometric muscle strength, a prospective study over five years with 67 nslbp - healthy persons described the extensor / flexor muscle strength ratio as the most sensitive parameter for the onset of nslbp. higher extensor than flexor muscle strength appears to be a resource preventing nslbp, but the literature reports inconsistent findings regarding the association between nslbp and trunk muscle function. however, individuals with nslbp were reported to have a pelvic floor muscle dysfunction compared to healthy individuals. in addition, a systematic review on prospective high quality controlled trials examined clinical interventions to prevent self - reported nslbp in working - age adults. the only treatment found to be effective was exercise. the aims of exercise mentioned in the included studies were to increase muscular strength, endurance, flexibility, and postural control. regarding the spinal muscle population, a systemic review on medical imaging studies revealed a paraspinal muscle wasting with reductions in fiber density, fiber atrophy, and fiber conversion from type i (slow - twitch fibers) to type ii (fast - twitch fibers) in individuals with chronic nslbp. meanwhile, in back healthy individuals, paraspinal muscles contained a high proportion of type i fibers, which played a crucial role in maintaining posture. it appears that the results from hultman and colleagues, describing thicker and more enduring back muscles, have influenced the formation of the systematic review by demoulin and colleagues. taken together, individuals without nslbp have many physical resources such as a better proprioception of the spine and overall better postural control. in addition, body perception and muscle strength seem to be different in individuals without nslbp. the absence of a physically heavy workload in relation to individuals without nslbp has been described in various studies [69, 98100, 127, 140, 164 ]. however, when prospectively comparing two different age groups over five years, only the older workers without nslbp (aged 5459 years) experienced a significantly lower physically heavy workload. nevertheless, this finding has been recently confirmed in a younger prospective subgroup as well : high physical workload was identified as the greatest risk factor for the onset of nslbp in 2,235 newly educated female health care workers without prior nslbp history, one and two years after graduation. further physical work resources, like an appropriate work posture or the absence of bent body positions, were quite often investigated by comparing individuals with and without nslbp (e.g., [99, 166 ]). when considering the duration of aversive postures at work, a period of less than two hours a day or the ability to change posture seems to prevent the onset of nslbp. individuals without nslbp perceived a high degree of freedom and higher levels of social support. additionally, social support was found to prevent the development from acute or subacute to chronic nslbp. moreover, two studies presented moderation effects of social support. in the first study, social support moderated the degree of chronic nslbp in that individuals with a very high subjective work load and high social support experienced significantly less chronic nslbp than individuals with low social support. the second study revealed that high social support buffered sickness absence at baseline as well as the impact of sickness absence one year later. thus, individuals with high sickness absence at baseline and high social support were no more absent from work after one year than the group with low baseline absence. however, the impact of high baseline sickness absence on individuals with poor social support resulted in high sickness absence after one year. individuals with a high to very high subjective work load and job satisfaction above the median level experienced less chronic nslbp than individuals with job satisfaction below the median level [96, 134 ], while high levels of job satisfaction buffered sickness absence at baseline as well as the impact of high sickness absence one year later. however, results for job satisfaction were controversial. first, the correlation between higher levels of job satisfaction and the absence of nslbp was only found in older workers when prospectively compared to a younger working group. the absence of psychological distress seems to be a resource for the absence of nslbp as well [101, 141 ]. however, psychological distress can be understood in broader associations like socioeconomic or psychologically demanding situations too. a high income category or good qualifications were resources for protecting against nslbp [140, 168 ]. similar findings were portrayed for the absence of psychologically demanding situations, like the inconsistency between job and educational level or excessive demands in the workplace [99, 164 ]. in line with these thoughts, pain - related fear or fear avoidance can also enhance psychological distress. thus, the absence of fear - avoidance beliefs or pain - related fear had a predictive effect on the absence of nslbp after one year [90, 167 ]. finally, the absence of a stressful job was mentioned as a resource against the onset of nslbp, although it was only found for the younger workers (aged 2534 years) and not for the older subgroup. again in line with a stressful job, the absence of a hectic work tempo or a blue collar job was revealed to be resources against the nonacute nslbp. taken together, personal, physical, and occupational resources that protect against the onset of nslbp or the transition from acute to chronic nslbp exist. back healthy individuals seem to be healthier overall, both physically and psychologically ; they appear to engage more often in proactive health behavior, demonstrate a higher sleep quality, and perform less risky health behaviors like smoking or drinking alcohol. the general positivity is recapitulated within the available physical resources like a good proprioception of the spine, good postural control, a better body perception, and higher muscle strength. also, there is absence of a physically heavy workload and awry body positions at work as well as the surplus of psychological work resources such as social support, high job control, or the absence of psychological distress. acute as well as chronic nslbp is a prominent and highly relevant personal and economic problem of our time. bone and joint decade 20002010 provoking a tremendous amount of research, nslbp remains a book with seven seals. a lot of questions still need to be answered. by taking a different point of view, the salutogenetic aspect of nslbp, the problem is tackled nonpathologically with the aim of looking for positive, health - enhancing perspectives. with regard to a resource - oriented approach to nslbp, some findings need to be further discussed. the comparison of a nslbp - resilient group to a case control group with momentary lbp confirmed previous findings. an univariate analysis identified for the nslbp group a better overall health state, fewer musculoskeletal as well as overall comorbidities [170, 171 ], a higher life satisfaction, a higher sleep quality [101, 138, 139 ], a better appreciation of their own health, and easier routine activities for lbp - resilient individuals [134, 135, 137 ]. however, with regard to vitality, the most prominent health factor differing between nslbp - resilient individuals and both control groups, no previous literature was found in the salutogenetic perspective. nevertheless, two recent studies included low vitality to predict a poor outcome for nslbp patients. the second study created distinct comorbidity clusters which predicted the nslbp or neck / shoulder pain diagnosis probability in an adolescent sample from australia. the clusters defined by the latter study included a healthy individuals ' cluster with a low probability of the diagnosis nslbp or any other medical condition. not only did vitality differ in a highly significant manner from the other three clusters, but beales and colleagues also related the groups ' resilience towards positive beliefs and appreciations about health. further investigations might validate the creation of four distinct clusters and might further examine prospective developments of nslbp resilience. when focusing on the case control group without momentary nslbp, univariate analysis revealed a higher vitality, greater personal importance of exercise, and lower alcohol consumption for the nslbp - resilient group. multivariate analysis further added significantly lower workload. before discussing workload, exercise and its implication towards nslbp although the importance of physical activity with regard to nslbp prevention is often implicitly assumed, scientific evidence is controversial. one systematic review clearly confirms this assumption, while another recent systematic review pointed out that intense physical exertion during leisure time was moderately associated with nslbp and that everyday physical activities prevented the onset of nslbp. the answer to physical activity as a resource for nslbp might be its frequency. with all of the evidence identified, the prognostic relevance for a low workload is beyond doubt [69, 99, 127, 140 ]. however, this significant segregation supplies an even heavier argument : workload even differs between lbp - resilient individuals and those without momentary nslbp of whom we do not know if and how often they have suffered from nslbp before. clearly, employers, and employees as well, carry a tremendous responsibility with regard to prevention measures by providing adequate and safe workplaces with high quality tools. it is, however, the responsibility of every individual to use the tools provided and to implement the health guidelines relating to lifting activities. rather, dissatisfaction with life was found to predict an nslbp incidence within a year. when compared to individuals without chronic disease, individuals with chronic nslbp demonstrated less life satisfaction, yet life satisfaction seemed to be relatively stable for individuals with chronic nslbp despite different treatment measures. even though it slightly improved over the course of four years, the baseline and follow - up scores of life satisfaction were very much alike between individuals treated with a lumbar fusion versus conservative cognitive interventions and exercise. however, life satisfaction is modifiable. working on acceptance strategies with the aim of increasing individuals ' ability to behave according to interfering pain and distress, life satisfaction improved significantly over the course of seven months. this might influence not only the treated persons ' perspective, but the treating persons ' perspective as well. first, the patients ' prospect will be discussed. a physical limitation like nslbp, but even more, chronic nslbp, can change individuals ' life. normal activities are forced into the background by upcoming limitations or disabilities due to nslbp and pain enters to the center of attention. in such a case, an individual might start feeling disabled, worthless, or lost in pain. this development might even be aggravated in individuals with a strong dichotomous attitude ; for example, being healthy and able to work is good, while being ill and disabled is bad. one might have learned to be worthy, recognized, and able to work despite the physical limitations. in short, resources might add a more sophisticated view of a persons ' life and of how to stay active despite pain. mccracken and colleagues, who investigated psychological acceptance of chronic pain, speak about psychological flexibility which may reduce the impact of chronic pain. this psychological flexibility not only is significant for the individual but also has implications for economic and health - political aspects, for example, work organizations as well as invalidity insurances. from the other point of view, the physician 's or clinical specialist 's side, working every day with individuals in pain might not be easy psychologically. treating persons in a health practitioner 's clinic, for example, might generate an individual strategic medical management that relies on tacit knowledge rather than on guidelines [181, 182 ]. this individual strategic medical management might include a feeling about who will pursue an easier healing process and with whom the situation might get difficult. by examining typical clinical situations that focus on preventing either the transition from acute to chronic nslbp or prolonged sickness absence, results might underline, confirm, or even supplement clinical tacit knowledge. since pain, disability, grief, or general (health) problems dominate the daily clinical business, a change from totally pathological towards a resource - added perspective might enhance work quality aspects of physicians or clinical specialists, might further simplify the physician / clinical specialist - patient relationship, and might finally empower patients pain self - efficacy. further research should investigate into resource - added treatments and possible outcomes as well as the implications for patients and treating persons with regard to work quality aspects. an international agreement has been reached for therapeutic aims and treatment measures for chronic nslbp. this includes a discouraged use of passive treatments like modalities, medication, or manipulation and motivates a focus on active measures like supervised exercise therapy, cognitive - behavioral therapy, or multidisciplinary treatments. however, two aspects should be pointed out. first, not all persons with chronic nslbp are thus treated ; second, with only moderate efficacy and not everybody benefiting from standardized treatments, trends tend to move in the direction of using different therapeutic measures for different patient subgroups [187, 188 ]. nevertheless, different therapeutic measures for different patient subgroups do not call into question the acquisition of resources. to retrieve resources as well as to enlarge the perception of pleasant life aspects should belong to an interdisciplinary, standardized, cognitive - behavioral based program (e.g.,). above and beyond, with regard to a salutogenetic approach to nslbp, some questions need to be asked. why should primary care staff like physicians, clinical specialists, care assistants or indeed medical specialists not benefit from experiences gained and learn from the knowledge acquired in interdisciplinary, specialized pain clinics ? should primary care treatment measures only address risk factors of nslbp and relating coping strategies ? why not peek towards a health- and resource - oriented perspective and include health - promoting measures in primary care ? focusing on resources despite nslbp might improve the patient - physician / therapist relationship and might moderate not only the therapeutic outcome (e.g.,) but also the nslbp patients ' therapeutic benefit, which is shown in the patient satisfaction. further research should address the moderating effects of resources, such as sensory perception, moderate activity, or social as well as therapeutic relationships, on function instead of impairment. increased knowledge on resource factors may enhance preventive behavior in personal and occupational settings, minimize work absenteeism, and decrease socioeconomic costs. in a more detailed view, a person being absent from work due to nslbp might decrease productivity or reduce available expertise in a working team. enhanced stress such as time pressures or isolation due to fewer interactions between workers, increased concerns, or difficult supervisor - employee relationships might arise in a team. one side of the answer includes the employee (or patient) view already mentioned above. resources might add a more sophisticated view of a persons ' life and of how to stay active despite pain. this would absolutely correspond to the fifth revision of the swiss disability insurance (fifth iv revision). a main objective of this revision is to integrate individuals at risk of disability and work absenteeism early. with regard to the employers ' side, the same question as mentioned above arises : why do employers or supervisors not benefit from experiences gained and learn from knowledge acquired in interdisciplinary, specialized pain clinics ? one answer might be because employers do not have a therapeutic mission. yet, regarding the fifth revision of the swiss disability insurance, it is not clear at all if employers are not told to take responsibility for their employees ' health and work ability. nevertheless, resources factors like social support at work or job satisfaction might not require vast investigations in new technology or better - quality material. simple things like a better supervisor - employee relationship (e.g.,), appreciation and valorization for the work done, or even a little financial recognition might improve job satisfaction. further research will have to prove these statements. the last point to mention is that scarce literature that deals with nslbp - asymptomatic individuals [101103, 127 ] exists. individuals experiencing no acute nslbp are compared as a control group to individuals with acute, subacute, intermittent, or chronic nslbp. a nslbp - healthy control group is a group of individuals without acute nslbp, yet nothing is known about the prior incidence or recurrence of nslbp. in order to facilitate the distinction between nslbp - asymptomatic individuals and individuals without current nslbp, the scientific community ought to realize first that differences exist. this requires more investigations with the intention to gain new insight about psychological, behavioral, physical, occupational, and even neurological characteristics of nslbp - resilient individuals in specific person - environment constellations. findings in such inquires might be included not only in medical and therapeutical, but also in pedagogial, as well as occupational settings in order to enhance nslbp resilience. however, before such specific person - environment constellations can be proven efficiently, there is still plenty of work ahead. also, one might not forget that the study sample will not be easy to collect since nslbp - resilient individuals are rather difficult to locate, especially in the age group of above 50 years. nonspecific low back pain is a dominant problem of our time with severe personal as well as occupational restrictions. the existence of health - promoting resources should be introduced gradually to the attention of the scientific community as well as the clinical staff at the patient front line and supervisors in their daily work although perspectives are often back breaking. | nonspecific low back pain (nslbp) is an important health issue of our time. personal as well as economic factors, like suffering pain and experiencing disability on the one hand and enormous and still increasing costs to the economy and society on the other hand, display the importance of the matter. tremendous research has been conducted in the last few decades on nslbp. a pubmed search (june 17, 2013) on low back pain provided 22,980 hits, and when specifying for low back pain, systematic review, 3,134 hits were still generated. most research has been done examining the development, risk factors, or therapeutic measures of nslbp, but hardly any literature exists on resources related to nslbp. the aims of this review are twofold. in order to shade light on the salutogenetic approach of nslbp, and thus to focus on health instead of illness, the first aim is to facilitate the understanding of which therapeutic measures enhance the ability to cope with chronic nslbp and enable (more) normal functioning in life. the second aim is to stimulate the understanding of resources protecting against the onset of nslbp or against the development of chronic nslbp and its resulting work absence. |
toxoplasmosis is a zoonosis caused by toxoplasma gondii which may be transmitted by blood transfusion (1). the acute invasion, characterized by parasitemia, is a transient stage followed by chronic invasion when parasites reside within different tissue in cysts (1 - 4). most of this infection is chronic without clinical symptoms in immunocompetent humans, although it may cause severe or fatal infection in immunodeficient patients (2). since t. gondii organism may be alive in the citrated blood at 5 c for up to 50 days and the buffy coat (5), so it appears likely that toxoplasmosis could be acquired via blood or leukocytes transfusions especially if parasitized leukocytes are transfused in a high concentration (5). multiple units of blood from different donors are regularly used in children with thalassemia, sickle cell anemia and aplastic anemia who need regular, frequent and multiple transfusions for survival. many studies showed high prevalence of t. gondii antibodies in healthy voluntary blood donors (6 - 13) whiles screening for t. gondii before transfusion blood has nott been considered yet. the aim of this study was to determine the prevalence of t. gondii infection in blood donors of shiraz city, iran and identify characteristics of blood donors associated with seropositivity. we performed a cross sectional study in the blood transfusion institute of shiraz city, iran in 2013. samples was collected from voluntary blood donors and were routinely tested for human immunodeficiency virus (hiv), hepatitis b virus, hepatitis c virus, anti htlv1, 2 and treponema pallidum (syphilis). samples included packed cells (pc) package (n = 138) and fresh frozen plasma (ffp) (n = 112). pc packages were centrifuged (2000 g, 10 min) and the plasma was separated and stored at 70 c. plasma samples were analyzed for anti- t. gondii igg and igm antibodies by elisa technology using commercially available kits (dsi, germany). anti- t. gondii igg antibodies levels of > 14.5 ul / ml were considered to be positive. anti- t. gondii igm antibodies levels were assessed in samples that igg anti - t. anti- t. gondii igm antibodies levels of > 1.1 ul / ml were considered to be positive. gondii antibodies were considered as a chronic toxoplasmosis, whiles those with both positive igg and igm anti - t. statistical analysis of and exact fisher s tests were performed to compare the prevalence of chronic toxoplasmosis for term of age, sex, educational level, residence place, marital status, occupation, blood types and kind of blood products. we performed a cross sectional study in the blood transfusion institute of shiraz city, iran in 2013. samples was collected from voluntary blood donors and were routinely tested for human immunodeficiency virus (hiv), hepatitis b virus, hepatitis c virus, anti htlv1, 2 and treponema pallidum (syphilis). samples included packed cells (pc) package (n = 138) and fresh frozen plasma (ffp) (n = 112). pc packages were centrifuged (2000 g, 10 min) and the plasma was separated and stored at 70 c. plasma samples were analyzed for anti- t. gondii igg and igm antibodies by elisa technology using commercially available kits (dsi, germany). anti- t. gondii igg antibodies levels of > 14.5 ul / ml were considered to be positive. anti- t. gondii igm antibodies levels were assessed in samples that igg anti - t. anti- t. gondii igm antibodies levels of > 1.1 ul / ml were considered to be positive. gondii antibodies were considered as a chronic toxoplasmosis, whiles those with both positive igg and igm anti - t. statistical analysis of and exact fisher s tests were performed to compare the prevalence of chronic toxoplasmosis for term of age, sex, educational level, residence place, marital status, occupation, blood types and kind of blood products. results were considered statistically significant if p < 0.05. analyses were performed using spss software version 16 (spss inc., the prevalence of chronic and acute toxoplasmosis was 23.2% and 0.4%, respectively. among these positive igg anti gondii antibody samples, just one sample of ffp was positive for igm anti - t. gondii antibody (table 1). in term of occupation, the prevalence of chronic toxoplasmosis was significantly high in workers, farmers, house wives, unemployed and free jobs (68.4%) than in mental activity job and employees (25.9%) (p=0.007). along with the abo blood type system the rate of chronic toxoplasmosis were 16%, 17%, 30% and 47% in o, a, ab and b types respectively. it was significantly higher in ab and b than the o and a types (p=0.000). there was no significant difference in prevalence of chronic toxoplasmosis between ffp (27%) and pc (24%) samples (p=0.0603). there were also no significant difference in prevalence of chronic toxoplasmosis regarding age, sex, marital and residency. in the present study, we have shown that of 250 samples 59 (23.2%) and one had chronic and acute toxoplasmosis respectively. in comparison to previous studies conducted in iran, it was similar to the result of sharif study (22%) (14). however the result is not similar to other studies in different parts of iran. it is lower than salahi- moghaddam (68.4%), assmar (51.8%) and gorbani (55.7%) reports (15 - 17) and higher than gorbani 17.7% study (18). the result is also similar to other studies in other countries (6 - 9, 19). prevalence of chronic toxoplasmosis was higher in the ffp as compared to the pc packages and these may depend on the way of ffp sample maintenance which transferred to freezer immediately and causes to preserve the antibody level better. high rate of prevalence shows that the risk to get infection by recipients might be high. the prevalence of acute toxoplasmosis was 0.4% in this study and it may indicate the possible recent infection and may remain undiagnosed (19) which is evidence for active t. gondii in blood. as t. gondii might be transmitted by blood supply, it may alarms for patients with different immunodeficient who are at highest risk of exposure to transfusion transmitted diseases. as nimir and coworker in malaysia mentioned there is an association between seropositivity in positive history of blood transfusion in patients with different malignancy and in this group seroprevalence was higher than who had negative history and this study suggests a larger sampling recommended to be able to determine these association before any conclusion (19) and in different studies suggested more attention before blood transfusion and mentioned the possible reactivation of this opportunistic parasite (6, 20 - 22). there was no significant difference in prevalence rate of anti- t. gondii antibodies regarding to age group, marital status, sex, residency and kind of blood product which is similar to the results of different studies (14,15). according to occupation, individuals included mental activity job and employee had the significant lower rate of positivity than other individuals included workers, farmers, housewives, unemployed and free jobs. it seems that the first group is less contact with risk factors which is similar to the results of previous studies (15, 23). according to educational level, people with academic education had significantly lower rate of positivity than who has low education level. it is similar to the result of the hashemi and saraei study (23). as regards to blood types, interestingly, individuals with ab and b types had significantly high rate of positivity than the other types. it suggests do to more survey about the status of sensitivity in different blood types against t. gondii to the best of our knowledge, this is the first. | backgroundthe prevalence of toxoplasma gondii infection in the blood donors has been poorly studied. the aim of this study was to assess the prevalence of acute and chronic toxoplasmosis in blood products.methodsa total of 250 blood products (112 fresh frozen plasma and 138 packed cells) in the blood transfusion institute, shiraz, iran were tested for specific t. gondii antibodies (igg and igm) by elisa method in 2013. positive igg anti - t. gondii samples were further tested for igm anti - t. gondii antibody. a positive igg test with the negative and positive igm test was interpreted as a chronic and acute toxoplasmosis, respectively. the relationship of jobs, blood types, sex, marital status and residency of participants with chronic toxoplasmosis prevalence were statistically analyzed by 2.resultsof 250 samples, 58 (23.2%) and one were positive for igg anti - t. t. gondii (chronic) and igm anti - t. t. gondii (acute) antibodies levels, respectively. twenty nine (25.9%) of fresh frozen plasma (ffp) samples were positive for igg anti - t. gondiiiand 1(0.89%) of them was positive for igm anti - t. gondiii antibody. thirty (21.74%) of packed cell samples were positive for igg anti - t. gondii antibody. the prevalence of chronic toxoplasmosis was significantly higher in workers, farmers, house wives, unemployed and free jobs (p=0.007), people with low education levels (p=0.035) and b type of blood abo system (p=0.0001). however, there were no significant differences regarding to age, sex, marital status, residency and type of blood products.conclusionsthere were chronic and acute toxoplasmosis in blood products and the prevalence of toxoplasmosis especially chronic form was high. therefore screening of blood for t. gondii antibodies may be considered. |
eight- to ten - week - old icr female mice (japan slc, hamamatsu, japan) were superovulated by injection of 5 iu of equine chorionic gonadotropin (ecg ; asuka pharmaceutical, tokyo, japan), followed by 5 iu of human chorionic gonadotropin (hcg ; asuka pharmaceutical) 48 h later. unfertilized eggs were harvested 14 h after hcg injection and placed in a 90-l droplet of htf supplemented with 4 mg / ml bsa (a3311 ; sigma - aldrich, st. spermatozoa were collected from the cauda epididymis of 11- to 15-week - old icr male mice (japan slc) and cultured for 2 h in 100-l of htf supplemented with 4 mg / ml bsa. after preincubation, sperm were introduced into fertilization droplets at a final concentration of 1 10 cells / ml. after a 3-h incubation, fertilized 1-cell embryos were collected and washed 3 times in ksom supplemented with amino acids and 4 mg / ml bsa and then used for microinjection. for construction of a hypbase expression vector, the hypbase orf was amplified from pcmv - hypbase by pcr using specific primers (5-gggaccggttaatacgactcactatagggaattcgccgccaccatgggc-3, 5-gggggtaccgaaacagctctggcacatgt-3), and the sv40 polyadenylation signal was added to the amplicon. the resultant dna fragment was used as a template for in vitro transcription. rna synthesis and poly(a) tailing were performed with a megascript t7 kit (invitrogen, carlsbad, ca, usa). approximately 510 pl of 0, 10, 30, 50 and 100 ng/l hypbase mrna and 30 ng/l ppb - cag - tagrfp in depc water (invitrogen) were microinjected into the cytoplasm of 1-cell embryos between 3 and 4 h after insemination. after injection, the embryos were cultured in ksom medium supplemented with amino acids and 4 mg / ml bsa under mineral oil (sigma - aldrich) at 37 c in an atmosphere containing 5% co2. to examine tagrfp fluorescence, embryos were observed at 38 and 108 h after insemination. at 108 h after insemination, embryos were collected and fixed in phosphate - buffered saline (pbs) containing 4% paraformaldehyde (sigma - aldrich) for 20 min at room temperature. after washing three times in pbs, nuclei were stained in pbs containing 10 g / ml hoechst 33342 (sigma - aldrich) for 10 min. stained embryos were mounted on slides in 50% glycerol / pbs, and fluorescent signals were detected using a fluorescence microscope (bx50, olympus, tokyo, japan). statistical analysis of the data was performed by analysis of variance (anova) with the student s t - test. all animal experiments were approved by the animal research committee of kyoto university (permit number : 2417) and performed in accordance with the guidelines of the committee. eight- to ten - week - old icr female mice (japan slc, hamamatsu, japan) were superovulated by injection of 5 iu of equine chorionic gonadotropin (ecg ; asuka pharmaceutical, tokyo, japan), followed by 5 iu of human chorionic gonadotropin (hcg ; asuka pharmaceutical) 48 h later. unfertilized eggs were harvested 14 h after hcg injection and placed in a 90-l droplet of htf supplemented with 4 mg / ml bsa (a3311 ; sigma - aldrich, st. spermatozoa were collected from the cauda epididymis of 11- to 15-week - old icr male mice (japan slc) and cultured for 2 h in 100-l of htf supplemented with 4 mg / ml bsa. after preincubation, sperm were introduced into fertilization droplets at a final concentration of 1 10 cells / ml. after a 3-h incubation, fertilized 1-cell embryos were collected and washed 3 times in ksom supplemented with amino acids and 4 mg / ml bsa and then used for microinjection. for construction of a hypbase expression vector, the hypbase orf was amplified from pcmv - hypbase by pcr using specific primers (5-gggaccggttaatacgactcactatagggaattcgccgccaccatgggc-3, 5-gggggtaccgaaacagctctggcacatgt-3), and the sv40 polyadenylation signal was added to the amplicon. the resultant dna fragment was used as a template for in vitro transcription. rna synthesis and poly(a) tailing were performed with a megascript t7 kit (invitrogen, carlsbad, ca, usa). approximately 510 pl of 0, 10, 30, 50 and 100 ng/l hypbase mrna and 30 ng/l ppb - cag - tagrfp in depc water (invitrogen) were microinjected into the cytoplasm of 1-cell embryos between 3 and 4 h after insemination. after injection, the embryos were cultured in ksom medium supplemented with amino acids and 4 mg / ml bsa under mineral oil (sigma - aldrich) at 37 c in an atmosphere containing 5% co2. to examine tagrfp fluorescence, embryos were observed at 38 and 108 h after insemination. at 108 h after insemination, embryos were collected and fixed in phosphate - buffered saline (pbs) containing 4% paraformaldehyde (sigma - aldrich) for 20 min at room temperature. after washing three times in pbs, nuclei were stained in pbs containing 10 g / ml hoechst 33342 (sigma - aldrich) for 10 min. stained embryos were mounted on slides in 50% glycerol / pbs, and fluorescent signals were detected using a fluorescence microscope (bx50, olympus, tokyo, japan). each experiment was repeated at least three times. statistical analysis of the data was performed by analysis of variance (anova) with the student s t - test. all animal experiments were approved by the animal research committee of kyoto university (permit number : 2417) and performed in accordance with the guidelines of the committee. | transgenic mice are important tools for genetic analysis. a current prominent method for producing transgenic mice involves pronuclear microinjection into 1-cell embryos. however, the total transgenic efficiency obtained using this method is less than 10%. here, we demonstrate that highly efficient transgenesis in mice can be achieved by cytoplasmic microinjection using a hyperactive piggybac system. in embryos in which hypbase mrna and ppb - cag - tagrfp dna were co - injected into the cytoplasm, tagrfp fluorescence was observed after the 2-cell stage ; when 30 ng/l ppb - cag - tagrfp dna and 30 ng/l hypbase mrna were co - injected, 94.4% of blastocysts were tagrfp positive. furthermore, a high concentration of hypbase mrna resulted in creation of mosaic embryos in which the tagrfp signals partially disappeared. however, suitable concentrations of injected dna and hypbase mrna produced embryos in which almost all blastomeres were tagrfp positive. thus, the hyperactive piggybac transposon system is an easy - to - implement and highly effective method that can contribute to production of transgenic mice. |
researchers in many biological fields are often confronted with classification problems concerning biological sequences. for example, analyzing promoter sequences often requires the classification in transcription factor binding sites and background sequence parts (1,2). for a given set of exons or splice sites one might be interested in predicting which of these are alternatively spliced (3,4). state - of - the - art machine - learning approaches extract various features from these sequences and perform classification on the feature vectors instead of the original sequences. bayesian networks (bn) have recently attracted considerable attention for data modeling and classification (5,6) since they can cope with features of various value ranges and can learn dependencies between features. bns have been successfully used for modeling of gene expression to derive genetic regulatory networks (79), for discovering pathogenic snps (10), for identifying missing enzymes in metabolic pathways (11), for protein folding (12), genetics and phylogeny analysis (5), as well as for predicting the effect of missense mutations (13). another large and rather new application area of bns are biological sequence data (2,1417). compared to profile hidden markov models (hmms) (18), which are often used to model conserved sequence families such as protein domains as in the pfam database (19), they allow for more modeling flexibility w.r.t. first, they allow a more flexible scheme of dependencies between variables. in profile hmms, in contrast, multiple dependencies are allowed in bns, and there is no fixed ordering of the variables. this has been shown to be especially important to model regulatory like tf binding sites (14). second, bayesian network allow to integrate arbitrary features, which is not possible for hmms. this has been shown to be important to integrate structural properties in the recognition of regulatory sequence (2,20). and third, the network structure (i.e. the set of all dependencies to be considered) must be given as an input to profile hmms, whereas they are automatically learned in the bn approach. to the best of our knowledge, there is no web - based application of bn modeling that is tailored to the analysis of biological sequences. to facilitate the use of bns in this context this web application allows to perform a wide spectrum of analysis from automatic feature generation and selection, to bn learning and application of the learned model to new input data and for probabilistic inference. furthermore, biobayesnet accepts any user - defined features as input, which extends its application range to various scientific areas. in this section, we briefly describe the methods applied by biobayesnet in the order in which they occur in the processing chain. a feature is a measurable property of a single input data sample (e.g. an input sequence). each feature has its own set of possible values which we denote as the feature range. for each feature, there is a well - defined feature value for each single input data sample. given that a class label is assigned to each input sample, biobayesnet tries to detect exactly that feature subset which is optimal in predicting the class label of so far unseen samples. the typical usage of biobayesnet assumes that the user defines a large bunch of features which might be useful for characterizing sequences of the different classes. for each sequence the value of every feature is calculated leading to a feature vector for each sequence. all further processing of the user input only requires the feature vectors, not the sequences. the next step is the search of a subset among all defined features which is optimal with respect to its ability to discriminate between feature vectors of different classes. for this purpose we apply the sequential feature subset selection algorithm (sffs) (21) which searches the space of feature subsets with respect to a special quality measure. starting from an initially empty subset, this algorithm successively adds that feature which best improves the quality measure. after each insertion step the algorithm deletes previously added features as long as this does not worsen the quality measure. these deletion steps are necessary for avoiding the search path being trapped in local optima since the whole set of defined features can contain redundant features. for instance, a single feature which has been added in the last step could perform better together with another selected feature and make a formerly selected third feature dispensable. the algorithm stops if neither insertion of another feature nor the deletion of features can improve the quality measure. in order to calculate the quality of a particular feature subset we perform a 10-fold cross validation. successively, 90% of the feature vectors are used to learn a bayesian network classifier. for the remaining 10% of the samples this value expresses the strength of evidence given by a feature vector for predicting its own class. finally, we obtain the quality measure value by summing up the information loss for the 10 runs of the cross validation. the core of biobayesnet is the probabilistic modeling of the resulting feature subset in bayesian classifiers (bcs) (22) which is a special class of bns. in general, a bn is a graphical representation of the joint probability distribution over a set of random variables. each feature f is represented by a discrete random variable which defines a probability distribution over the feature range of f. formally, a bn is a pair b = (g, p). its first component g is an annotated directed acyclic graph whose vertices correspond to random variables f1, f2, it contains probability parameters pfi | fi = pb (fi = fi | fi = fi) for each possible value fi of random variable fi and each configuration fi of the set of parent variables fi. thus, a bn b defines a unique joint probability distribution over all concerned random variables f = { f1, f2,, fd } given by beside random variables for the features, a bc also contains an additional variable, the class variable c, which is parent of every feature variable. obviously, the range of this class variable is the set of the different class labels c1,, ck. for a given feature vector f = f1,, fd (i.e. observations of values for all considered features), a bc classifies with respect to the conditional probabilities of having a sample of class ck. thus, class c is predicted so that we further restrict the structure (i.e. the edges) of the bn in allowing at the most one parent feature variable for each feature. these specially structured networks are called tree - augmented networks (tan) (21). the restriction is done due to the higher robustness of the learning procedure when confronted with small data sets and the existence of efficient structure learning algorithms for this subclass of bns. learning a bayesian classifier from a set of feature vectors comprises two steps : (i) the structure learning and (ii) the probability parameter estimation. for structure learning, we apply the algorithm chow and liu (23) which reduces that problem to the finding of a minimal spanning tree using the conditional mutual information content (mic) between the distributions of two features as edge weights. to avoid the insertion of edges between features which only show weak correlation we slightly have modified this procedure by setting up a mic threshold and only including edges with weights above this threshold. once the structure of the network is determined, the (conditional) probability distributions over the feature values of each feature given the class label and optionally the value of the parent feature are estimated straightforward from count statistics derived from learning data. since the usage of bns requires that there do not occur zero probabilities, we use dirichlet priors for smoothing the probability distributions. the conditional probability in the previously illustrated bc - decision rule is an instance of what is called bayesian inference, the querying of probabilities for some variable value in presence of observed values for other (not necessarily all) variables. it is one advantage of bns that such queries (marginalizations) can be approximately calculated by efficient algorithms. in biobayesnet optionally, one may specify a subsequence (for example, a protein binding site within an entire promoter sequence) which allows to use relative positions in the next step. to generate the features from these sequences, the user is redirected to step 1.2, where the server allows the selection of a wide range of features. there are five main groups of features : nucleotides at particular positions : features of this group all have the same range, namely the four different nucleotides. a nucleotide feature for position i is the analogue of the ith column of a position weight matrix (pwm).dna structural parameters which express the sequence - dependent local variation of geometrical or physiochemical dna properties at a subsequence. examples are the average helical twist between two base pairs, the dna bendability or the average melting temperature of the subsequence. a feature value for a subsequence is calculated as the mean of all dinucleotide steps in this subsequence. we provide 38 different dna properties that can be calculated from a user - defined subsequence.rna single - strandedness measures the probability for a given rna subsequence to be completely single - stranded (i.e. not part of a secondary structure). for that we use rnaup from the vienna rna package (27).subsequence nucleotide contents : these features measure the fraction a subset of nucleotides in a user - defined subsequence. an example is the fraction of pyrimidines in the subsequence from position 10 to 20.consensus matches : features of this group decide whether there is a match of a given subsequence to a given consensus sequence. nucleotides at particular positions : features of this group all have the same range, namely the four different nucleotides. a nucleotide feature for position i is the analogue of the ith column of a position weight matrix (pwm). dna structural parameters which express the sequence - dependent local variation of geometrical or physiochemical dna properties at a subsequence. examples are the average helical twist between two base pairs, the dna bendability or the average melting temperature of the subsequence. a feature value for a subsequence is calculated as the mean of all dinucleotide steps in this subsequence. we provide 38 different dna properties that can be calculated from a user - defined subsequence. rna single - strandedness measures the probability for a given rna subsequence to be completely single - stranded (i.e. not part of a secondary structure). subsequence nucleotide contents : these features measure the fraction a subset of nucleotides in a user - defined subsequence. an example is the fraction of pyrimidines in the subsequence from position 10 to 20. consensus matches : features of this group decide whether there is a match of a given subsequence to a given consensus sequence. features of all groups can be restricted to particular subsequences or positions in the sequences. if a subsequence is specified the positions refer to a location relative to the subsequence. for example, position -5 refers to the 5 nucleotides upstream of the start of the specified subsequence. features of groups 2, 3 and 4 describe continuous properties of sequences. in order to derive a finite feature range, the continuous ranges are discretized using the entropy - based, supervized discretization algorithm by fayyad and irani (28). this procedure finds a partition of the continuous range which best separates the different classes. the second possibility is to input user - given feature vectors for each data sample in c4.5 format (29). this allows full flexibility as the user can input any prior computed feature. for example, one might input pre - computed features about protein sequences and/or structures to analyze protein data. the first file contains the class labels and feature names with possible feature values, whereas the second file contains the data samples (table 1). table 1.an example of user - given feature vectors describing potentially discriminative features of alternatively and constitutively spliced exonsfile 1 : class labels and feature namesfile 2 : data samplesalternative, constitutive.3.4, 100, high, yes, alternative.donor_splice_site_score : continuous.5.7, 67, medium, no, alternative.exon_length : continuous.7.4, 167, high, yes, alternative.flanking_intron_conservation : high, medium, low.13, 231, low, no, constitutive.length_divisible_by_3 : yes, no.9.5, 189, medium, yes, constitutive.7.8, 345, low, no, constitutive.the first line of the first file has to contain the class labels (alternative and constitutive). the features are given in the order of the first file and the class label is given at the end of each line. an example of user - given feature vectors describing potentially discriminative features of alternatively and constitutively spliced exons the first line of the first file has to contain the class labels (alternative and constitutive). the features are given in the order of the first file and the class label is given at the end of each line. after the data input, the user can select which features are used to learn the bn (step 2). apart from manual selection, this process is assisted by an automatic feature selection method (see methods section), which selects the most discriminative features from all generated or user - given features. this step also provides an overview of the value ranges and the empirical probability distribution. the selected features are used in the next step (step 3) to learn a bayesian classifier with tan structure. after learning, this includes two quality measures (information loss function and the average posterior probability for the correct class) and the final classification of the input data. furthermore, the power of the individual features is estimated by computing the loss of quality if this feature is omitted during learning. the server also produces a graphical representation of the network structure, which allows the exploration of learned dependencies between the features (figure 2). figure 2.graphical overview of a bn and the dependencies between feature variables. besides this graphical overview, an interesting information is the distribution of a single feature, given particular values for some of the other features (variables). to this end, our tool allows to set some variables to particular values and query the a posteriori probability distribution of another variable given this setting. furthermore, one can view the feature values for each data sample and how these samples were classified by the bn. the final bn can be downloaded as a file in the bayesian interchange format (bif) to use it for further data classification or to use it in bayesian network software such as javabayes (30). in the next optional step (step 4), if a bn has been learned in advance, the server also allows classification after the upload of the bn in bif format. in each case, the user has to upload new input data (either fasta sequences or feature vectors). after the data input, the user can select which features are used to learn the bn (step 2). apart from manual selection, this process is assisted by an automatic feature selection method (see methods section), which selects the most discriminative features from all generated or user - given features. this step also provides an overview of the value ranges and the empirical probability distribution. the selected features are used in the next step (step 3) to learn a bayesian classifier with tan structure. this includes two quality measures (information loss function and the average posterior probability for the correct class) and the final classification of the input data. furthermore, the power of the individual features is estimated by computing the loss of quality if this feature is omitted during learning. the server also produces a graphical representation of the network structure, which allows the exploration of learned dependencies between the features (figure 2). figure 2.graphical overview of a bn and the dependencies between feature variables. graphical overview of a bn and the dependencies between feature variables. besides this graphical overview, an interesting information is the distribution of a single feature, given particular values for some of the other features (variables). to this end, our tool allows to set some variables to particular values and query the a posteriori probability distribution of another variable given this setting. furthermore, one can view the feature values for each data sample and how these samples were classified by the bn. the final bn can be downloaded as a file in the bayesian interchange format (bif) to use it for further data classification or to use it in bayesian network software such as javabayes (30). in the next optional step (step 4), the user can classify new input data using the learned bn. if a bn has been learned in advance, the server also allows classification after the upload of the bn in bif format. in each case, the user has to upload new input data (either fasta sequences or feature vectors). the user interacts with this application via html pages which are dynamically generated using java server pages (jsp). input given by the user is directed to java servlets which validate the input and generate objects which are conducted to the algorithmic layer of the application. the servlets further take the result objects of the algorithmic layer and redirect it to java server pages which again produce html output for the user. as a java - based web application, the implementation of the bns and related algorithms partly rely on third - party apis, namely javabayes (30) and jbnc (32). we have developed the web server biobayesnet that enables an easy use of bayesian network models for the analysis of biological sequence data. we are working on extending the set of automatically generated features, especially to include protein - related features and a greater variety of rna structural features. | biobayesnet is a new web application that allows the easy modeling and classification of biological data using bayesian networks. to learn bayesian networks the user can either upload a set of annotated fasta sequences or a set of pre - computed feature vectors. in case of fasta sequences, the server is able to generate a wide range of sequence and structural features from the sequences. these features are used to learn bayesian networks. an automatic feature selection procedure assists in selecting discriminative features, providing an (locally) optimal set of features. the output includes several quality measures of the overall network and individual features as well as a graphical representation of the network structure, which allows to explore dependencies between features. finally, the learned bayesian network or another uploaded network can be used to classify new data. biobayesnet facilitates the use of bayesian networks in biological sequences analysis and is flexible to support modeling and classification applications in various scientific fields. the biobayesnet server is available at http://biwww3.informatik.uni - freiburg.de:8080/biobayesnet/. |
it is well established that individuals with serious mental illness (smi), which include diagnoses like schizophrenia and bipolar disorder, have a much higher rate of morbidity and mortality in comparison to the general population. specifically, diabetes impacts approximately 25% of individuals with smi, 2 to 3 times the rate than the general population. while this increased rate in diabetes is a multifaceted issue, factors such as lower utilization of preventive care and use of newer antipsychotic medications are likely to be associated with higher prevalence of diabetes in this population. with a growing emphasis on preventive and population - based care set forth by the affordable care act (aca), new avenues of health care delivery are being promoted. through the aca, under section 2703, these health home programs were designed to provide services to reduce barriers to care for those with chronic conditions. given the chronic impairments and difficulties faced by those with smi, it has been hypothesized that the use of the health home will improve early recognition of developing problems and provide access to much needed preventative somatic care. furthermore, given the psychiatric nature of the impairments faced by those with smi, it was hypothesized that placing the health home within the mental health clinic these individuals receive the majority of their care would be the ideal setting to maximize exposure to health home services. this case study presents some preliminary results on a new behavioral health home (bhh), which focused on the use of population - based care to identify abnormal hemoglobin a1c (hba1c) levels and clarify the severity of such findings thus permitting enhanced, individualized interventions for individuals with smi. it was hypothesized that through the use of bhh services, patients would receive better quality of somatic care and that gaps in care would be readily identified. this bhh was embedded in the division of community psychiatry at the university of maryland school of medicine. the division consists of 2 large outpatient mental health clinics, adult and child assertive community treatment (act) team, and an adult psychiatric rehabilitation program (prp). in total, these settings serve 2000 individuals annually. although efforts to enhance somatic care for patients with smi had been a top priority of the division since 2005, it was in 2013 that the state of maryland approved funding that allowed for the creation of the bhh. behavioral health home referrals were made to patients who were considered to require the most assistance. this was determined by selecting persons with smi who were enrolled in the more intensive act or prp services of the division. individuals in these intensive services were referred to the bhh staff by their mental health provider, and for those who consented, an intake was conducted by bhh initiate services. once enrolled, the bhh provided a minimum of 2 bhh services per month, coordinated by a nurse case manager. a wide variety of services qualified for bhh services, including care coordination, comprehensive transitional care, and health promotion. one service per month was also allowed to be delivered in a group format. given this allowance, a population - based health management program was created to target highly prevalent somatic conditions for individuals with smi (eg, diabetes and hypertension). while initially designed to alternate the somatic condition being tracked every month (eg, may diabetes, june hypertension), it was quickly realized that the high prevalence of these conditions required more monitoring time for a more comprehensive assessment. it was determined that monitoring a specific health condition for several consecutive months provided a better understanding of the population s care needs and impact of interventions that included additional education about the illness, nutritional guidelines, and value of regular exercise. patients who did not have their hba1c level drawn within 6 months of the monthly chart review were identified to be not in compliance with clinic recommendations for individuals prescribed atypical antipsychotics. changes in, as well as severity of, hba1c levels (eg, normal, prediabetes, or type 2 diabetes) were assessed during monthly chart reviews. this bhh was embedded in the division of community psychiatry at the university of maryland school of medicine. the division consists of 2 large outpatient mental health clinics, adult and child assertive community treatment (act) team, and an adult psychiatric rehabilitation program (prp). in total, these settings serve 2000 individuals annually. although efforts to enhance somatic care for patients with smi had been a top priority of the division since 2005, it was in 2013 that the state of maryland approved funding that allowed for the creation of the bhh. behavioral health home referrals were made to patients who were considered to require the most assistance. this was determined by selecting persons with smi who were enrolled in the more intensive act or prp services of the division. individuals in these intensive services were referred to the bhh staff by their mental health provider, and for those who consented, an intake was conducted by bhh initiate services. once enrolled, the bhh provided a minimum of 2 bhh services per month, coordinated by a nurse case manager. a wide variety of services qualified for bhh services, including care coordination, comprehensive transitional care, and health promotion. one service per month was also allowed to be delivered in a group format. given this allowance, a population - based health management program was created to target highly prevalent somatic conditions for individuals with smi (eg, diabetes and hypertension). while initially designed to alternate the somatic condition being tracked every month (eg, may diabetes, june hypertension), it was quickly realized that the high prevalence of these conditions required more monitoring time for a more comprehensive assessment. it was determined that monitoring a specific health condition for several consecutive months provided a better understanding of the population s care needs and impact of interventions that included additional education about the illness, nutritional guidelines, and value of regular exercise. patients who did not have their hba1c level drawn within 6 months of the monthly chart review were identified to be not in compliance with clinic recommendations for individuals prescribed atypical antipsychotics. changes in, as well as severity of, hba1c levels (eg, normal, prediabetes, or type 2 diabetes) were assessed during monthly chart reviews. a total of 120 (73 males and 47 females) patients with smi were enrolled and receiving population - based health management services within 10 months of starting the bhh. the average age of the population was 50 years (standard deviation = 12.75 years). of the 120 patients, 54 (45%) were identified who did not have hba1c levels drawn per the clinic s recommended guideline of having hba1c drawn every 6 months for those prescribed antipsychotic medications. thirty - three patients had a diagnosis of type 2 diabetes, 4 of which were newly diagnosed as a result of the population - based health management (phm) initiative. another 39 patients were identified as having prediabetes (hba1c levels between 5.7 and 6.4 ; table 1). abbreviations : bhh, behavioral health home ; hba1c, hemoglobin a1c. of the 33 patients identified as having type 2 diabetes, of the 16 patients, 4 within their target goal achieved this range after initiating in bhh services. two patients who were originally in the prediabetes range when starting bhh services had higher hba1c levels and were in the type 2 diabetes after 10 months. another 7 patients had improvements in hba1c but remained above their target goal, and 4 patients had elevated hba1c since initiating bhh services. behavioral health home services were able to identify 54 individuals who did not have their hba1c levels checked according to the program s recommended guidelines for individuals being prescribed antipsychotic medications. this screening is particularly important for individuals with smi because, consistent with the literature, approximately 27% of this sample had a diagnosis of diabetes. also, another 4 patients were identified to be within diabetic range but did not have any diagnosis of diabetes. preliminary data indicate that the use of bhh services may be particularly useful in identifying gaps in care such as laboratory monitoring or missed diagnoses and allow for the delivery of preventative and individualized proactive care. in regard to health improvements, the data for the 33 patients who were within the type 2 diabetes range are mixed. slightly over 12% (n = 4) of patients improved to be within the recommended guidelines for hba1c levels. another 21% (n = 7) showed improvements in their hba1c levels, albeit still above the target goal. however, 6% (n = 2) of individuals who were originally considered to be in the prediabetes range moved within the diabetic range, and another 12% (n = 7) had higher hba1c levels in comparison to when they started receiving services. given identified problems with traditional primary care settings for this population, new models of care that can deliver patient - centered primary care within the mental health setting are needed to decrease the high rates of mortality and morbidity. while more empirical data are needed, preliminary results indicate that the use of a bhh may be particularly useful in understanding the unique characteristics of a specific population. this allows for earlier recognition of common medical disorders through more careful monitoring according to program guidelines and promotes proactive preventative care to prevent advancement of disease. as a result of these findings, a center for disease control and prevention diabetes prevention program group has been started within the mental health clinic and closer monitoring of clinician practice with regard to monitoring and referral to specific services is being done. it is important to note that the current report is limited by the fact that not all patients received equal amounts of phm services, given that enrollment occurred throughout the entirety of the 10 months. also, given the lack of a comparison group, meaningful statistical tests could not be conducted. future research examining the utility of bhh services would benefit from investigating both a clinical and a control sample. furthermore, given that these services are designed to provide more patient - centered care for individuals with smi, an examination of patient satisfaction with bhh and phm services is warranted. while the current data are mixed, the bhh may provide a cost - effective way to provide patient - centered care that helps identify gaps in somatic care for individuals with smi and provide this population with much need interventions. | objective : individuals with serious mental illness (smi) have higher rates of preventable diseases such as diabetes in comparison to the general population. while multifaceted, these high rates of preventable diseases in the population with smi may be partially attributed to limited access to primary care. a new program, the behavioral health home (bhh), which allows for the delivery of somatic care coordination and population - based care, may provide this population with the much needed somatic coordination and education it requires.methods:the impact of the population - based health management program of the bhh identification and severity rating of glucose metabolism disorders was assessed during the initial 10 months of the bhh.results:multiple patients were identified who either were not having hemoglobin a1c (hba1c) levels drawn per recommended guidelines for individuals prescribed antipsychotic medications or were within diabetic range but did not have a diagnosis of diabetes. mixed results occurred in regard to patients hba1c levels while engaging in the bhh.conclusion:this case study provides some initial evidence for the utility of the bhh in regard to identifying patients who need preventive care. |
anandamide and the other " endocannabinoids " are endogenous ligands for " brain - type " (cb1) and " spleen - type " (cb2) cannabinoid receptors. also " non - classical " cannabinoid receptors and vanilloid receptors are activated by aea, which plays a number of roles with potential clinical relevance, both in the central nervous system and in the periphery. in view of the broad implications of aea as central and peripheral modulator, its quantification is of utmost importance, also for the consideration that inhibition of aea degradation via uptake and hydrolysis might open new therapeutic perspectives. aea content has been measured in pig, sheep and cow brain, in rat brain and rat peripheral tissues [5 - 7 ], in human brain and human cells in culture, and in mouse brain. most quantitative analyses of aea content have been made by gas chromatography / mass spectrometry (gc / ms) after prepurification of the lipid extracts, and normalized to the fresh weight of the samples. this quantification has yielded concentration values in the range 0.2 30 pmol / g fresh weight [4 - 7 ]. recently, we have developed a gc / ms procedure for the direct analysis of lipid extracts without prepurification steps. moreover, we normalized the amounts of aea to the protein content of the samples, in order to better compare different tissues and cells, and also in consideration of the fact that determination of proteins is much more reliable than that of fresh weight. using this direct gc / ms analysis we found concentrations of aea in the 340 400 pmol / mg protein range. here, we show that, besides the procedure adopted, the normalization of aea content is a critical factor, which might induce significant differences in the same sample. most notably, we show how applying the same normalization procedure very similar or even identical amounts of aea are found in tissues and in cells independently of their origin, suggesting that this compound is evenly distributed in animals. aea was determined in rat brain, mouse brain, human neuroblastoma chp100 cells and human lymphoma u937 cells, and its content was then normalized to 1 mg of protein, dna or lipid phosphorus, to fresh weight, or to the cell number. while it is obvious that a different normalization method may yield very different values of aea in the same sample (~12 to 50-fold lower when aea was normalized to fresh weight or cell number, as compared to protein or dna content), it is noteworthy that normalization to a common parameter yielded similar or even identical levels of aea in all samples tested (table 1). in particular, normalization to dna content yielded the same amounts of aea in rat and mouse brain, but approximately half that value in both tumor cells (table 1). this finding seems in agreement with the fact that these cells are rapidly growing, and hence they have more dna. values of ~40%, in line with previous reports [4 - 7 ], whereas normalization to the other parameters showed s.d. impact of different normalizations on the quantification of anandamide in brain, and in human cells values are reported as the mean s.d. pi, lipid phosphorus ; fw, fresh weight ; n.d., not determined. taken together, the present observation might represent a caveat to researchers when they report on the content of a metabolite, also by using newly developed liquid chromatography / mass spectrometry techniques. on the other hand, the present results suggest that the most reliable reference to quantify aea and congeners from different sources is the protein content. in fact, it is a parameter common to cells and tissues, which is easy and rapid to measure with accuracy and reproducibility, even in tiny samples. the authors wish to thank drs natalia battista and valeria gasperi for their skilful assistance. | anandamide (n - arachidonoylethanolamine, aea) is an endogenous lipid that binds to cannabinoid receptors in the central nervous system and in peripheral cells. quantitative analysis of aea is generally based on the normalization to the fresh weight of the samples. here, we show that the normalization procedure of aea content is such a critical factor, that it might introduce per se significant discrepancies in the quantification of aea even in the same sample. we suggest that a rapid, accurate and most reliable reference to quantify aea and congeners from different sources is the protein content, a common parameter to cells and tissues. |
they carry genes that help their host adapt to new niches and stresses, playing a key role in bacterial evolution. plasmids frequently code for antibiotic resistance genes, being responsible for the spread of antibiotic resistance and multiresistance among pathogenic bacteria, which is currently a major concern for public health. one of the central questions about plasmid biology is how plasmids can be stably maintained in bacterial populations in the long term. specifically, (i) plasmids produce a cost to the host bacteria, causing a competitive disadvantage and (ii) plasmids can be lost during cell division (even if it is at a very low rate). taken together, these 2 factors predict a constant decline of the plasmid frequency in the population over time. however, there are factors that counteract the effect of cost and segregational loss and contribute to plasmid maintenance in bacterial populations, such as (i) selection for plasmid - encoded traits, (ii) horizontal transfer of the plasmid between bacteria (mainly by conjugation) and (iii) compensatory mutations alleviating the cost produced by the plasmid. the balance among all these factors determines the fate of a given plasmid in a bacterial population. previous theoretical studies have argued that plasmids can only be maintained in a bacterial population when they are able to conjugate. however, recent advances in genome sequencing have shown that, paradoxically, a large fraction of plasmids seem to be non - transmissible by conjugation according to their sequence. the mechanisms that allow non - transmissible plasmids to persist are poorly understood. in a recent study we used mathematical modeling, functional genomics and experimental evolution to investigate this problem. we developed a model system based on the opportunistic pathogen pseudomonas aeruginosa pao1 carrying the small non - conjugative plasmid pnuk73, which produces a particularly big fitness cost in this strain (approximately 20% reduction in relative fitness). pnuk73 confers resistance to neomycin, producing an increase in the minimal inhibitory concentration (mic) of approximately 60-fold in pao1. after 30 daily passages (300 generations) the plasmid - bearing subpopulation presented compensatory mutations that completely compensated for the cost of plasmid carriage. these mutations putatively inactivated 3 particular chromosomal genes : a helicase carrying an uvrd - like helicase c - terminal domain (pa1372) and 2 contiguous putative serine / threonine protein kinases (pa4673.15 and pa4673.16). however, the plasmid - free subpopulation was also adapting to the environmental conditions and therefore their fitness also increased relative to the parental strain. this was due to the acquisition of generally beneficial mutations for adaptation to the laboratory conditions. these mutations targeted primarily the diguanylate cyclase gene wspf (pa3703) in our experimental system. thus, the plasmid - bearing lineage continued to go extinct, albeit at a slower rate. as a result, the population size of the plasmid - bearing lineage was too small for the generally beneficial mutations to fix. this led to an increase in plasmid - bearing cells population size which allowed generally beneficial mutations to fix. therefore, we found that positive selection and compensatory adaptation interact to stabilize pnuk73 : positive selection increases the probability of compensatory adaptation by increasing the population size of plasmid - bearing lineages, improving therefore the chances of new adaptive mutations in the plasmid - bearing cells. compensatory adaptation, in turn, increases the effect of positive selection on plasmid stability by slowing the rate at which the plasmid is lost between episodes of positive selection. our previous results show that compensation and positive selection help stabilize non - transmissible plasmids over a few hundreds of generations. however, the long - term maintenance of these plasmids remains challenging to understand. in this commentary we expand the results of our population genetics model to analyze the effects of different regimes of selection as well as the impact of horizontal gene transfer in the long - term stability of these plasmids. the mechanisms that drive plasmid dynamics in a population, for instance conjugation and segregational loss, are noisy processes that can be studied using a stochastic modeling approach. the mathematical model presented in san millan. we use this approach because it allows us to numerically simulate a system of ordinary differential equations to identify the ecological and evolutionary forces that drive plasmid dynamics. for instance, this simple population genetics model predicts that a consequence of having a dynamic population structure with different bacterial types having different stability patterns is that the rate of decay of the plasmid in a mixed population also has to be a dynamic property of the system. furthermore, differences in susceptibility patterns for different bacterial types imply that the use of antibiotics has the effect of modifying allele frequency in the population and, as a result, the plasmid can become more stable after being exposed to antibiotics. in our previous work we observed that rare events of selection slowed down plasmid loss but could not halt it completely. here we used the same model based on pnuk73 and p. aeruginosa to investigate the effect of different selective regimes in the stability of the plasmid and try to find the conditions necessary for long term maintenance. in order to quantify the stability of the plasmid we will use the time elapsed before the plasmid frequency in the population is below a threshold represented with a parameter > 0 (in the numerical simulations presented in this paper we considered 1%, but found no qualitative differences when evaluating different values of). now, let us denote with t the duration of the observation (in our evolutionary experiment t = 32 days) and with t the interval of time before the frequency of plasmid - bearing cells is below. we say the plasmid persists when t > t, that is when the frequency of plasmid - bearing cells at the end of the experiment is larger than. figure 1 shows that if we vary the strength (the antibiotic dose) and the frequency (interval of time between periodic exposures) of the selective pressure, there is a region (at high - doses and high - frequencies) in the dose - frequency plane where a non - transmissible plasmid can be maintained indefinitely. furthermore, if the selective pressure is applied frequently, very low concentrations are able to maintain the plasmid in the population. in agreement with this prediction, a recent experimental study shows that very low concentrations of antibiotics or heavy metals are sufficient to maintain resistance plasmids. conversely, if the dose is too low or the interval of time between antibiotic exposures too far in between, the plasmid becomes unstable (fig. 2014 with parameter values determined from experimental data and an extinction threshold of = 0.01. (a) frequency of plasmid bearing cells under 2 different selection regimes : same antibiotic dose (25% above the mic of the plasmid - free cells) but with different frequencies of drug exposure (antibiotic use is represented with yellow bars). note how when the antibiotic is used once weekly the plasmid - bearing population is driven to extinction (red dot), while if the drug is used twice - weekly the plasmid is able to persist indefinitely. (b) heat map representing the time to extinction of the plasmid - bearing population when exposed to increasing drug concentrations and different periods of drug exposure (from daily exposure, every 2, d etc.). note that plasmid persistence is observed only at certain combinations of dose and frequency of antibiotic exposure (black region). the positive selection regimes used to obtain the time - series illustrated in (a) are represented with white boxes in the period - dose plane. 2014 with parameter values determined from experimental data and an extinction threshold of = 0.01. (a) frequency of plasmid bearing cells under 2 different selection regimes : same antibiotic dose (25% above the mic of the plasmid - free cells) but with different frequencies of drug exposure (antibiotic use is represented with yellow bars). note how when the antibiotic is used once weekly the plasmid - bearing population is driven to extinction (red dot), while if the drug is used twice - weekly the plasmid is able to persist indefinitely. (b) heat map representing the time to extinction of the plasmid - bearing population when exposed to increasing drug concentrations and different periods of drug exposure (from daily exposure, every 2, d etc.). note that plasmid persistence is observed only at certain combinations of dose and frequency of antibiotic exposure (black region). the positive selection regimes used to obtain the time - series illustrated in (a) are represented with white boxes in the period - dose plane. in our previous work we demonstrated that compensatory adaptation and positive selection interact to stabilize non - conjugative plasmids on a time scale of hundreds of generations. moreover, in the previous section of this paper we showed, using the same model, that very low concentrations of frequent positive selection could theoretically stabilize non - transmissible plasmids in the short term. however, the maintenance of these plasmids over longer time scales remains enigmatic for several reasons. first, although compensatory adaptation removes the cost of plasmid carriage rapidly, it is only under episodes of early positive selection that the plasmid - bearing lines are able to reach the same fitness as the plasmid - free cells, removing the effective cost of plasmid carriage (by acquiring extra general beneficial mutations). second, even if plasmid - bearing cells have no competitive disadvantage compared to plasmid - free cells, segregational loss will gradually reduce the frequency of plasmids in the population (as observed in our experiments). third, plasmid - encoded genes could theoretically move to the chromosome if they were under constant selection, rendering the plasmid redundant. and fourth, even if the pervasive presence of positive selection can explain the maintenance of part of the non - transmissible plasmids, it could not justify the persistence of the small non - transmissible cryptic plasmids carrying no selectable genes, which are also common in nature. so, how are non - transmissible plasmids maintained in the population in the long - term ? (2010) that predicted that approximately 50% of plasmids are non - transmissible by conjugation is based on detecting the presence of genes involved in conjugation and mobilization on the plasmids. therefore, and as the authors of this study have pointed out, these analyses may underestimate the potential for the horizontal spread of plasmids by other means, such as transduction or natural transformation. in addition, some of the plasmids lacking conjugation - related genes may still be able to undergo conjugation by co - integration into conjugative elements (also known as conduction). in particular, we considered that plasmid mobilization occurs through conjugation and, similar to previously published models, we included a mass action term representing the rate at which plasmid - free cells acquire plasmids from plasmid - bearing donor cells. in our model, the parameter 0 will denote the rate of conjugation. although we are modeling the horizontal transmission by conjugation, we argue that modeling other forms of horizontal gene transfer of the plasmid such as transduction or transformation will produce similar results. in agreement with previous studies, our numerical simulations show we can stabilize a plasmid in a selection regime where the plasmid would be unstable by considering a non - zero probability of horizontal transmission. for instance, if we numerically simulate our model considering a daily usage of antibiotics, then we can identify a range of drug concentrations where the plasmid is unstable if the rate of horizontal transmission is below a certain threshold. indeed, figure 2a shows how the plasmid - bearing population is driven to extinction in less than 12 d if we consider the plasmid is non - conjugative (that is, = 0) but with a horizontal transmission rate as low as = 5 10 the plasmid - bearing subpopulation recovers and fixes. theoretical scenario where antibiotics are used every day at a constant dose and where the rate of horizontal transmission () is allowed to change. (a) comparison between plasmid dynamics for a non - conjugative plasmid (black line) and (blue line) when the rate of horizontal transmission is low (but non - zero) and the antibiotic is deployed at a dose corresponding to 12.5% the mic of the plasmid - free cells. note how if = 5 10 the plasmid - bearing population recovers after an initial decrease in frequency, but if = 0 the plasmid - bearing population is driven to extinction. (b) by numerically simulating our evolutionary model under different rates of conjugation and intensities of positive selection we can characterize different plasmid stability regions. theoretical scenario where antibiotics are used every day at a constant dose and where the rate of horizontal transmission () is allowed to change. (a) comparison between plasmid dynamics for a non - conjugative plasmid (black line) and (blue line) when the rate of horizontal transmission is low (but non - zero) and the antibiotic is deployed at a dose corresponding to 12.5% the mic of the plasmid - free cells. note how if = 5 10 the plasmid - bearing population recovers after an initial decrease in frequency, but if = 0 the plasmid - bearing population is driven to extinction. (b) by numerically simulating our evolutionary model under different rates of conjugation and intensities of positive selection we can characterize different plasmid stability regions. the minimum horizontal transmission rate that stabilizes the plasmid can be estimated numerically for different antibiotic concentrations (it is important to highlight that these values should not to be considered as quantitative predictions of the model, as this would be beyond the objectives of this simple model). of course, at high antibiotic concentrations (close or above the mic of plasmid - free subpopulation), selection for the plasmid is so high that even a non - conjugative plasmid can be maintained in the population during the duration of our experiment. but, interestingly, the critical value of that maintains the plasmid is relatively low even when selection for the plasmid is zero, a prediction in agreement with a recently published mathematical analysis showing that a plasmid carrying deleterious genes can still present a non - zero fixation probability. this result is based on the observation that when the frequency of plasmid - bearing cells is low, then almost all individuals in the population are potential recipients of the plasmid and thus the probability of a horizontal transmission event occurring is increased. in summary, both our theoretical model and our experimental results support the tenet that it is very difficult for strictly non - transmissible plasmids to persist over the long - term in a bacterial population. these findings tantalizingly suggest that such plasmids may need to experience episodes of horizontal gene transfer in order to persist. in addition, although conjugation is arguably the main route for plasmid horizontal transfer, transformation and especially transduction may play an important role in lower rates of plasmid dissemination. it is therefore possible that all plasmids are transmissible, only at extremely different rates. the frequency of transmission will probably determine the fate of the plasmid and will depend on multiple factors such as the genetic composition of the plasmid itself (presence of conjugative genes), the host bacterium genetic background (coexistence of other mobile elements such as plasmids or phages) and environmental factors (inducing or facilitating conjugation or transduction). further studies will be required to assess the mobility of the a priori non - transmissible plasmids and to identify the underlying horizontal transfer mechanism. in order to achieve this goal, we could use stochastic models of plasmid dynamics to assess the temporal variability observed in the data and use fluctuations in plasmid - bearing frequencies to make inferences about different mechanisms of horizontal transfer driving plasmid dynamics. furthermore, we conceive 2 possible approaches to determine the actual potential and frequency of transmission of these plasmids : first, the direct experimental observation of plasmid transmission between donor and recipient strains in vitro or in vivo. this is the classical approach used to determine the transfer rate of conjugative elements and has already been successfully applied to the phage - mediated mobilization of plasmids and to plasmid transformation. the main advantage of this method is that one can directly observe the transfer events and accurately quantify the frequencies under very controlled experimental conditions. the principal disadvantage of this approach is that the experimental settings usually differ to the real world conditions, and this may bias the results. also, these methods are time - consuming and low - throughput, since each plasmid - host combination has to be tested individually.second, the use of phylodynamic methods have played a big role in our understanding of the transmission of viruses between humans. this approach is based on inferring viral transmission dynamics from viral genomic data, using bioinformatics and sophisticated statistical methods. the movement of mobile genetic elements between bacterial strains is analogous to the viral transmission among patients so phylodynamic methods could be adapted to analyze this problem. this approach presents several benefits : the utilization of the increasingly available bacterial and plasmid genomes and existing statistical methods, the possibility of integrating temporal dynamics and ecological contexts in the analysis of the spread of the plasmid (coalescent theory analysis) and the potential for high - throughput analysis. the main problem of this approach could be that the resolution of the method is hampered by low mutation rate. while viruses mutate at a much higher rate than their human hosts, bacteria and plasmid have much lower mutation rates than viruses, and this could decrease the power of this approach. finally, alternative approaches such as network modeling coupled with phylogenetic analysis could also provide valuable information about the rate of transmission of plasmids between bacterial strains. first, the direct experimental observation of plasmid transmission between donor and recipient strains in vitro or in vivo. this is the classical approach used to determine the transfer rate of conjugative elements and has already been successfully applied to the phage - mediated mobilization of plasmids and to plasmid transformation. the main advantage of this method is that one can directly observe the transfer events and accurately quantify the frequencies under very controlled experimental conditions. the principal disadvantage of this approach is that the experimental settings usually differ to the real world conditions, and this may bias the results. also, these methods are time - consuming and low - throughput, since each plasmid - host combination has to be tested individually. phylodynamic methods have played a big role in our understanding of the transmission of viruses between humans. this approach is based on inferring viral transmission dynamics from viral genomic data, using bioinformatics and sophisticated statistical methods. the movement of mobile genetic elements between bacterial strains is analogous to the viral transmission among patients so phylodynamic methods could be adapted to analyze this problem. this approach presents several benefits : the utilization of the increasingly available bacterial and plasmid genomes and existing statistical methods, the possibility of integrating temporal dynamics and ecological contexts in the analysis of the spread of the plasmid (coalescent theory analysis) and the potential for high - throughput analysis. the main problem of this approach could be that the resolution of the method is hampered by low mutation rate. while viruses mutate at a much higher rate than their human hosts, bacteria and plasmid have much lower mutation rates than viruses, and this could decrease the power of this approach. finally, alternative approaches such as network modeling coupled with phylogenetic analysis could also provide valuable information about the rate of transmission of plasmids between bacterial strains. we would like to thank ben s cooper for his help developing the mathematical model. | in theory, plasmids can only be maintained in a population when the rate of horizontal gene transfer is larger than the combined effect of segregational loss and the decrease of fitness associated with plasmid carriage. recent advances in genome sequencing have shown, however, that a large fraction of plasmids do not carry the genes necessary for conjugation or mobilization. so, how are so - called non - transmissible plasmids able to persist ? in order to address this question, we examined a previously published evolutionary model based on the interaction between p. aeruginosa and the non - transmissible plasmid pnuk73. both our in silico and in vitro results demonstrated that, although compensatory adaptation can decrease the rate of plasmid decay, the conditions for the maintenance of a non - transmissible plasmid are very stringent if the genes it carries are not beneficial to the bacterial host. this result suggests that apparently non - transmissible plasmids may still experience episodes of horizontal gene transfer occurring at very low frequencies, and that these scattered transmission events are sufficient to stabilize these plasmids. we conclude by discussing different genomic and microbiological approaches that could allow for the detection of these rare transmission events and thus to obtain a reliable estimate of the rate of horizontal gene transfer. |
glial cell line - derived neurotrophic factor (gdnf) is a small protein that potently promotes the survival of many types of neurons and is able to prevent apoptosis of motor neurons induced by axotomy. the detection of gdnf is vital to monitoring the survival and maintenance of sympathetic and sensory neurons.16 several approaches have so far been devised for the detection of gdnf. conventional antibody - based methods, including western blotting, dot blotting, immunohistochemistry, and enzyme - linked immunosorbent assay, are widely used analytical approaches in both clinical diagnosis and biological research.7 however, these methods always suffer from the limitations of long assay time, complex experimental procedures, high cost, and low sensitivity. therefore, continuing efforts have been made to seek ideal methods for rapid, sensitive, low - cost, and user - friendly detection of gdnf. a molecular translator can be employed as a signal transducer by highly converting the input target molecules into unique output deoxyribonucleic acid (dna) for signal amplification. for example, picuri have developed a novel nucleic acid detection method through converting any target nucleic acids to a predesigned output dna. li and zhang,13 have designed a molecular translator base on binding induced dna assembly for fluorescence detection of protein, including prostate - specific antigen and platelet - derived growth factor (pdgf). although these approaches are effective, most of them are time - consuming, laborious, and high cost with low sensitivity. recently, lateral flow biosensors (lfbs) have attracted considerable research interest because of their simple assay procedure, user - friendly platform, short assay time, and cost - effectiveness. the result of the assay can be observed by naked eyes within 15 minutes, and quantitative analysis can be realized by recording the color intensity of the red band on the test zone with a portable lfb reader.14,15 isothermal strand - displacement polymerization reaction (isdpr) is a commonly used approach for dna signal amplification due to its robustness and simplicity.16 in this work, for the first time, we propose an lfb for detecting gdnf with a molecular translator and isdpr. we employ a molecular translator to convert the protein signal to a dna signal, which can be further amplified by isdpr. gdnf and rabbit anti - gdnf polyclonal antibody, bovine serum albumin (bsa), streptavidin, and gold nanoparticles (aunps) (5 nm, 10 nm, and 20 nm) were purchased from sigma - aldrich (steinheim, germany). the polymerase klenow fragment exo- and deoxynucleoside triphosphates (dntps) were obtained from new england biolabs (ipswich, ma, usa). all buffer solutions used in this study were prepared in our laboratory with ultrapure water. the oligonucleotide sequences were synthesized and purified through high - performance liquid chromatography by shanghai sangon biological engineering technology & services co. ltd (shanghai, people s republic of china). the conjugates were prepared by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and n - hydroxysuccinimide (edc / nhs) activity. briefly, 20.6 mg edc and 11.5 mg nhs were mixed in 1 ml of water for 10 minutes. from this solution, 10 l was added to 200 l of 500 g / ml rabbit anti - gdnf polyclonal antibody, rabbit antitransforming growth factor [tgf]-, anti - tgf-, antiepidermal growth factor [egf ], anti - insulin - like growth factor [igf]-i, and antifibroblast growth factor [fgf ]) polyclonal antibodies solution in carbonate buffer, ph 11, and reacted for 2 hours. the solution was then centrifuged for 5 minutes at 4,000 r / min and washed twice with 200 l of water for 5 minutes at 4,000 r / min before being brought up to a final volume of 200 l with water. the biosensor consisted of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. the sample pad (1.730 cm) was prepared by soaking a glass fiber pad in sample pad buffer (ph 8.0) (1% triton, 2% bsa, 3% glucose, and 50 mm boric acid). the sample pad was then dried at room temperature. the conjugate pad (fiberglass : 0.830 cm) was prepared by dispensing a desired volume of aunp antidigoxin conjugates onto the glass fiber using a dispenser. the conjugate pad was dried at room temperature for 12 hours and stored at 4c. streptavidin (1 mg / ml) and rabbit antimouse immunoglobulin g (igg) antibody (30 l, 1 mg / ml) were dispensed onto the nitrocellulose membrane to form a test zone and a control zone, respectively, with a lateral flow dispenser. finally, the four components of the lateral flow dna biosensor were assembled on plastic adhesive backing (630 cm). each part overlapped by 2 mm to ensure solution migration through the strip during the assay. strips were cut into 3 mm width with a paper cutter. a dna probe for the molecular translator was prepared at a final concentration of 5 m by mixing 20 l 50 m mouse anti - gdnf antibody conjugated dna1 with 13.3 l 50 m output dna3 in 166.7 m tris ethylenediaminetetraacetic acid (te) buffer (containing 10 mm mgcl2 and 0.05% tween 20), heating to 90c for 5 minutes, and allowing solution to cool down to 25c slowly in a period of 3 hours. strand displacement of molecular translator was performed in 20 l te buffer, 10 nm dna1/dna3 complex, 10 nm dna2. isdpr was performed in a 25 l 50 nm tris - hcl (ph 8.0) buffer containing 50 nm short primer, 3u polymerase klenow fragment exo-, 50 m dntps, 6% dimethyl sulfoxide, 0.1% bsa, 1 mm dtt, 5 mm mgcl2, and 2 l of product of strand displacement of molecular translator. the red bands were observed within 5 minutes, and the biosensor was scanned with a portable lfb reader. the optical intensities of the test zone and the control zone were recorded simultaneously by the reader, which automatically located the red bands in a fixed reaction area and then measured strip parameters such as peak height and area integral. for the process of molecular translator and strand - displacement amplification, dna1 conjugated with the gdnf antibody is initially hybridized to dna3 to form a stable dna1/dna3 duplex. dna2 sequences are designed in such a way that the complementary sequences between dna1 and dna2 are 4-nt shorter than the complementary sequences between dna1 and dna3. in the absence of gdnf, the strand displacement activity is poor between dna2 and dna3 at 25c, and the displacement of output dna3 by competing dna2 is extremely minimal. however, in the process of gdnf, the binding of the same target protein to its antibodies that are coupled with dna1 and dna2 brings dna1 and dna2 to a close proximity, resulting in an increasing in their local effective concentration. this process triggers the strand - displacement reaction between dna2 and dna3. as a result, the output dna3 is released for the next signal amplification (figure 1a). the releasing output dna3 recognizes and hybridizes with the loop region of the hairpin probe, which is composed of an eleven base pair stem, a 25-nt loop, and a biotin at the 5 end, causing the hairpin probe to undergo a conformational change and leading to stem separation. the digoxin - coupled primer (8-nt) complementary to the stem region of the hairpin probe at the 3 end thus anneals with the open stem and triggers a polymerization reaction in the presence of dntps and dna polymerase. in the process of primer extension, the output dna3 is released by dna polymerase with strand - displacement activity, after which complementary dna is synthesized, leading to the formation of a hairpin dna complex. to start the next cycle, the releasing output dna3 hybridizes with another hairpin probe, triggering another polymerization reaction. through this cyclic reaction comprising release of output dna3 and hybridization of output dna3 with remaining hairpin probe, a great amount of duplex dna products with a biotin and digoxin tag at the 5 end are synthesized. the hairpin probe retains its original stem - loop structure in the absence of gdnf, the primer is unable to anneal to the hairpin to initiate a polymerization, and no duplex dna is produced (figure 1b). an lfb consists of four components : sample pad, conjugate pad, nitrocellulose membrane, and absorption pad. streptavidin and goat antimouse igg antibody (secondary body) are dispensed on the nitrocellulose membrane, respectively, to form the test zone and the control zone. the sample solution containing the isdpr product and running buffer is applied on the sample pad of the lfbs. the solution migrates along the lfb by capillary action to the conjugate pad, where antidigoxin aunp conjugates have been deposited. antidigoxin antibody couples with digoxin of the isdpr duplex dna product to form a biotin duplex dna the complexes are captured at the test zone by the specific reaction between preimmobilized streptavidin and biotin. the accumulation of aunps on the test zone is then visualized as a characteristic red band aunp conjugates continue to migrate and are captured at the control zone by immunoreactions between antidigoxin antibody and the preimmobilized secondary antibody on the aunp surface, thus forming a second red band. in the absence of gdnf, no isdpr duplex dna product is produced ; therefore, no red band is observed at the test zone. in this case, the red band at the control zone indicates that the lfb is working properly. visual detection is performed by observing the color change caused by the accumulation of aunps on the test zone, and quantitative detection can be realized by recording the color intensity of the red band on the test zone with a portable lfb reader (figure 1c). the data in this study were analyzed with spss statistics software version 19.0 (ibm corporation, armonk, ny, usa). gdnf and rabbit anti - gdnf polyclonal antibody, bovine serum albumin (bsa), streptavidin, and gold nanoparticles (aunps) (5 nm, 10 nm, and 20 nm) were purchased from sigma - aldrich (steinheim, germany). the polymerase klenow fragment exo- and deoxynucleoside triphosphates (dntps) were obtained from new england biolabs (ipswich, ma, usa). all buffer solutions used in this study were prepared in our laboratory with ultrapure water. the oligonucleotide sequences were synthesized and purified through high - performance liquid chromatography by shanghai sangon biological engineering technology & services co. ltd (shanghai, people s republic of china). the conjugates were prepared by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and n - hydroxysuccinimide (edc / nhs) activity. briefly, 20.6 mg edc and 11.5 mg nhs were mixed in 1 ml of water for 10 minutes. from this solution, 10 l was added to 200 l of 500 g / ml rabbit anti - gdnf polyclonal antibody, rabbit antitransforming growth factor [tgf]-, anti - tgf-, antiepidermal growth factor [egf ], anti - insulin - like growth factor [igf]-i, and antifibroblast growth factor [fgf ]) polyclonal antibodies solution in carbonate buffer, ph 11, and reacted for 2 hours. the solution was then centrifuged for 5 minutes at 4,000 r / min and washed twice with 200 l of water for 5 minutes at 4,000 r / min before being brought up to a final volume of 200 l with water. the biosensor consisted of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. the sample pad (1.730 cm) was prepared by soaking a glass fiber pad in sample pad buffer (ph 8.0) (1% triton, 2% bsa, 3% glucose, and 50 mm boric acid). the sample pad was then dried at room temperature. the conjugate pad (fiberglass : 0.830 cm) was prepared by dispensing a desired volume of aunp antidigoxin conjugates onto the glass fiber using a dispenser. the conjugate pad was dried at room temperature for 12 hours and stored at 4c. streptavidin (1 mg / ml) and rabbit antimouse immunoglobulin g (igg) antibody (30 l, 1 mg / ml) were dispensed onto the nitrocellulose membrane to form a test zone and a control zone, respectively, with a lateral flow dispenser. finally, the four components of the lateral flow dna biosensor were assembled on plastic adhesive backing (630 cm). each part overlapped by 2 mm to ensure solution migration through the strip during the assay. a dna probe for the molecular translator was prepared at a final concentration of 5 m by mixing 20 l 50 m mouse anti - gdnf antibody conjugated dna1 with 13.3 l 50 m output dna3 in 166.7 m tris ethylenediaminetetraacetic acid (te) buffer (containing 10 mm mgcl2 and 0.05% tween 20), heating to 90c for 5 minutes, and allowing solution to cool down to 25c slowly in a period of 3 hours. strand displacement of molecular translator was performed in 20 l te buffer, 10 nm dna1/dna3 complex, 10 nm dna2. isdpr was performed in a 25 l 50 nm tris - hcl (ph 8.0) buffer containing 50 nm short primer, 3u polymerase klenow fragment exo-, 50 m dntps, 6% dimethyl sulfoxide, 0.1% bsa, 1 mm dtt, 5 mm mgcl2, and 2 l of product of strand displacement of molecular translator. finally, the isdpr product was loaded onto the sample pad together with 20 l pbs. the red bands were observed within 5 minutes, and the biosensor was scanned with a portable lfb reader. the optical intensities of the test zone and the control zone were recorded simultaneously by the reader, which automatically located the red bands in a fixed reaction area and then measured strip parameters such as peak height and area integral. for the process of molecular translator and strand - displacement amplification, dna1 conjugated with the gdnf antibody is initially hybridized to dna3 to form a stable dna1/dna3 duplex. dna2 sequences are designed in such a way that the complementary sequences between dna1 and dna2 are 4-nt shorter than the complementary sequences between dna1 and dna3. in the absence of gdnf, the strand displacement activity is poor between dna2 and dna3 at 25c, and the displacement of output dna3 by competing dna2 is extremely minimal. however, in the process of gdnf, the binding of the same target protein to its antibodies that are coupled with dna1 and dna2 brings dna1 and dna2 to a close proximity, resulting in an increasing in their local effective concentration. this process triggers the strand - displacement reaction between dna2 and dna3. as a result, the output dna3 is released for the next signal amplification (figure 1a). the releasing output dna3 recognizes and hybridizes with the loop region of the hairpin probe, which is composed of an eleven base pair stem, a 25-nt loop, and a biotin at the 5 end, causing the hairpin probe to undergo a conformational change and leading to stem separation. the digoxin - coupled primer (8-nt) complementary to the stem region of the hairpin probe at the 3 end thus anneals with the open stem and triggers a polymerization reaction in the presence of dntps and dna polymerase. in the process of primer extension, the output dna3 is released by dna polymerase with strand - displacement activity, after which complementary dna is synthesized, leading to the formation of a hairpin dna complex. to start the next cycle, the releasing output dna3 hybridizes with another hairpin probe, triggering another polymerization reaction. through this cyclic reaction comprising release of output dna3 and hybridization of output dna3 with remaining hairpin probe, a great amount of duplex dna products with a biotin and digoxin tag at the 5 end are synthesized. the hairpin probe retains its original stem - loop structure in the absence of gdnf, the primer is unable to anneal to the hairpin to initiate a polymerization, and no duplex dna is produced (figure 1b). an lfb consists of four components : sample pad, conjugate pad, nitrocellulose membrane, and absorption pad. streptavidin and goat antimouse igg antibody (secondary body) are dispensed on the nitrocellulose membrane, respectively, to form the test zone and the control zone. the sample solution containing the isdpr product and running buffer is applied on the sample pad of the lfbs. the solution migrates along the lfb by capillary action to the conjugate pad, where antidigoxin aunp conjugates have been deposited. antidigoxin antibody couples with digoxin of the isdpr duplex dna product to form a biotin duplex dna the complexes are captured at the test zone by the specific reaction between preimmobilized streptavidin and biotin. the accumulation of aunps on the test zone is then visualized as a characteristic red band aunp conjugates continue to migrate and are captured at the control zone by immunoreactions between antidigoxin antibody and the preimmobilized secondary antibody on the aunp surface, thus forming a second red band. in the absence of gdnf, no isdpr duplex dna product is produced ; therefore, no red band is observed at the test zone. in this case, the red band at the control zone indicates that the lfb is working properly. visual detection is performed by observing the color change caused by the accumulation of aunps on the test zone, and quantitative detection can be realized by recording the color intensity of the red band on the test zone with a portable lfb reader (figure 1c). the data in this study were analyzed with spss statistics software version 19.0 (ibm corporation, armonk, ny, usa). the molecular translator is composed of target recognition elements (dna1 and dna2) and a signal output element (dna3), which is illustrated in figure 1a. the output dna3 triggers isdpr for signal amplification by producing a large amount of duplex dna (figure 1b). figure 1c illustrates the schematic illustration of the strip biosensor for visual detection of duplex dna. we investigated the optical response of 10 nm dna3 on the lfb with and without isdpr amplification. in the case without isdpr, there was no polymerase in the reaction buffer. as shown in figure 2, the response of the sample with isdpr was 35 times higher compared with that without isdpr or the blank group (p<0.01). the experimental parameters, such as isdpr temperature and reaction time that could affect the analytical performance, were optimized systematically. we compared the signal to noise (s / n) ratio of 10 ng ml and 1 ng ml gdnf with four different isdpr temperatures (25c, 37c, 42c, and 50c). as shown in figure 3a, the s / n ratios increased with increasing reaction temperature in the range from 25c to 42c. as shown in figure 3b, the optical response elevated gradually with the augmenting of reaction time from 10 minutes to 30 minutes. the peak area of the 10 ng / ml gdnf and 1 ng / ml gdnf was significantly higher compared with the blank group (figure 3b, p<0.05). further increasing the reaction time led to the increasing background signal. a longer reaction time at 42c may cause stem separation of the hairpin probe, even in the absence of output dna3, resulting in a high background signal. the results indicated that 30 minutes is the optimal reaction time (figure 3b). we have examined the sensitivity and dynamic range of the lfb using different concentrations of gdnf under optimal conditions. as shown in figure 4a, the color intensities of the red bands on the test zone increased with the increasing of gdnf concentrations. no distinct red band was observed on the test zone in the absence of gdnf. for quantitative detections, the optical responses were further confirmed by recording the color intensities of the red bands by a portable strip reader. the peak areas versus the incremental concentrations of gdnf have been plotted in figure 4b, which followed a sigmoid increase. the resulting calibration curve shows that the peak areas are proportional to the logarithm of gdnf concentrations in the range from 1 pg ml to 10 ng ml (figure 4c). the specificity of the lfb for gdnf was studied by testing the responses of the assay to other kinds of growth factors, including pdgf, tgf-, tgf-, egf, igf - i, and fgf, which are similar to gdnf. as shown in figure 5a, 1 ng / ml gdnf produced a visible bright red band on the test zone of lfb, whereas all other growth factors at a concentration of 100 ng / ml did not yield a red band on the test zone of lfb. the prepared lfbs were sealed and stored at 37c for the following time periods : 12 hours, 24 hours, 14 days, and 1 month. as shown in figure 5b, there were no significant performance differences among different storage times, indicating that the stability of the assay is acceptable. the molecular translator is composed of target recognition elements (dna1 and dna2) and a signal output element (dna3), which is illustrated in figure 1a. the output dna3 triggers isdpr for signal amplification by producing a large amount of duplex dna (figure 1b). figure 1c illustrates the schematic illustration of the strip biosensor for visual detection of duplex dna. we investigated the optical response of 10 nm dna3 on the lfb with and without isdpr amplification. in the case without isdpr, there was no polymerase in the reaction buffer. as shown in figure 2, the response of the sample with isdpr was 35 times higher compared with that without isdpr or the blank group (p<0.01). the experimental parameters, such as isdpr temperature and reaction time that could affect the analytical performance, were optimized systematically. we compared the signal to noise (s / n) ratio of 10 ng ml and 1 ng ml gdnf with four different isdpr temperatures (25c, 37c, 42c, and 50c). as shown in figure 3a, the s / n ratios increased with increasing reaction temperature in the range from 25c to 42c. as shown in figure 3b, the optical response elevated gradually with the augmenting of reaction time from 10 minutes to 30 minutes. the peak area of the 10 ng / ml gdnf and 1 ng / ml gdnf was significantly higher compared with the blank group (figure 3b, p<0.05). further increasing the reaction time led to the increasing background signal. a longer reaction time at 42c may cause stem separation of the hairpin probe, even in the absence of output dna3, resulting in a high background signal. the results indicated that 30 minutes is the optimal reaction time (figure 3b). we have examined the sensitivity and dynamic range of the lfb using different concentrations of gdnf under optimal conditions. as shown in figure 4a, the color intensities of the red bands on the test zone increased with the increasing of gdnf concentrations. no distinct red band was observed on the test zone in the absence of gdnf. for quantitative detections, the optical responses were further confirmed by recording the color intensities of the red bands by a portable strip reader. the peak areas versus the incremental concentrations of gdnf have been plotted in figure 4b, which followed a sigmoid increase. the resulting calibration curve shows that the peak areas are proportional to the logarithm of gdnf concentrations in the range from 1 pg ml to 10 ng ml (figure 4c). the specificity of the lfb for gdnf was studied by testing the responses of the assay to other kinds of growth factors, including pdgf, tgf-, tgf-, egf, igf - i, and fgf, which are similar to gdnf. as shown in figure 5a, 1 ng / ml gdnf produced a visible bright red band on the test zone of lfb, whereas all other growth factors at a concentration of 100 ng / ml did not yield a red band on the test zone of lfb. the prepared lfbs were sealed and stored at 37c for the following time periods : 12 hours, 24 hours, 14 days, and 1 month. as shown in figure 5b, there were no significant performance differences among different storage times, indicating that the stability of the assay is acceptable. a molecular translator can be employed as a signal transducer by highly converting the input target molecules into a unique output dna for signal amplification.17,18 isdpr is a commonly used approach for the dna signal amplification.1618 therefore, we combined the molecular translator and isdpr method to detect the gdnf in cells. in this work, isdpr was used to produce a large amount of digoxin- and biotin - coupled duplex dna for visual detection of gdnf. compared with the solutions without isdpr, the response of the sample with isdpr was 35 times higher. figure 3a shows that the s / n ratios increased with increasing reaction temperatures in the range from 25c to 42c, however s / n ratios decreased with temperatures over 42. these results could be explained by conformational change of the hairpin probe at high temperature, which leads to the stem separation of the hairpin probe. the primer can anneal with the open hairpin probe and trigger a polymerization reaction, even in the absence of output dna3, resulting in a high background signal. usually, the isdpr can be enhanced with increasing the reaction time. in this study, 30 minutes was selected as the reaction time throughout the experiments to obtain the highest signal. as shown in figure 4, the red band on the test zone was observed with as little as 1 pg ml of gdnf, which can be considered the limit of detection. based on the molecular weight of gdnf (13.5 kda), the limit of detection was calculated to be 74 fm. the detection limit of our proposed approach is two orders of magnitude better than the antibody - based immunoassay.7 generally, human basal serum gdnf concentrations are between 20 pg / ml and 30 pg / ml. this lfb holds great promise in monitoring gdnf by naked eyes with its superior detection sensitivity and large dynamic range. from the results of figure 5a, 1 ng / ml gdnf produced a visible bright red band, while the other growth factors (such as tgf-, tgf-, egf, igf - i, and fgf) did not yield a red band even at a concentration of 100 ng / ml. these results indicated that our constructed lfb exhibited excellent selective response to gdnf. our results also showed that this method is very stable after different periods of storage. in summary, we have successfully fabricated an lfb for amplified detection of gdnf using a molecular translator for converting the input protein to output dna signal and isdpr for signal amplification. compared with other reported antibody - based immunoassays, the sensitivity of the lfb developed here is higher. by using this assay, gdnf as low as 1 pg ml the response of the optimized assay is highly linear over a range of 1 pg / ml to 10 ng / ml. this new gdnf detection method is expected to be a great potential platform for the detection of protein, which would be valuable for clinical therapy for some diseases related to gdnf. | backgroundglial cell line - derived neurotrophic factor (gdnf) is a small protein that potently promotes the survival of many types of neurons. detection of gdnf is vital to monitoring the survival of sympathetic and sensory neurons. however, the specific method for gdnf detection is also un - discovered. the purpose of this study is to explore the method for protein detection of gdnf.methodsa novel visual detection method based on a molecular translator and isothermal strand - displacement polymerization reaction (isdpr) has been proposed for the detection of gdnf. in this study, a molecular translator was employed to convert the input protein to output deoxyribonucleic acid signal, which was further amplified by isdpr. the product of isdpr was detected by a lateral flow biosensor within 30 minutes.resultsthis novel visual detection method based on a molecular translator and isdpr has very high sensitivity and selectivity, with a dynamic response ranging from 1 pg / ml to 10 ng / ml, and the detection limit was 1 pg / ml of gdnf.conclusionthis novel visual detection method exhibits high sensitivity and selectivity, which is very simple and universal for gdnf detection to help disease therapy in clinical practice. |
bronchopleural fistula (bpf) is a well - known complication among the several pulmonary conditions. although bpf is a common postoperative complication of pulmonary resection, in developing countries it commonly occurs due to tuberculosis. postoperative fistulas need open surgical closure and central bpf will usually require occlusion devices if they persist even after surgery. we present a case that offers a novel method of closure of a chronic postoperative broncho - pleuro - cutaneous fistula by a patent ductus arteriosus (pda) occluder device. a 25-year - old lady presented to our hospital with complaints of productive cough and streaky hemoptysis for the last 4 weeks. she had been treated in the past for smear positive pulmonary tuberculosis in 2000 for 3 months and again in 2005 for 9 months with first - line antituberculous (att) drugs with poor drug compliance. she was diagnosed to have relapse in november 2007, and was being treated with second - line att drugs. her detailed workup showed radiographic evidence of large cavitary lesion in the right middle zone and fibrocystic opacities in the right upper zone. reports of sputum smear for acid - fast bacilli (afb) and culture for mycobacterium tuberculosis were negative. second - line att drugs were continued and, the patient was reviewed on a monthly basis in our outpatient department (opd). she had a persistent large cavity in the superior segment of the right lower lobe after 18 months of treatment and bronchiectasis in right upper lobe. the patient underwent right upper lobectomy and excision of cavity in right lower lobe but developed a discharging sinus with air leak from the thoracotomy wound after 1 month. the patient was bronchoscopically evaluated and was found to have a fistula at the stump of right upper lobe bronchus. the fistulous tract was delineated by contrast injection under fluoroscopy. in view of poor general condition, the patient was not a candidate for surgical closure and was offered device occlusion of the fistula with duct occluder device. it was decided that the approach to the site of bpf would be transcutaneous route and monitoring of the release of a pda duct occluder device will be done under bronchoscopy and fluoroscopy. the bpf was measured to be 8 mm in diameter and it was decided that the size of the occluder device will be increased to achieve good wall approximation and to avoid postdeployment migration. a extra- stiff wire 0.035 (cook medical, united states of america) wire was passed through the working channel of the bronchoscope under conscious sedation to gain access into the bpf opening and onwards to the pleural space and then the sinus track. the bronchoscope was withdrawn from the wire to be repositioned by the side of the wire for better visual control of the procedure. the wire was torqued to guide it along the sinus track till the skin wound and was made to exit by pull - and - push technique under local anesthesia. an 8f pda delivery sheath was passed transcutaneously over the wire and its tip was positioned in the bpf under fluoroscopic and bronchoscopic control. after removing the wire, nitinol pda device (lifetech, shenzhen, pr china) of size 10 mm (proximal waist), 8 mm (distal waist), 7 mm (length), and retention skirt of 2 mm on either side was selected and loaded into the delivery sheath. the device was pushed up to the proximal level of fistula and released under fluoroscopic and bronchoscopic control to assume the predetermined sizes and to occlude the fistula snugly in the shape of a mushroom [figure 1 ]. the bpf before and after the closure with duct occluder device in the right upper lobe bronchus the device position was confirmed and there was complete cessation of air leak and cough experienced by the patient earlier. patient was managed with broad spectrum antibiotics and anti - inflammatory drugs. on the fourth postprocedure day, the device was bronchoscopically checked and found in situ with good approximation of the device well - aerated right lower lobe with no clinical or radiological evidence of air leak or device migration [figure 2 ]. the cutaneous opening has completely healed, and the nutritional status and effort tolerance level of the patient have improved. post - pneumonectomy empyema (pne) occurs in approximately 5% of pneumonectomy patients, and bpf appears in more than 80% of cases. occasionally, small, uncomplicated bpfs may heal spontaneously, and in 20% of the patients, bpfs will close with drainage only. however, the remaining 80% of patients whose bpfs persist will require additional operative procedures. various surgical procedures such as direct repair, thoracoplasty, myoplasty, omental transposition, and completion pneumonectomy have been described. surgical option appears to provide the definite closure but not always, as in the case we encountered. endobronchial occlusion methods are now performed routinely with different bronchoscopes and a long list of embolic agents such as blood clots, gelatin, silicon rubber plugs, lead fishing sinkers, various sponge materials, tetracyclines, fibrin glue, cyanoacrylate, detachable balloons, metallic coils, stents, and septal occluder device with successful outcomes. in our case, we resorted to use an occluder device as bpf did not close in spite of conservative measures. we selected the nitinol duct occluder device and the transcutaneous route for its delivery as it was easier and more maneuverable. fruchter. have published their experience of bronchoscopic closure of bpfs using amplatzer devices with 5 f7 f delivery sheath under fluoroscopic visualization. the delivery sheath used by us was 8 f in size and was not compatible with the working channel of the bronchoscope. the occluder is shaped into a mushroom plug with a collar to secure the duct occluder in the stump of the right upper lobe bronchus. the transcutaneous delivery of device ensured that the larger proximal part is placed into the bronchus, thus preventing distal migration due to gravity into the pleura. to the best of our knowledge, this is the first report where pda device has been used transcutaneously to close the bpf. fruchter. deployed amplatzer devices bronchoscopically for the closure of bpf in patients without cutaneous fistula. in conclusion, this endobronchial technique seems to be safe and effective to manage large bpfs. some authors have reported the successful use of fibrin sealant in video - assisted thoracoscopic technique as well as in computer tomography (ct)-guided percutaneous transthoracic application to seal pulmonary air leaks but the drawback of these techniques is the need of an additional invasive procedure. we applied a simple endobronchial technique without general anesthesia, rigid bronchoscopy, thoracoscopy, or bronchography. if similar results are seen with ongoing experience, it may represent a major step forward in the management of a debilitating and difficult - to - treat complication after pulmonary resections in the resource - constrained settings. | bronchopleural fistula (bpf) is a well known complication of several pulmonary conditions posing challenging management problem and is often associated with high morbidity and mortality. though no consensus exists on a definite closure management algorithm, strategies for closure widely include various methods like tube thoracostomy with suction, open surgical closure, bronchoscopy directed glue, coiling and sealants which now also includes use of occlusion devices. we report a case in which a novel method of delivery and closure of recurrent post - operative broncho - pleuro - cutaneous fistula by a duct occluder device was done transcutaneously which has not been previously described in literature. |
turkey ear is a descriptive term only used in lupus pernio of earlobe which can occur as one of the skin manifestations of sarcoidosis. lv lesions are mostly reported on the head and neck region, rarely on earlobes. although the diagnosis of lv can be established according to the clinical, histological and bacteriological criteria, the most confusing clinical appearance with lupus vulgaris of earlobe is lupus pernio. a 65-year - old man presented with an asympthomatic erythematous and swelling plaque lesion on his right ear, which had been gradually progressing for 15 years. on examination, red - brown infiltrated two plaques were palpated on the earlobe and just above [figure 1 ]. the dorsum of the ear was particularly livid in color and atrophic cicatricial appearance was prominent [figure 2 ]. apple - jelly nodules were seen on diascopy and also fine - needle test was positive. there were no associated constitutional symptoms, history of previous trauma bacille calmette - gurin (bcg) vaccination or medical history of systemic tuberculosis. skin biopsy from the plaque lesion on hematoxylin and eosin stain revealed an atrophic epidermis overlying caseating tuberculoid granulomas, consisting of lympho - histiocytes, epitheloid cells and langhans giant cells in the papillary and upper reticular dermis [figure 3 ]. no acid - fast bacilli were demonstrated on ziehl - neelsen staining but the polymerase chain reaction (pcr) was positive for mycobacterial dna. the diagnosis of plaque form of lv was depending on clinical and histopathological findings and the patient was administered conventional antituberculous therapy of rifampin, isoniazid, pyrazinamide and ethambutol for 6 months. red brown infiltrated plaques on the right earlobe and just above atrophic cicatricial appearance on the dorsum of the right ear lympho - histiocytes, epitheloid cells and langhans giant cells in the papillary dermis (h and e stain, 200) the development of resistance to antitubercular drugs and the increase in diseases and conditions associated with immunodeficiency such as aids and chemotherapy have caused tuberculosis to increase recently. as a result lv is the most common morphological variant of cutaneous tuberculosis with an average prevalence of 0.37% among the general skin patients. it is usually reinfection tuberculosis of the skin, which originates from tuberculosis focus in the body, spreading by hematogenous, lymphatic or contagious way. however, in many cases the exact way in which lv develops is difficult to assess. classically well - demarcated soft reddish - brown papules or small plaques are often seen in the head and neck region. coloration on diascopic examination.[17 ] conventional morphological patterns of lv are papular, nodular, plaque, ulcerative, vegetating and tumid forms. unusual variants are frambesiform, gangrenous, ulcerovegetating, lichen simplex chronicus, myxomatous and sporotrichoid types. in the myxomatous form, huge soft tumors occur predominantly on the earlobes, which become grossly enlarged. because of its rarity over 80% of lesions are on the head and neck, particularly around the nose. lv lesions tend to be chronic, with new plaques appearing at the site of regressed atropic ones, and require appropriate therapy for complete healing the diagnosis of lv can be established according to a combination of clinical, histological and bacteriological criteria. lv is seen in individuals with moderate immunity and a high degree of tuberculin sensitivity. in addition, its diagnosis remains bothersome, because detecting mycobacteria in skin lesions using conventional laboratory examination remains difficult and cultures are often negative. concomitant diagnosis by both culture and detection of mycobacterial dna using pcr has been reported and may be useful where small numbers of mycobacteria are present. histopatologically, typical caseating necrotic granulomatous tubercule with langhans giant cells, epitheloid cells and mononuclear infiltrate can be seen. lv is treated by conventional antitubercular therapy consisting of rifampicin, isoniazid, pyrazinamide and ethambutol for 6 months. if not properly treated, it presents a progressive chronic development whose long- term complications include cutaneous neoplasms. lv is often confused with various cutaneous disorders and some other granulomatous processes of the skin as lupus pernio, tuberculoid leprosy, lupoid leishmaniasis, deep - seated mycosis, cutaneous lymphoma and even granuloma annulare.[1468 ] however, the most confusing diagnosis with lupus vulgaris of earlobe is lupus pernio. lupus pernio is a relatively common skin manifestation of sarcoidosis characterized by slowly progressive bluish - red or violaceous indurated plaques and nodules that usually affect the nose, cheeks, ears, fingers, hands and toes. williams. reported a case of lupus vulgaris, which had turkey ear appearance and suggested if turkey ear could be thought as a clinically descriptive term. in our case, localization and duration of the lesion were very typical for lv, in which the diagnosis was supported by tuberculin hypersensitivity and histopathological findings. atrophic cicatricial changes at the dorsum of the ear were also significant for lupus vulgaris. the clinical appearance was very similar to turkey ear, which was previously described once as a sign of lupus vulgaris. this is the second reported case of turkey ear as a manifestation of cutaneous tuberculosis. thus, the term turkey ear is supposed to be used not only for sarcoidosis but also for lupus vulgaris. thus, the term turkey ear may be also used for lupus vulgaris of earlobe. | lupus vulgaris is the most common morphological variant of cutaneous tuberculosis. classical lupus lesions are often seen in the head and neck region. turkey ear is a clinically descriptive term, previously being used for the earlobe with reddish indurated plaque lesions, which recently can be a sign for lupus vulgaris. a 65-year - old man presented with lupus vulgaris of the earlobe. the diagnosis was confirmed by conventional laboratory investigations and the patient showed well response to antituberculous therapy. this is the second reported case of turkey ear as a manifestation of cutaneous tuberculosis. |
permanent prostate brachytherapy using i for patients with low- or intermediate - risk prostate cancer is an effective treatment option, and can achieve high biochemical progression - free survival [1, 2 ]. however, there are some cases of locoregional recurrence. currently, there are few recommendations for a relapse of prostate cancer, except for palliative androgen deprivation therapy (adt). despite its antineoplastic activity on prostate cancer,, adt was found to be associated with cardiac sequelae, including coronary heart disease, myocardial infarction (mi), and sudden cardiac death. considering the site of recurrence in cases of subsequent biochemical relapse, the development of new therapeutic approaches (salvage re - irradiation) will be necessary. there are few reports on salvage re - irradiation using i permanent prostate brachytherapy for local recurrence after seed implantation. the purpose of this study was to evaluate the effectiveness of partial salvage re - implantation to develop an effective treatment option for local recurrence after permanent prostate brachytherapy. between january 2010 and september 2015, 12 patients were treated with partial salvage i brachytherapy for local recurrence after permanent prostate brachytherapy. the 12 patients underwent initial permanent prostatic implantation (monotherapy) using i radioactive seeds for low- or intermediate - risk prostate carcinoma at our institution between september 2004 and october 2008. eligibility criteria for focal partial salvage re - implantation were as follows : 1) biochemical failure was defined using the phoenix criteria (prostate - specific antigen [psa]-nadir + 2.0 ng / ml) ; 2) a systematic bilateral template - guided prostate / seminal vesicle trans - perineal mapping biopsy with a 5 mm sampling frame (more than 20 biopsies) was performed for patients with biochemical failure, and locoregional clinical failure was diagnosed by a positive template biopsy ; 3) pre - retreatment psa 144 gy was prescribed to the target area and was delineated on mri combining biopsy results. hsu. reported that the prescription dose for implants was 144 gy for i, 125 gy for pd, and 108 gy for i with imrt (40 gy). future investigations for prescription dose recommendations are required to estimate salvage re - implantation as a curable treatment. langley. reported that the f - ctv is delineated as the gross visible or clinically demonstrable location and the extent of the targeted cancer plus clinically insignificant disease ; it was expanded to create the focal ptv to compensate for uncertainties in image registration and treatment delivery, such as movement, and the focal ptv contours would be restricted to oar contours such as those of the urethra and rectum. reported that the application of a margin of 2 voxels (voxel size 2.5 2.5 2.5 mm ; 5 mm margin) to an mr - based tumor delineation in five patients with biopsy - proven prostate cancer (psa : 14 - 29 ng / ml ; pathological gleason : 6 - 8 ; pathological t stage : t2a - t3a) enables the coverage of approximately 85 - 100% of the tumor. sasaki. reported that the clinical target volume (ctv) was manually delineated as a region of local recurrence with biopsy positivity and initial under - dosage according to mri / initial ct dosimetry, and the ptv included the ctv plus a 5 mm margin (5 mm posterior margin). peters. reported that the treatment margins were expanded up to half of the prostate to account for uncertainties in the definition and delineation of the gross tumor volume (based on t2-weighted magnetic resonance imaging [t2w - mri ]), combining biopsy results and multi - parametric mri and the uncertainty in matching of the mri and ultrasound images. hsu. demonstrated mri - guided focal partial salvage permanent prostate implantation for local recurrent prostate cancer after initial implantation, and they defined the ctv as defined as an area of local recurrence with initial under - dosage. in the current study, f - ctv was not delineated on imaging modalities such as mri but rather on positive biopsy areas in a 5 mm mapping biopsy, combining the cold spots on post - implant dosimetry and taking into account microscopic carcinoma in the cold spot with a negative biopsy. we performed mri in 5 of 12 cases to delineate the recurrent gross tumor ; however, it was impossible to identify the location of the tumor in 5 cases. if the needle biopsy did not sufficiently cover the apex to the base of the prostate gland or the cold spot in initial brachytherapy located in only the apex or base side of the prostate, we divided the biopsy areas into two parts (apex and base sides) and detected the presence of malignancy in each part. the f - ctv will not necessarily include the cold spot with a negative biopsy, thus, it is difficult to conclude that our f - ctv is adequate. regarding the ptv margin size, the f - ctv was expanded by 3 mm to create the ptv as a margin to compensate for uncertainties in image registration and treatment delivery. in the re - implantation study, a 3 mm margin to both the positive biopsy areas and cold spots may also be a practical first step. without a systematic comparison of margin distances biochemical progression is due to either local recurrence or distant metastasis, and the psa doubling time was identified as a predictive factor of distant metastases [5, 6, 25 ]. demonstrated that, due to less time to biochemical recurrence / a shorter psa doubling time, some patients failed to reflect probable (micro) metastatic disease at the time of focal salvage [11, 12 ]. in the present study, one patient who had a shorter psa doubling time at the time point of partial salvage had psa progression after salvage treatment. a shorter psa doubling time may imply the existence of regional or distant recurrence, even if locoregional clinical failure was diagnosed by a positive biopsy, and no lymph node or distant metastases were diagnosed on pelvic - ct or bone scan. partial salvage re - implantation for patients with biochemical failure after permanent brachytherapy was effective and safe. a large clinical trial regarding such a treatment may be warranted. in that case, careful follow - up after re - irradiation is important, including prevention of constipation - reducing rectal toxicity, because a previous prospective phase ii study on whole prostate salvage ldr brachytherapy after ebrt or brachytherapy reported 30% grade 3 - 4 gi toxicities and 13% grade 3 - 4 gu toxicities. partial salvage low dose rate brachytherapy for local recurrence after permanent prostate brachytherapy is well tolerated, with high biochemical response rates. this treatment can be not only a method to delay chemical castration but also a curative treatment option in cases of local recurrence of prostate carcinoma after seed implantation. | purposeto investigate the treatment results for focal partial salvage re - implantation against local recurrence after permanent prostate brachytherapy.material and methodsbetween january 2010 and september 2015, 12 patients were treated with focal partial salvage re - implantation for local recurrence after low - dose - rate brachytherapy using 125i seeds. the focal clinical target volume (f - ctv) was delineated on positive biopsy areas in a mapping biopsy, combining the cold spots on the post - implant dosimetry for initial brachytherapy. the f - ctv was expanded by 3 mm to create the planning target volume (ptv) as a margin to compensate for uncertainties in image registration and treatment delivery. the prescribed dose to the ptv was 145 gy. the characteristics and biochemical disease - free survival (bdfs) rates were analyzed. genitourinary (gu) and gastrointestinal (gi) toxicities were evaluated using the common terminology criteria for adverse events version 4.resultsthe median prostate - specific antigen (psa) level at re - implantation was 4.09 ng / ml (range : 2.91 - 8.24 ng / ml). the median follow - up time was 56 months (range : 6 - 74 months). the median rd2cc and ud10 were 63 gy and 159 gy, respectively. the 4-year bdfs rate was 78%, which included non - responders. biochemical recurrence occurred in two patients after 7 and 31 months, respectively. the former was treated with hormonal therapy after biochemical failure, and the latter underwent watchful waiting (psa at the last follow - up of 53 months : 7.3 ng / ml) at the patient 's request. no patients had grade 3 gu / gi toxicities or died after salvage re-implantation.conclusionsthe partial salvage low - dose - rate brachytherapy used to treat local recurrence after permanent prostate brachytherapy is well - tolerated, with high biochemical response rates. this treatment can be not only a method to delay chemical castration but also a curative treatment option in cases of local recurrence of prostate carcinoma after seed implantation. |
the scapula is an important link between the trunk and the upper extremities and it also provides proximal stability for functional activity of the upper extremities2. a change in the scapular position and motion influences the change in muscle length attached to the scapula and eventually causes shoulder pathologies such as impingement1, 2. lin jj.3 reported that increased upper trapezius muscle activity is caused by scapular elevation and inferior angle tipping. another study showed that lower trapezius weakness is produced by an anterior tilt of the scapula4. the proper alignment and muscle balance of the scapula is essential for acquiring stability of the shoulder girdle and functional shoulder movement1. in recent reviews, the dynamic hug exercise was recommended for effective strengthening of the scapulothoracic musculature5, 6. we developed a multi air - cushion biofeedback device (mabd) for assisting the performance of the dynamic hug exercise. in the present study, we investigated the effects of mabd on shoulder muscle activities during the dynamic hug exercise. twelve males, aged 2232 years with a mean height and weight of 172.5 6.7 cm and 68.2 6.8 kg, respectively, participated in this study. the subjects had no history of musculoskeletal disorders or pain associated with the upper extremity in the past 6 months. emg signals were collected for 30 seconds, sent to the data acquisition unit of a mp150 system (biopack system, santa barbara, ca, usa), and expressed relative to the maximum voluntary contraction (mvc). the surface electrodes were attached to the right upper trapezius, right lower trapezius, and right serratus anterior muscles. we developed a multi air - cushion biofeedback device (mabd) for assisting the performance of the dynamic hug exercise. the mabd consists of three 15 10 cm air cushions and a 100 100 cm back support board with containing pressure detecting sensors (ap - series pressure sensor, keyence, japan). visual feedback was provided to each participant by linking the pressure sensors to display devices. the three air cushions were positioned over the left and right scapulas and the cervical spine and were attached to the back support board. the subjects were instructed to maintain the pressure based on the air pressure data of the initial standard leaning posture. the axis of the pulley was placed at the level of each subject 's acromion by inserting plastic plates under his feet. the subjects stood with their feet shoulder - width apart, elbows flexed at 90 and internally rotated at 90, and with the shoulders abducted at 90. the subjects pushed the handle using horizontal shoulder adduction and elbow extension6. the statistical package (spss, chicago, il, usa) was used to conduct paired t - tests to analyze the significance of differences in the dynamic hug exercise with and without mabd. the serratus anterior muscle activity significantly increased during the dynamic hug exercise performed with mabd (39.815.9%) compared to without mabd (33.216.0%) (p<0.05). the lower trapezius muscle activity significantly increased during the dynamic hug exercise performed with mabd (28.89.2%) compared to without mabd (19.012.2%) (p<0.05). the upper trapezius muscle significantly decreased during the dynamic hug exercise performed with mabd (18.18.8%) compared to without mabd (26.5 9.0%) (p<0.05). we investigated the effects of mabd on the shoulder muscles during the dynamic hug exercise. the serratus anterior and lower trapezius muscles activities significantly increased during the dynamic hug exercise with mabd. the serratus anterior and lower trapezius muscles are the major scapulothoracic muscles associated with normal scapular alignment and functional stability and mobility1, 5. previous researchers have suggested exercises for selective activation of the serratus anterior and lower trapezius for therapeutic assessment of shoulder rehabilitation4,5,6. decker.6 described the dynamic hug as a combined movement involving horizontal shoulder adduction and scapular protraction. in recent reviews, the dynamic hug exercise was recommended for strengthening the scapulothoracic musculature5, 6. biofeedback, in particular, has been reported to be an effective intervention for re - educating posture and reducing altered activation of the upper trapezius muscles7. in the present study, the upper trapezius muscle activity significantly decreased during the dynamic hug exercise performed with the mabd compared to the same exercise without mabd. higher activation of the upper trapezius has been observed in patients with shoulder disorders, and previous research has suggested that inhibition of the upper trapezius effectively restores the normal pattern of the scapulothoracic muscles8. the mabd changes the dynamic hug exercise, which is an open chain exercise, to a closed chain exercise. tucker.9 investigated scapular muscle activities in closed kinetic chain exercises and showed that these could affect the scapular muscle activities in symptomatic subjects. the dynamic hug exercise with mabd also provides a labile support for the left and right scapular and cervical spine. recently, de oliveira.10 suggested that a labile surface enhances the activities of the scapulothoracic muscles. therefore, our opinion is that performance of the dynamic hug exercise the mabd would help to improve scapular stability during the dynamic hug exercise. | [purpose ] we developed a multi - air - cushion biofeedback device (mabd) to assist the dynamic hug exercise, and investigated the effects of mabd on the shoulder muscles during the dynamic hug exercise. [subjects ] twelve males aged 2232 years were recruited. [methods ] we measured the right side serratus anterior, lower trapezius, and upper trapezius muscle activities during the dynamic hug exercise with and without mabd. [results ] the serratus anterior and lower trapezius muscles activities significantly increased during the dynamic hug exercise with mabd compared to without it. the upper trapezius muscle significantly decreased during the dynamic hug exercise with mabd compared to without it. [conclusion ] the results suggest that the dynamic hug exercise with mabd is an effective scapular stability exercise. |
you are working in the intensive care unit (icu) and you get a call from the infection control nurse, who tells you that one of your newly admitted icu patients has tested positive on a nasal swab for methicillin - resistant staphylococcus aureus (mrsa). the nurse informs you that it is essential that the patient be moved to a single room, the door be closed, and entrance into the room be monitored. those entering should wear a gown and gloves, and disinfect their hands on entry and exit. you worry about the impact of these procedures on the patient 's care and wonder whether they are really that important. to minimize nosocomial mrsa transmission and thereby nosocomial mrsa infection rates, isolation appears to be essential in patients colonized with mrsa. this seems important because mrsa infections have been associated with significantly greater prolongation of hospital stay and greater mortality than methicillin - susceptible s aureus infections, after adjustment for patients ' underlying severity of illness. many studies have reported significantly better control using surveillance cultures and contact precautions, including multiple studies in icus ; the consistency of evidence in different studies conducted by different investigators and in different populations was one of the criteria for causal inference advocated by hill. high strength of association, reversibility, a dose response relation, and specificity have also been demonstrated, and these features also suggest causality. although most of those studies used historical controls, multiple studies have used concurrent controls. to date, all cost - effectiveness studies have concluded that it is less expensive to pay for detection and control than it is to use inadequate measures and let mrsa spread. randomized controlled trials (rcts) are usually optimal for demonstrating reversibility with an intervention because they minimize selection bias (i.e. they ensure that patients cared for using an intervention are similar to controls), but such studies have not been conducted regarding the surveillance cultures / contact precautions approach to mrsa control. first, the us centers for disease control and prevention have recommended since 1983 that colonized patients be cared for with contact precautions, and there was a lack of support from the us national institutes of health or the centers for disease control and prevention for such studies from 1970 to 2000. also, many studies have reported control using surveillance cultures / contact precautions, and there are ethical concerns about randomizing controls to what many studies suggest may be suboptimal protection against potentially lethal infection. finally, with respect to cost, rcts are in general expensive, and the expense is greater still for rcts that must take account of widespread intrafacility and interfacility transmission by making large facility clusters the unit of randomization. recent meta - analyses of rcts found that their results showed as much and sometimes more variability than did those from unrandomized studies examining the same question, which neither overestimated nor underestimated the central tendency of the rct results. a recent, unrandomized study reported a statistically significant increase (i.e. 31 versus 15 per thousand patient - days) in some adverse effects among mrsa isolation patients (mostly falls, pressure sores, and fluid / electrolyte disorders), but no increase in numerous other types of adverse effects or deaths. far more studies have demonstrated the adverse effects of inadequate isolation for important nosocomial infections (e.g. one mrsa neonatal icu outbreak continued for 51 months, resulting in 75 mrsa bacteremias and 14 deaths). the worldwide emergence of virulent mec iv strains of mrsa that are able to spread in the community as well as in hospitals has led some to believe that it is no longer worthwhile trying to contain nosocomial mrsa spread. recognized for almost a decade, mec iv strains have not yet resulted in population - based studies demonstrating a high mrsa national prevalence in any country, nor has their documented presence in northern europe (where surveillance cultures / contact precautions are used routinely) resulted in failure to control nosocomial mrsa infections to very low levels. this suggests that control of nosocomial mrsa infections is still possible and as important as it ever was. to reduce the spread of mrsa, i agree that universal precautions, including gloves, hand - washing, disposable aprons, cleaning equipment and the environment, and regular surveillance cultures, are important and all are practiced in our icu. i question, however, the validity of isolation or cohort nursing to further prevent mrsa transmission, and whether this a safe strategy in the critically ill. numerous articles have proposed isolation / cohort nursing in addition to universal precautions [11 - 13 ], and many concluded that isolation reduces mrsa transmission. when closely examined, however, isolation / cohort nursing were in all but one case introduced as part of a varied package of measures, including closure of units, surveillance, re - emphasis on hand washing, reduction in overcrowding, infection control nurses, and addition of other treatments. a recent review of 46 studies of isolation policies in the management of mrsa by cooper and coworkers concluded that it was usually impossible to adjust for any variation in mrsa reservoir in different phases of these studies and most lacked proper statistical analysis. furthermore, few studies were prospective, and many seemingly prospective studies occurred in response to new high mrsa levels. of those that provided the strongest evidence, four suggested that infection control measures, including isolation, were effective whereas two implied that isolation failed to prevent endemic mrsa. mathematical models also propose that isolation should be effective, but where infection is endemic they show that, despite effective control measures, the status quo can be maintained by new admissions. this suggests that the prevalence of mrsa is an important part of the equation. across the world this varies enormously, with 60% of icus in germany reporting no mrsa, contrasting with 11.4% acquiring mrsa in icu in australia and a point prevalence of 16.2% in uk icus [16 - 18 ]. not only is the evidence weak that isolation is effective, but also there is evidence that it may be detrimental. evans and coworkers observed that, despite isolated patients being more dependent than non - isolated ones, they had less contact time with clinicians. furthermore, isolation frequently necessitates moving critically ill patients, with well recognized associated risks. in view of this we recently conducted a study in two london icus to determine prospectively the effect of removing isolation / cohort nursing on mrsa transmission. for the following 6 months mrsa - positive patients were not moved and then for the final 3 months patients were again isolated. the primary outcome was time to acquisition of mrsa, and the cox proportional hazards model showed no evidence of increased transmission associated with non - isolation, and neither was there any increase in individual hospitals, even after adjusting for colonization pressure. hence, i believe that where the background incidence of mrsa is high there is no evidence that isolating mrsa - positive patients reduces cross - infection, and it may indeed restrict patient care inappropriately. mrsa infections have been more deadly than nosocomial mssa infections, mrsa is usually acquired only by spread, and the vast majority of studies have shown significantly better mrsa control with surveillance cultures and contact isolation than with standard precautions. (this includes the review by cooper and coworkers, cited prominently by dr bellingan, which noted studies ' methodologic shortcomings but concluded that surveillance cultures and isolation work and should be used.) without mentioning statistical power, the proportion excluded because their stay in the icu was shorter than 48 hours, and the proportion refusing consent to participate, dr bellingan cites his own negative, unpublished and unrandomized, historical comparison study, which does little to counterbalance scores of studies showing the opposite. effective infection control policies for mrsa, including active surveillance and contact precautions, are essential. what i question is whether this can be achieved through rigorous adherence to universal precautions alone or whether we also must physically isolate icu patients, with attendant risks of reduced carer input and increased adverse events. no studies have, until now, specifically addressed the efficacy of single room isolation, and our study throws new light on current recommendations. it highlights the continued importance of universal precautions while suggesting single room isolation confers no additional benefit. icu = intensive care unit ; mrsa = methicillin - resistant staphylococcus aureus ; rct = randomized controlled trial. | antibiotic - resistant bacteria are an increasingly common problem in intensive care units (icus), and they are capable of impacting on patient outcome, the icu 's budget and bed availability. this issue, coupled with recent outbreaks of illnesses that pose a direct risk to icu staff (such as sars [severe acute respiratory syndrome ]), has led to renewed emphasis on infection control measures and practitioners in the icu. infection control measures frequently cause clinicians to practice in a more time consuming way. as a result it is not surprising that ensuring compliance with these measures is not always easy, particularly when their benefit is not immediately obvious. in this issue of critical care, two experts face off over the need to isolate patients infected with methicillin - resistant staphylococcus aureus. |
stool samples from 311 children hospitalized with acute gastroenteritis at sheba medical center, tel hashomer, israel, during may 15, 2005may 15, 2006, were suspended in saline. rotavirus dipsticks (hy laboratories ltd, rehovot, israel) were used to identify rotavirus - positive stool samples. (for comparison with rotavirus elisa with dako reagents [dako diagnostics ltd, glostrup, denmark ], diagnostic dipsticks from hy - laboratories and novamed [novamed, jerusalem, israel ] had sensitivities of 98.5% 3% and 92.3%, and specificities of 92.5% 1% and 93.5%, respectively [l.m. shulman., unpub data ].) the section of the dipstick containing the rotavirus - specific bands from 14 rotavirus - positive dipsticks that had been stored at room temperature for at least 1 week were excised and placed into 120 l saline. rotaviral rna was extracted from these rotavirus - specific bands and from the corresponding saline stool suspensions by using qiamp viral rna mini kits (qiagen, valencia, ca, usa). rna amplified from rotavirus - specific bands from dipsticks archived at room temperature 5 years earlier was similarly extracted. pcr (rt - pcr) (qiagen onestep rt - pcr kits, qiagen) followed by heminested pcr (amplitaq gold, applied biosystems, foster city, ca, usa) with genotype - specific primers to identify g and p genotypes as described by gouvea. rt - pcr products were gel purified (qiaquick gel extraction kit, qiagen) or purified directly (hipure pcr product purification kit ; roche applied science, indianapolis, in, usa) from the pcr mix and sequenced by using an abi prism dye deoxy terminator cycle sequencing kit (applied biosystems) and the genotype - specific primer and common primer from the heminested pcr reaction described. reaction mixtures were analyzed on applied biosystems model 373 dna automatic sequencing system to confirm the rt - pcr genotyping results and for phylogenetic analysis. sequences of the same genotype were truncated to the longest common length by using the sequencher program (genecodes, ann arbor, michigan, usa). nearest - neighbor phylogenetic trees were constructed from these sequences after the data were bootstrapped 1,000 times with clustal x (13). sequences have been deposited in european molecular biology laboratory / genbank / dna data bank of japan under accession nos. this study was approved by the institutional review board of chaim sheba medical center (smc-7606 - 09). all personal identification was removed from the remnants of fecal samples sent to the national center for viral gastroenteritis for rotavirus analysis. the viral rna used for this study to assess the suitability of rna extracted from dipsticks for determining the vp7 genotype, we first determined the vp7 g genotype of rotavirus in rotavirus - positive clinical samples from children hospitalized during may 15, 2005may 15, 2006. of 311 stool samples, 61 were determined to be rotavirus positive by using diagnostic rotavirus dipsticks. the g genotype and p genotype of rna in 54 of the 61 samples was determined by the size of the heminested pcr amplification product as described. mixed infections, e.g., > 1 g (6 samples) and p genotypes (2 samples) in the same fecal suspension, were found in 8 (15%) of the 54 positive samples. taking into account mixed infections, we found g1, g2, g3, and g9 g genotypes in 79%, 5%, 2%, and 15%, respectively, of the 62 rotaviruses identified in the 54 clinical samples. the frequencies of associated g and p genotypes for isolates from the 20052006 rotavirus season were 3% g1p, 76% g1p, 5% g2p, 2% g3p, 3% g9p, and 11% g9p. we chose 18 rotavirus - positive clinical samples with which to study the feasibility of recovering molecular information from dipsticks. thirteen were from children infected with 1 g genotype of rotavirus and 5 from children simultaneously infected with rotaviruses belonging to 2 different g genotypes. of the 13 infections by a single g genotype, 8 were g1, 3 were g2, and 1 each were g3 and g9. all 5 mixed infections, rov-24_isr05, rov_41_isr05, rov_56_isr06, rov_57_isr06, and rov_60_isr06, were simultaneous infections with g1 and g9 genotypes. mixed infections were identified by specific product size on gels and confirmed by partial sequencing of the g1 product for all 4 patients whose g9 sequences appear in the figure. neighbor - joining phylogenetic trees for viral protein (vp) 7 g1, g2, g3, and g9 genotypes of hospitalized children in israel, including sequences recovered from archived rotavirus dipsticks. representative isolates for lineages and sublineages of vp7 genotypes g1 (a), g2 (b), g3 (c), and g9 (d) were chosen from phan. (18), respectively, and the sequences were downloaded from the european molecular biology laboratory / genbank / dna data bank of japan. these sequences were aligned with israeli sequences by using the sequencher program (genecodes, ann arbor, mi, usa) and truncated to the longest segment common to all sequences in the alignment ; 515 nt for g1, 547 nt for g2, 274 nt for g3, and 207 nt for g9. each of the 4 phylogenetic trees was prepared by using clustalx (13) for data bootstrapped 1,000 and was analyzed with njplot (14). the genotype, lineage, and, where relevant, sublineage of each isolate appears in brackets after the name of the isolate : for example, ky3303[g2.2a ] is vp7 genotype g 2, lineage 2, sublineage a for isolate ky3303. a letter at the end of the name of the israeli sequences indicates the source of the rna (d for dipstick or s for fecal suspension). rna was extracted from rotavirus - positive bands of dipsticks from the 18 clinical samples. the quality of the rna from all 18 extractions was sufficient to enable determination of vp7 g genotypes by heminested rt - pcr. the genotypes obtained from the rna extracted from dipsticks were identical to those from rna extracted from corresponding saline suspensions for 14 of the 18 rotavirus - positive stool samples, including 3 samples from children with dual infections. in 2 mixed g1g9 infections, g9, but not g1 rna, was recovered, and g1 rna was recovered from dipstick rna in addition to g9 from a g9-infected child. a fourth dipstick from a g3 sample yielded a g3 and an equivocal g1 that did not appear in subsequent replicate heminested amplifications. in other words, 21 (92%) of 23 genotypes identified from rna extracted from 18 fecal suspensions were also identified from rna extracted from dipsticks, including 8 of 10 genotypes from 5 samples simultaneously infected with rotaviruses of 2 different genotypes. rotaviral vp7 rna suitable for genotyping was also recovered from all 5 air - dried rotavirus - positive dipsticks that had been stored at room temperature for 5 years. the g genotypes of the rotavirus in the fecal suspensions from the 5 archived dipsticks from 2002 were unknown, and the suspensions were no longer available for comparison. one of the dipsticks was manufactured by hy laboratories ; the remaining 4 were manufactured by novamed. initial amplification with generic g - type primers yielded generic g genotype amplification products of expected size (1,062 nt) upon gel electrophoresis. three of the dipsticks yielded g3 rna ; the other 2 yielded g1 rna. as proof of concept that p genotypes could be determined in addition to g genotypes, the p genotypes of rna recovered from 2 of the 5-year - old rotavirus dipsticks with g3 rna were also determined by direct sequencing of the rt - pcr products by using the generic p - type primers. both were p by sequence analysis (table a1). the g genotypes of 3 g1s, 2 g2s, 1 g3, and 1 g9 rna extracted from dipsticks were confirmed by sequencing their heminested amplification products. moreover, the sequences of the rna extracted from each of the 7 dipsticks were identical to the sequence of the rna extracted directly from the corresponding stool suspension. the lineages and sublineages of these and other isolates from the 200506 season were inferred from phylogenetic comparisons to equivalent segments of the 11 lineages of g1, the 2 lineages of g2, the 4 lineages of g3, and the 6 lineages of g9 described by phan. the g3 phylogenetic tree also includes 3 sequences obtained from the 5-year - old dipsticks and 4 g3 isolates from among those with highest homology to g3 isolates from israel identified in blast searches (http://blast.ncbi.nlm.nih.gov/blast.cgi) (19). specifically, all vp7 sequences for a given g genotype were aligned and truncated to the longest common segment. four separate phylogenetic trees (figure) were constructed because of the size difference between g genotype specific heminested amplification products. the lineage or sublineage of each isolate from israel (table a) was inferred from the relative difference of each sequence compared with equivalent segments of previously determined lineages. for each genotype, a new lineage or sublineage was suggested when the sequence from israel failed to closely group with the equivalent segment of > 1 reference strains and the nucleotide differences between the strain from israel and the reference strains were similar or greater than the differences among reference lineages and sublineages. the existence of such new lineages and sublineages would need to be confirmed with longer full - length open reading frame sequences. finally, sequences from rna recovered from dipsticks and/or from saline suspensions were used in blast searches to identify the most similar contemporary sequences. g1 isolates were most similar to isolates from europe, the far east, and south america ; g2 isolates to isolates from the far east ; and g3 isolates to isolates from europe and the far east (figure). g9 isolates were most similar to isolates from africa, europe, and the far east. the g1p genotype predominated in central israel during the 200506 rotavirus season as it did throughout israel during 19911994 (20) and in northern israel during 20072009 (2,10). for example, g4p, absent during 20052008, was present in 32.3% of samples a decade earlier and reappeared in 200809 (2). conversely, g9 genotypes, absent a decade earlier, were present in 15.7% of the 200506 isolates and 9.3% of the 200607 isolates. this substantial prevalence of g9 mirrors the global emergence of g9 among hospitalized children in the mid-1990s (21,22). in addition, double infections indicated by > 1 g or p genotype rose from < 2% during 19911994 (20) to 16.4% for the isolates in this study and 27% during 200708 (2). as indicated by muhsen (2), evidence is good for rotavirus reassortants emerging in vivo from appropriate double infections. phylogenetic analysis in the present study suggested that some g2 and g9 rotaviruses from israel belonged to new sublineages or lineages, respectively. in conclusion, air - dried affinity - concentrated virus from rotavirus - positive bands of dipsticks yielded rna suitable for g genotyping and sequencing, even for dipsticks stored at room temperature for 5 years. even though p genotyping and sequencing was performed only on rna isolated from 2 of the 5-year old dipsticks, it is reasonable to assume that p genotyping and sequencing would have been possible for the rest based on the equivalent quality of the rna extracted from the dipsticks and rna extracted from stool suspensions. thus, dipsticks can be used for recovery of molecular epidemiologic data by reference laboratories from diagnostic tests routinely performed in many clinical laboratories and point - of - care facilities. rna extracted from these archived dipsticks will enable centralized laboratories to easily recover epidemiologically useful data from the samples of other laboratories and from regions lacking adequate laboratory facilities. in addition, centralized laboratories will be able to assess whether the introduction of universal rotavirus vaccination changed the distribution of rotavirus genotypes associated with severe rotavirus - associated acute gastroenteritis. | genotyping circulating rotaviruses before and after introduction of rotavirus vaccine is useful for evaluating vaccine - associated changes in genotype distribution. we determined frequency of rotavirus genotypes among 61 rotavirus - positive children hospitalized in israel during the 200506 rotavirus season. accurate molecular epidemiologic data were recovered from affinity - concentrated rotavirus immobilized in rotavirus - positive bands from air - dried, diagnostic rotavirus rapid test strips (dipstick) stored at room temperature from 1 week to 5 years. g genotypes were identical for 21 paired dipsticks and suspensions, whereas dipsticks or suspensions detected an additional g genotype in 2 samples. rna sequences from 7 pairs were identical. phylogenetic analysis suggested previously unreported g2 sublineages and g9 lineages. the ease with which dipsticks can be stored at local facilities and transported to central reference laboratories can reverse increasing difficulties in obtaining geographically representative stool samples and expand surveillance to regions lacking adequate laboratory facilities. |
the survival and success of dental implants have been found to be influenced by various factors. the factors such as bone quality and quantity, primary stability of implant, implant surface characteristics are few of the factors, which influence osseointegration of the implant. however, the long - term success of implants is determined by factors such as the bone quality, type of prosthesis, occlusal loading, oral hygiene, overlying soft tissue, and regularity of recall visits. the stability of the marginal bone levels has long been used as a determining factor while describing the survival and success of dental implants. bone loss of 0.51 mm in the 1 year postrestoration was considered as physiological and was attributed to the bone remodeling that happens after implant placement surgery, placement of restoration, and initial occlusal loading of the restoration along with the implant design and surface. the role of the peri - implant mucosa as one of the factors determining the stability of marginal bone levels has received little attention as compared to various other factors that have been studied. cochran. stated a need of 3 mm of peri - implant mucosa for a stable epithelial, connective tissue attachment. stated that the biologic width around implants serves as a protective mechanism for the underlying bone. evaluated the formation of biologic width around dental implants and concluded that excision of overlying soft tissue leads to a compensatory bone resorption meaning that the amount of soft tissue thickness has to be re - established irrespective of sacrificing the bone levels. most of the studies assessing the impact of the soft tissue on the marginal bone levels have evaluated the width of the attached mucosa, measured as the distance from the free mucosal margin to the muco - gingival junction. however, the change in soft tissue thickness, which describes the bulk of the tissues as measured from the surface of the mucosa to the body of the implant / abutment has not been evaluated and so has been its effect on the marginal bone levels around implants. this study was thus conducted to evaluate the change in soft tissue thickness around the dental implants and co - relate the same with the marginal bone levels over a 1-year postrestorative period. the study was conducted in the department of oral implantology and periodontics after getting the approval from the institutional review board. patient inclusion criteria were : patients willing to participate in the study and give a written informed consent, patients requiring placement of implants for replacement of missing teeth, nonsmokers, patients with good general health, patients not on any medications that would influence the gingival status. all the patients to have a well - healed partially edentulous site and a healthy overlying mucosa., each variable was assessed to evaluate if the parameters were normally distributed and parametric statistical tests could be applied. as the variables appeared to be normally distributed, frequencies were calculated, and thus the sample size was determined. a total of 22 patients (10 males and 12 females) were enrolled in this study and received a total of 36 implants. all the implants were placed by the same operator (sk) and were in the range of 3.754.2 mm diameter. the implants were placed with a conventional protocol, that is, submerged placement at the marginal level in well - healed sites at a minimum of 3 months after extraction. the second stage procedure was done at 3 months after implant placement and 15 days later the final impressions were made. all the abutments had a finish line set at 1.5 mm from the implant mouth. the following parameters were evaluated : (1) mucosal thickness, (2) radiographic bone level. these parameters were evaluated by a single examiner who was trained to measure the mucosal thickness and the radiographic bone levels. at the implant site, local anesthetic was administered after which the tissue thickness was measured using an endodontic reamer (size 20-yellow) with a stopper [figure 1 ]. the thickness of the tissues was determined by the depth of penetration of the reamer from the external surface of the mucosa to the point where bony resistance could be felt. the stopper was then adjusted, and the depth of penetration / thickness was measured in millimeters on a geometric scale / ruler. the measurement of soft tissue thickness the measurements were done at three points on the crest of the edentulous ridge namely mesial, mid and distal, and on the buccal surface of the ridge at a point 3 mm apical the crestal measurements and 1 mm coronal to the muco - gingival junction. sites with a mucosal thickness of 2.0 mm or more at the baseline, that is, at implant placement were categorized as a thick biotype case group and those 0.6) thus reproducibility of the measurements through the study was ensured. a value of 2 mm of soft tissue thickness at the time of implant placement was used as the benchmark to group the patients into thick and thin biotype cases. in a study similar to the one used in the study had a soft tissue thickness around natural teeth ranged from 0.56 mm to 1.02 mm. as the edentulous ridges were evaluated in the study, mucosal thickness measured was thicker than around natural teeth. a reduction in the tissue in both the thick and thin biotype patients from the time of implant placement to restoration stage and from the second stage to restoration was observed and a subsequent increase in the thickness was observed after the placement of the final restorations. the reduction in thickness till cementation and the concomitant increase later is attributable to the surgical intervention at implant placement as well at implant uncovering at second stage and to the formation and organization of the supramarginal connective tissue, morphology of the peri - implant mucosa, and establishment of the biologic width around the implants, respectively. the thicker biotype cases showed a lesser reduction in thickness from placement to restoration and higher rebound from restoration to the 1-year follow - up, as compared to the thin biotype cases. this is due to the higher amount of the connective tissue and vascularity in thicker as compared to the thin biotype tissues, and thus the ability of these tissues to re - organize is better than thinner tissues. this study also evaluated the effect of tissue thickness change on the marginal bone levels as the stability of the marginal bone levels has been used as a benchmark for implant success. the bone levels were measured on serial radiographs taken throughout the course of study, where the measurements were carried out from the implant shoulder to the first thread exposed. bone loss was observed in both the thick and the thin biotype cases, over the duration of the study. this bone loss happening around the implants can be divided into two parts, the first being the one occurring from the time of implant placement to time of restoration and second one from the time of restoration to the end of the follow - up period. the bone loss from the baseline to the period of first 6 months of implant placement can be attributed to the surgical trauma and bone remodeling that happens soon after the implant placement. the bone loss that was observed from 6 months to 12 months postcementation, can be attributed to various factors such as stress at the marginal bone which may cause micro - fracture or overload, or even the biomechanical adaptation of the bone to the occlusal load resulting in marginal bone loss in the 1 year in function. the amount of bone loss was more in thin biotype cases as compared to thick. the difference between the two biotypes can be attributed to the fact that the thick tissues formed the biologic width by proliferating laterally or coronally, which is unlike to that observed in thin biotype cases wherein the bone around the implants underwent remodeling to accommodate the soft tissue biologic width. the present study was carried out only for a period of 1-year, and it can be hoped that marginal bone levels would stabilize at the end of 1-year post cementation and achieve a however, it should be kept in mind that ongoing marginal bone loss is a factor affecting the outcome of implant treatment. the peri - implant sulcus forms after the second stage procedure and is due to the formation and stabilization and organization and maturation of the connective tissue fibers of the overlying soft tissue, and depending on the nature of the overlying soft tissue, resorption of the bone at the crest may occur to accommodate the biologic width around the implants. though the probing depth around dental implants is influenced by various factors such as probing force and angulation, probe tip diameter, roughness of the implant type of abutment / restoration, inflammatory state of the periodontium and firmness of the marginal tissues, and access to probing, deeper probing depth often indicate bone loss. an increase in the probing pocket depth in both the biotypes from the time of restoration to 1-year was observed in the study. this can also be related to the bone loss occurring to re - establish the biologic width, coronal movement of the marginal tissue due to an increase in the thickness or a combination of both. in the present study, the probing pocket depth roughly co - related to the combined values of the mucosal thickness and bone loss in both the thick and thin biotype cases (thick biotype probing pocket depth of 3.39 mm 0.59 mm = soft tissue thickness of 3.22 mm 0.22 mm, + bone loss of 0.61 mm 0.36 mm, thin biotype probing pocket depth of 3.38 0.56 = soft tissue thickness of 1.59 mm 0.22 mm and bone loss of 1.70 mm 0.36 mm). thus, it can be concluded that the soft tissue undergoes a change in its thickness from implant placement to restoration and till 1-year postrestoration. the changes are more pronounced in thin biotype cases as compared to the thick biotype ones, and these changes also influence the marginal bone levels around the implants. | background : the peri - implant mucosa undergoes surgical and bacterial assaults in various stages of implant therapy, however, the literature on changes occurring in the peri - implant mucosa is minimal. this study was thus conducted to evaluate the change in the peri - implant mucosal thickness and its effect on the marginal bone levels around dental implants treated in a conventional two - stage implant therapy.materials and methods : a total of 36 implants were placed in 22 subjects. two subjects dropped out. thirty - three implants in 20 subjects were then evaluated. initial mucosal thickness, marginal bone levels on radiographs, pain, and exudation were evaluated. all these parameters were recorded at the time of implant placement, at the time of cementation of final restoration, 6 months and 12 months post cementation / restoration.results : the peri - implant mucosal thickness reduced from implant placement to second stage and till restorations and was statistically significant, in both the thick and thin biotypes, however, at 12 months there was a rebound of the tissue thickness, which was more in the thick biotype (p < 0.05). at 1-year follow - up, there was a reduction in the marginal bone levels, which was more in the thick biotype as compared to the thin biotype (p < 0.05).conclusion : the mucosa at implant sites undergoes a reduction in thickness from the time of implant placement till the placement of final restorations. the placement of the final restorations and then end of active therapy leads to a rebound of the tissue thickness. sites with thicker tissues preoperatively have a lesser bone loss and better rebound as compared to thinner tissues. |
upper urinary tract urothelial cancer (uut uc) is a rare lesion accounting for approximately 10% of all renal and 5% of all urothelial tumours [1, 2, 3 ]. there is ongoing debate whether multifocal presenation of urothelial cancer (uc) suggests panurothelial (wide spread) cancerous changes in the upper and lower urinary tract. previous / synchronous bladder cancer appears to have a significant effect on the recurrence free rate and outcomes following the treatment of uut uc [4, 5, 6 ]. there is an association of bladder cancer (bc) with ureteric and multifocal uut uc. liang suggested that previous or concomitant non - muscle invasive bladder cancer was a significant risk factor of high grade uut uc confirmed in a nephroureterectomy specimen. the presence of more advanced uut uc has been reported if concomitant bc is confirmed. synchronous bc does not influence cancer - specific survival following treatment of uut uc. it does however increase the risk of intravesicle recurrences [8, 9 ]. it is therefore essential to perform a panurothelial endoscopy in patients under surveillance following treatment of uut uc. although bc is one on the most important predictors of uut uc treatment outcome, its detection relies on white light cystoscopy. to increase detection, the european association of urology (eau) guidelines recommended pdd cystoscopy if cis or high grade bc is suspected. the use of pdd cystoscopy is advised as part of the routine inpatient endoscopic surveillance to confirm the efficacy of treatment and to identify any previously missed or recurrent tumours. the principle of pdd is the interaction between light and a fluorophore such as protoporphyrin ix, which has high accumulation in tumour cells. the light is absorbed by the fluorophore and then re - emitted at a longer wave length which could then be easily detected [13, 14, 15 ]. two photosensitizers have been used for the photodynamic diagnosis of bladder cancer : 5 aminolevulinic acid (5-ala) and its ester, hexyl aminolevulinate (hal). both agents have been found to be similar in terms of sensitivity and specificity [13, 16 ]. when investigating possible bladder tumours the photosensitizer is usually instilled into the bladder directly, however, this can be time consuming. this method only allows the use of pdd in the bladder whereas the use of systemic (oral) photosensitizer is needed when investigating the upper tract. we have previously demonstrated our success in investigating uut uc with 5-ala given orally 34 h before the planned ureterorenoscopy [17, 18 ]. therefore, we believe it is feasible to detect synchronous multifocal upper and lower urinary tract tumours using this method. white light cystoscopy, a standard tool in the assessment of the bladder at the time of primary diagnosis of uut uc, remains insufficient to depict all urothelial lesions within the bladder. photodynamic diagnosis is acknowledged to achieve a better bladder mucosal visualisation and improves the detection of exophytic lesions by 20% and carcinoma in situ (cis) by 39%. the aim of the study is to assess the detection rate of concomitant bc during photodynamic diagnostic ureterorenoscopy. we investigated the diagnostic accuracy of detecting bladder tumours concurrently and / or incidentally found during diagnosis and surveillance of patients with suspected / proven uut uc using oral 5-ala. retrospective review of all patients who underwent photodynamic diagnostic ureterorenoscopy (pdd - furs) was carried out. between july 2009 and june 2013, sixty nine patients underwent bladder biopsies at the time of pdd endoscopic inspection of uut. twenty five underwent initial diagnostic ureterorenoscopy and forty four had surveillance ureterorenoscopy for uut uc. cytological assessment of urine sampled from the bladder is of questionable value in the detection of uut uc. patients demographics uc urothelial cancer ; cis carcinoma in situ caldicott approval was granted and the lead consultant (collecting data) was registered with the national information commissioner s office. each patient received 20 mg / kg body weight of oral 5-ala (medac, scion house, stirling university innovation park, stirling, uk) dissolved in 50 ml of water, 34 hours before surgery. for palatability, the mixture was further added to 100 ml of juice. each procedure was started with rigid cystoscopy under standard white light followed by blue light endoscopy. after an assessment of the lower urinary tract, the flexible ureterorenoscopy (furs) was performed with the similar pattern of white light and blue light inspection. all suspicious bladder lesions where biopsied and all tumours were treated regardless of the white or blue light use. biopsies were taken from small lesions first to avoid being missed due to photobleaching effect. d - light system (karl storz, tuttlingen, germany) was used to detect fluorescence (olympus pdd cystoscope with 30-degree telescope and karl storz pdd flex - x ureterorenoscope). special fluorescence excitation light source (d - light - c, karl storz gmbh & co. kg, tuttlingen, germany) was used with a protoporphyrin ix excitation filter permitting the blue - violet light (380430 nm) and a fluid light cable (495 fs, karl storz gmbh & co. kg, tuttlingen, germany) to ensure a much higher transmission, mainly in the blue spectral range in comparison to a standard glass fiber bundle. an ocular of cystoscope / ureterorenoscope has a protoporphyrin ix cut - off filter (long pass filter) which blocks light below 450 nm in pdd - mode reducing almost the complete blue excitation light that is diffusely backscattered by the tissue. the capable fluorescence camera (tricam ii sl pdd pendulum camera head, karl storz gmbh & co. kg, tuttlingen, germany) allows detection of the red fluorescence light with an increased sensitivity especially in the range between 600700 nm. a control on the camera head for the cystoscope and ureterorenoscope allowed for switching between the white and blue lights with ease. the diagnostic accuracy was calculated for white light (wl) and blue light (bl) cystoscopy by visualisation of the lesion and by correlation with the biopsy results to obtain true positive (tp), true negative (tn), false positive (fp), or false negative (fn) values. the sensitivity, specificity and accuracy for the correct detection of the lesion for each wl and bl were calculated. the resulting diagnostic accuracy figures where compared between the two groups (wl and bl cystoscopy). the analysis was done using the meta - analysis of diagnostic and screening tests 1.4 programme (unidad de bioestadistica clinica, hospital ramon y cajal, madrid). in total, 43.5% (30/69) patients were found to have bladder lesions, of which 43.3% (13/30) were proven to be cis. all the patients in this study were planned to have pdd ureterorenoscopy with only one bladder lesion detected on computed tomography urography (ctu) prior to the procedure. twenty five patients underwent diagnostic pdd ureterorenoscopy (table 2) for the abnormal findings on ctu (n = 21), persistent frank hematuria (n = 1), suspicion of distal ureteric cancer on turbt (n = 1), abnormal cytology (n = 1) and for the assessment of contralateral uut before nephroureterectomy (n = 1). ten (40%) patients were diagnosed with bc, of which seven (70%) were concomitant to uut - uc. five (50%) of bcs were missed on white light cystoscopy of which four were concomitant to uut. four of undetectable lesions under white light was reported as cis and one a solitary tiny high grade uc ptag3. white light cystoscopy also missed a dysplasia (not classified as cis) lesion concomitant to uc ptag2. two of bladder lesions missed by the white light were solitary (normal uut). three of them were diagnosed with bc concomitant to uut - uc and two had solitary uut - uc on pdd ureterorenoscopy. findings on diagnostic (suspicion of upper urinary tract urothelial cancer) photodynamic diagnostic ureterorenoscopy in the surveillance group (n = 44), twenty (45.4%) patients were diagnosed with bc, of which fourteen had a past history of bc. ten (50%) white light missed eleven (55%) of all bladder cancers (9 of cis and 2 small ptag2 lesions) and one dysplasia lesion (not classified as cis) concomitant to uc ptag2. blue light was statistically more sensitive in detecting bladder lesions, however, less specific (tables 3, 4). overall, cystoscopy during pdd ureterorenoscopy detected significantly more malignant lesions compared with the standard white light ureterorenoscopy (table 4). in addition, blue light panurothelial endoscopy detected significantly more lesions, while white light missed all but one cis bladder lesion (table 4). there was no statistical difference between the two modalities regarding detection of urothelial cell cancer (table 4). diagnostic accuracy was 0.68 for white light and 0.75 for pdd cystoscopy, respectively (table 3). despite this, white light missed six of the eighteen lesions and blue light detected all of them (18/18). diagnostic findings of photodynamic diagnosis (pdd) and white light cystoscopy comparison of white and blue light diagnostic accuracies ci confidence interval ; n number ; tn total number ; cis carcinoma in situ ; uc urothelial cancer a study of patients who underwent radical nephroureterectomy between 1995 and 2010 revealed 9.4% incidence of concomitant and 12.5% of past history of bladder cancer. of which, 31.4% of patients developed bladder cancer within 37.5 months after nephroureterectomy. furthermore, 2829% of patients diagnosed with uut uc have past history of bladder cancer [9, 16 ]. another study reported that 8090% of patients develop metachronous bc within 2 years from uut uc diagnosis. the high rate of intravesicle recurrences is either a consequence of missed bladder cancer on initial diagnosis or panurothelial manifestation of uc (panurothelial field defect). there is no evidence to support the higher risk of seeding into the bladder following diagnostic ureterorenoscopy. the reported bladder recurrence free survival for nephroureterectomy following diagnostic ureterorenoscopy was (60%) compared to (58.7%) in patients undergoing nephroureterectomy alone. endoscopic ablation of the uut uc also does not appear to increase the risk of seeding into the bladder. it is well established that white light cystoscopy detects approximately 50% of cis lesions only. cis is undersampled during routine treatment of bc and photodynamic diagnosis improves detection rate from 2368% (white light alone) to 9197%. missing cis during cystoscopy may result in progression and higher risk of cancer specific death in up to 2083% of patients. in our report we confirm inability of standard white light cystoscopy during ureterorenoscopy to visualise cis lesion, which is a high grade cancer with potential risk of progression and development of metastases. precise detection of cis during diagnostic or surveillance ureterorenoscopy will have a significant impact on the treatment and follow - up. pdd increased the detection rate of cis in these patients, hence giving a higher accuracy for superficial cancer detection. the use of pdd as a method for the detection of bladder cancer has been accepted by the eau since 2006. moreover, the use of photosensitizers during transurethral resection of bladder tumours allows for more complete resection and reduces the recurrence rate. the photosensitizer, 5-aminolevulinic acid (5-ala) can be administered as an intravesical installation or as an oral solution. inoue. found that both routes were equally effective in detecting bladder lesions that were otherwise invisible when using the white light endoscopy. intravesical 5-ala was installed for 1 to 3 hours while the oral solution is given to the patient 3 to 4 hours prior to the procedure. in our cohort 5-ala was only given orally as all of our patients were planned to undergo pdd visualisation of the uut. it can be argued that oral administration of the photosensetizing agent would be preferable because this method does not require prior catheterisation of the patient. on the other hand the other photosensitizer, hexaminolevulinate (hal) is a hexyl - ester of 5-ala with potential to produce at least twice the fluorescence of ala in a lower concentration 24. hal, however, is a topical photosensitizer and can not be used systemically and so therefore has a limited use for panurothelial endoscopy. the advantage of using oral 5-ala is the ability to perform a simultaneous upper and lower urinary tract pdd endoscopy in patients who have a high risk of multi - focal disease. the false positive findings of pdd in the bladder is usually caused by different factors including hyperplasia, inflammation, previous intravesical treatment and inexperience in the use of pdd. patients who have had catheters or ureteric stents could develop areas of mucosal reaction which have a higher uptake of the photosensitising agent. none of our patients had urinary tract instrumentation or a ureteral stent for at least 4 weeks prior to the procedure and all procedures were carried out by a senior endourologist with experience in pdd. a single dose intravesicle instillation of mitomycin c following nephroureterectomy reduces the risk of bladder recurrences, with an absolute reduction in risk by 11% and the relative reduction by 40%. it is hypothesised that mitomycin c prevents the implantation of the urothelial cancer cells exfoliated from the upper urinary tract. studies have suggested the ability of intravesicle chemotherapeutics to ablate solitary non - muscle invasive low grade bladder cancer. post nephroureterectomy instillation of mitomycin c may be able to ablate small urothelial cancer lesions, which are not visualized on preoperative cystoscopy. there are no studies investigating the incidence of bladder cancer concomitant to upper urinary tract urothelial cancer on enhanced visualization. all together 43.5% of patients who underwent pdd ureterorenoscopy were found to have bladder lesions, of which 43.3% were proven to be cis. we found that bladder inspection under blue light during pdd ureterorenoscopy with oral 5-ala had higher sensitivity than white light cystoscopy (96.7%. though it did not reach statistical significance, pdd had a much higher detection rate, and so accuracy in the detection of cis and dysplasia lesions which was clinically significant. this is mainly due to high false positives which could be attributed to a number of factors, including the irritation from the catheter or stents, infections and inflammations, previous intravesical treatment or even inexperience in the use of pdd. this is not unexpected as cystoscopy was done in the nave bladder in 33.3% cases (n = 23) only. our report is the first to present the results of blue light inspection of the bladder in a group of patients undergoing pdd ureterorenoscopy. although the number of procedures is small, there is a raised concern about missing small bladder cancer / carcinoma in situ lesions on initial diagnosis or surveillance of uut uc. our data suggest the value of photodynamic diagnosis. systemic administration of 5-aminolevulinic acid as a photosensitizer for pdd - furs allows complete assessment of the upper and lower urinary tract urothelium (panurothelial endoscopy). the technique has a higher sensitivity and detection rate for cancerous lesions (especially for carcinoma in situ) of the lower, as well as, upper urinary tract compared with the white light alone. higher than the previously reported rate of concomitant bc may suggest utilisation of pdd to ensure cancer free status of the bladder, but this needs to be ratified in a multi - institutional randomised trial. | introductionthere is an ongoing debate on panurothelial changes in the upper and lower urinary tract as multifocal presentation of urothelial cancer is well recognised. concurrent bladder cancer impacts the outcome of the upper urinary tract urothelial cancer treatment, while its detection still relies on the white light cystoscopy.material and methodswe retrospectively reviewed all patients who underwent photodynamic diagnostic ureterorenoscopy, choosing those who had synchronous bladder biopsies. each patient received 20 mg / kg body weight of oral 5-aminolevulinic acid around 34 hours before endoscopy. all procedures were performed by a single endourologist experienced in photodynamic diagnosis and flexible ureterorenoscopy.resultsbetween july 2009 and june 2013, 69 patients underwent bladder biopsies at the time of photodynamic diagnostic endoscopic inspection of the upper urinary tract. in total, 43.5% (30/69) patients were found to have bladder lesions, of which 43.3% (13/30) were proven to be carcinoma in situ. white light inspection of the bladder missed bladder cancer in 16 (23.1%) patients, of which 12 were carcinoma in situ. there were 14 bladder cancer lesions missed under white light which were concomitant to the upper urinary tract urothelial cancer. twelve (17.4%) patients developed minor complications relevant to the photosensitizer.conclusionsthe study raises a concern about missing small bladder cancer / carcinoma in situ lesions on the initial diagnosis or in surveillance of the upper urinary tract urothelial cancer. higher than previously reported, the rate of concomitant bladder cancer may suggest utilisation of photodynamic diagnosis to ensure the cancer free status of the bladder, but this needs to be ratified in a multi - institutional randomised trial. |
green anoxygenic bacteria comprise three phylogenetically unrelated families of photosynthetic bacteria : green sulfur bacteria (family chlorobiaceae) and green filamentous bacteria (families chloroflexaceae and oscillochloridaceae) [13 ]. in 2000, the genus oscillochloris was excluded from the family chloroflexaceae, and a new family oscillochloridaceae was proposed based on phylogenetic data and unique physiological, biochemical, and chemotaxonomical properties. the photosynthetic apparatus of green anoxygenic bacteria has a particular molecular organization and contains chlorosomes, unique extramembrane light - harvesting antennae structures [4, 5 ]. the group of chlorosome - containing bacteria was enlarged by the recently discovered new phototrophic chlorosome - containing organism candidatus chloracidobacterium thermophilum from the phylum acidobacteria, and it is a surprising fact. chlorosomes are ellipsoid oblong bodies of about 70260 nm long, 30100 nm wide, and 1030 nm thick (depending on the species) attached to the inner surface of the cytoplasmic membrane. they are enveloped by a protein - lipid monolayer of 2 - 3 nm width. the bulk of light - harvesting pigments (including various types of bacteriochlorophylls (bchl) c and/or d or e and carotenoids) is located within chlorosomes. the organization of the major bchl c / d / e in chlorosomes is based upon pigment - pigment interactions and not upon pigment - protein interactions as in other photosynthetic antenna systems [8, 9 ]. these bchl c / d / e oligomers form either rod- (with a diameter of 510 nm) [1013 ] or lamellar - like structures [1416 ], arranged parallel to the longer chlorosome axis. recently, using computational integration of two different bioimaging techniques, solidstate nmr and cryoem, it was concluded that pairs of alternating syn - anti - ligated bchl c and d stacks form concentric helical nanotubes in chlorosomes from a cba.. a minor amount of bchl a is also present in the chlorosome [18, 19 ]. this bchl a is located in the baseplate, observed in freeze - fracture electron - micrographs of chlorosomes from chloroflexaceae and chlorobiaceae species as a 5 - 6 nm thick paracrystalline layer [10, 11 ]. it should be noted that the baseplate thickness was 3 nm according to recent cryo - electron tomography of cfx. the baseplate is believed to be a pigment - protein complex that is located at the base of the chlorosome. the b798 light - harvesting baseplate of the chlorosome antenna complex of chloroflexus aurantiacus was isolated and characterized. the isolated baseplate consists of bchl a, -carotene, and the 5.7 kda csma protein. the baseplate is in contact with the cellular cytoplasmic membrane and mediates excitation energy transfer to reaction centers located in the cytoplasmic membrane. in green sulfur, bacteria an additional layer (not found in chloroflexus) composed of a water - soluble bchl a - protein, the fenna - matthews - olson (fmo) protein, is situated between the baseplate and the cytoplasmic membrane [5, 23 ]. aurantiacus have been reported to contain three major proteins, csma, csmm, and csmn (which are the products of the csma, csmm, and csmn genes, resp.), with molecular masses 5.7, 11, and 18 kda [2426 ]. additional minor proteins, 6 kda protein, csmo (9,5 kda), csmy (22 kda), and csmp (20 kda), were also observed [26, 27 ]. tepidum, ten chlorosome proteins have been identified : csma (6,2 kda), csmb (7,5 kda), csmc (14,3 kda), csmd (11,1 kda), csme (6,7 kda), csmf (7,6 kda), csmh (21,6 kda), csmi (25,9 kda), csmj (21,8 kda), and csmx (24,0 kda), all of which are located in the protein - lipid envelope of the chlorosome [19, 28 ]. the 6.2-kda csma accounts for about half of the protein present in the chlorosome. besides, gel electrophoresis and immunoblotting experiments have shown that the same set of ten chlorosome proteins are present in chl. strong evidences has been obtained that only csma is absolutely necessary for the normal assembly and organization of bchl c and bchl a within the chlorosome. all functional genes responsible for each of the nine other chlorosome proteins could be eliminated with a little phenotypic effect. besides, csma is a bchl a - binding protein in both cfx. csma binds one bchl a molecule and one or two carotenoid molecules per monomer [21, 26, 33 ] and probably forms an oligomeric, paracrystalline csma bchl a complex [33, 34 ]. the precise function of the chl. aurantiacus is clearly related in sequence to a protein found in the chlorosome envelopes of the green sulfur bacteria [19, 27 ]. at present, it is not known how the bchl a subantenna in the chlorosomes from the green anoxygenic mesophilic filamentous bacteria from the recently discovered family oscillochloridaceae is organized. our previous results indicated that unlike chloroflexaceae species, the photosynthetic apparatus of oscillochloridaceae characterized by a very large size of bchl c chlorosomal antenna similar to that in chlorobiaceae species, so that the absorption of bchl c practically completely shields the absorption of other light - harvesting pigments in the near - infrared region of the absorption spectra in intact cells [35, 36 ]. the absorption spectra of isolated chlorosomes oscillochloridaceae exhibited no bchl a component found in isolated chlorosomes from two other families of green bacteria, chloroflexaceae and chlorobiaceae [35, 37 ]. however, fluorescence spectra of chlorosomes and absorption spectra of acetone - methanol extracts of isolated chlorosomes from osc. trichoides revealed the presence of very small amounts of bchl a in chlorosome samples [35, 38 ]. this allowed us to propose the existence of an intermediate - energy subantenna to interface the chlorosomal bchl c and the membrane bchl a ones. nevertheless, neither use of bchl c synthesis inhibitors nor cultivation of this culture at high light intensity allowed us to identify visually some additional subantenna in absorption spectra of oscillochloridaceae chlorosomes. at the same time, the biological expedience of such intermediate - energy subantenna in the light - harvesting system of this family was shown by us theoretically. in this work, trichoides was probed by room- and low - temperature absorbance and fluorescence spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) analysis of alkaline - treated and untreated chlorosomes. we showed that the baseplate bchl a subantenna does exist in oscillochloridaceae chlorosomes as a complex of bchl a with the 5.7 kda csma protein. trichoides (327 km mgu), was grown, as described earlier, in batch cultures with stirring under anaerobic conditions at 30c on a modified dgn medium at a moderate light intensity (50 e m s) from incandescent lamps. cells of filamentous thermophilic green bacterium chloroflexus aurantiacus strain ok-70-fl (collection belonging to leiden university, the netherlands) were cultivated anaerobically in batch cultures with stirring at 55c on a standart medium at light intensity 50 e m s. the electron microscopic observations were made with a hitachi-12 (japan), operating at 75 kv. for ultrathin sectioning, oscillochloris cells were fixed for 30 min in the culture medium at 30c by adding 25% glutaraldehyde to a final concentration of 1% and then at room temperature for 60 min. the samples were postfixed with 1% oso4 for 90 min, embedded in epon-812, and ultrathin - sectioned by standard methods. for negative staining osc. trichoides chlorosomes were dialyzed against 10 mm tris - hcl - buffer (ph 8.0) to remove sucrose, were fixed by glutaraldehyde at a final concentration of 0.1%, and, after that, were negatively stained on formvar - coated copper grids with 2% uranyl acetate. trichoides cells in a twofold successive continuous sucrose gradient (55%20% and 45%15%) in the presence of 10 mm sodium ascorbate and 2 m sodium thiocyanate as described earlier [35, 37 ]. absorption spectra were recorded at room and 77 k temperature with a hitachi-557 spectrophotometer (japan). glycerol was added (60% v / v) to the samples for measurements at 77 k to obtain clear samples upon cooling. fluorescence excitation and fluorescence emission spectra at both room temperature and 77 k were recorded using a hitachi-850 spectrometer. samples were prepared by diluting a sample in 50 mm tris buffer (ph 8.0) to obtain an optical density 0.2 at the chlorosome peak 750 nm. before fluorescence measurements, the chlorosomes were incubated 60 min with 20 mm sodium dithionite at 4c to ensure strongly reduced conditions (up to 400 mv). glycerol was added (60% v / v) to the samples for measurements at 77 k. the fluorescence emission spectra recorded at excitation wavelength 720 nm. quantitative bchl a and bchl c contents were determined according to the method developed by. samples were extracted for 20 min in the dark at 4c with a 25-fold volume of an acetone - methanol mixture (7 : 2, v / v). the absorbance of the clarified supernatant was measured at 769 nm for bchl a and 663 nm for bchl c with a hitachi-557 spectrophotometer. calculations were based on molar extinction coefficients,, of 68.6 and 74 mm cm for bchl a and bchl c, respectively. chlorosomes were treated with alkali by adding 0.1 volume of 10 m naoh to a chlorosome suspension in a 10 mm potassium phosphate buffer, ph 7.0 (final a750 was 1), and incubating the suspension at 40c for 30 min (final ph was 12.7). after incubation, two volumes of a 1.0 m potassium phosphate buffer, ph 6.0, were added to obtain a final ph of 7.2. the suspension was further diluted with the 10 mm potassium phosphate bufer and the chlorosomes were pelleted by centrifugation (180000 g for 90 min at 4c). the chlorosomes were washed twice with the 50 mm tris - hcl bufer (ph 8.0), resuspended in the same buffer, and stored at 70c. proteins were collected by centrifugation and dissolved in sample buffer (50 mm tris - hcl (ph 8.6), 24% (v / v) glycerol, 8% (w / v) sds, 2% (v / v) 2-mercaptoethanol, and 0.1% (w / v) bromophenol blue). the samples were boiled for 1 min before being loaded onto gels containing 16.5%, 10%, and 4% acrylamide as separating, spacer, and stacking gel, respectively, as described. after electrophoresis, the gels were stained with coomassie brilliant blue r-250 (cbb) or with cbb and silver. trichoides clearly showed an electron - dense area (3.55.0 nm thick) between the chlorosome and membrane which could be interpreted to be the baseplate that anchors the chlorosome to the membrane (figure 1(a)). on the micrographs of chlorosomes negatively stained with 2% uranyl acetate chlorosomes appear to be cross - linked bodies probably as a result of the interactions between their baseplates possessing the hydrophobic nature (figure 1(b)). these data clearly show that each chlorosome consists of two spatially separate compartments pressed to each other. trichoides exhibited a single peak of bchl c at 750 nm in the near - infrared region of the absorption spectra at room temperature. at 77 k, the 750-nm peak of osc. trichoides was red - shifted to 758 nm and was sharper and more narrow (fwhm 35 nm) than the room temperature peak (fwhm 47 nm) (figure 2, solid line). trichoides chlorosomes at 77 k showed, in addition to the near - infrared absorption band at 758 nm due to bacteriochlorophyll c, a weak shoulder near 805 nm, which may be attributed to bchl a. the presence of bchl a in chlorosomes was clearly visualized only by fluorescence spectroscopy measured at 77 k or by pigment extraction [3538 ]. the main light - harvesting pigment in the chlorosomes was identified as bchl c and the minor pigment as bchl a [35, 37 ]. these results suggest there was no possibility to observe changes in the bchl a content in osc. trichoides chlorosomes by measuring absorption spectra of chlorosomes at room and 77 k temperature (figure 2). actually, there were over 100 bchl c molecules per one bchl a molecule in osc. figure 3(a) (dotted line) shows the effect of alkaline treatment on the absorption spectrum of the osc. obviously, the absorption bands of bchl c, the main light - harvesting pigment in osc. trichoides chlorosomes, were practically not affected by alkaline treatment. trichoides chlorosomes, measured at room temperature and 77 k, are shown in figure 4. at room temperature, fluorescence excitation spectra of bchl a (figure 4(a)) resembles the absorbance spectrum of bchl c (fwhm 47 nm) (figure 2, dotted line), and positions of the maxima in both spectra are identical (750 nm). at 77 k, fluorescence excitation spectra of bchl a (figure 4(b)) also resembles the absorbance spectrum of bchl c (fwhm 35 nm) (figure 2, solid line), and positions of the maxima in both spectra are identical (758 nm). as it was shown earlier, bchl a emission (in contrast to absorbance) could be discerned in the fluorescence emission spectra of osc. trichoides chlorosomes at 77 k but not at room temperature [35, 37 ]. the fluorescence emission spectra of the isolated chlorosomes, when measured at 77 k, showed mainly a broad band at 780 nm, due to bchl c, together with another band near 820 nm, due to bchl a, suggesting that a baseplate is probably associated with the chlorosome (figure 5(b)). trichoides chlorosome antenna exhibited a highly redox - dependent bchl c fluorescence similar to chlorobiaceae species [3537 ]. for this reason, fluorescence emission spectra of untreated and alkaline - treated chlorosomes were measured at room temperature and 77 k under both aerobic and reducing conditions (dithionite-20 mm) after excitation in the qy - band of bchl c at 720 nm. trichoides showed different response on redox conditions at room temperature and 77 k. at room temperature, the bchl c emission intensity was about tenfold higher under reducing conditions than that under aerobic conditions (figure 5(a)), while at 77 k, the intensity of the bchl c and bchl a emission increased only two- and threefold, respectively, under reducing conditions in comparison with aerobic conditions (figure 5(b)). alkaline treatment had some (but also different) influence on the intensity of bchl c fluorescence emission both at room temperature and 77 k in osc. trichoides chlorosomes (figures 5(c) and 5(d)). at room temperature, alkaline treatment increased slightly (1,6-fold) the bchl c emission under aerobic conditions but decreased twofold the bchl c emission under reducing conditions (figure 5(c)), as compared to untreated chlorosomes (figure 5(a)). at 77 k, alkaline treatment increased slightly (1,3 - 1,4-fold) the bchl c emission both under aerobic and reducing conditions (figure 5(d)). fluorescence from bchl a could no longer be seen in alkaline - treated chlorosomes from osc. trichoides under either reducing or aerobic conditions (figures 5(c) and 5(d)). thus, in both aerobic and reducing medium, alkaline treatment strongly decreases steady - state fluorescence intensity in the 820 nm spectral region. it is obvious that the disappearance of bchl a emission is caused by the removal or destruction of bchl a in the baseplate. at room temperature in osc. trichoides alkaline - treated chlorosomes under aerobic conditions, the fluorescence intensity of bchl c increases only slightly and decreases about threefold on going from aerobic to reducing conditions (figure 5(c)) in comparison with untreated chlorosomes (figure 5(a)). at 77 k, changes in bchl c fluorescence intensity under different redox conditions were identical in untreated (figure 5(b)) and alkaline - treated chlorosomes (figure 5(d)). figure 6(a) demonstrates that under reducing conditions at room temperature, untreated chlorosomes from green filamentous bacterium cfx. aurantiacus exhibited the bchl c emission intensity more than threefold higher than that under aerobic conditions. alkaline treatment resulted in approximately 2 - 3-fold reduction of the bchl c fluorescence intensity under both aerobic and reducing conditions (figure 6(b)). aurantiacus chlorosomes are very much alike : depletion of bchl a by alkaline treatment led to small or moderate effects on bchl c fluorescence intensity. trichoides chlorosomes led to a selective degradation of bchl a in the baseplate and caused dramatic changes in the fluorescence spectra of chlorosomes while leaving bchl c in a form that is spectrally indistinguishable from that in untreated chlorosomes. these results are in agreement with conventional ideas about the organization of chlorosome pigments : the bchl c and bchl a pigments housed within two different (but neighboring) substructures. selective degradation of bchl a would be expected if it is located outside of the chlorosomal bchl c body in the contact with cytoplasmic membrane, whereas bchl c is organized in rod or lamellar aggregates within the bchl c body of chlorosomes. the relative contents of bchl c and bchl a pigments were determined in acetone methanol extracts of osc. the absorption spectra of acetone - methanol extracts of untreated and alkaline - treated osc. trichoides chlorosomes are shown in figure 3(b). in the absorption spectra of untreated chlorosomes, two bands, at 663 nm (corresponds to monomeric bchl c) and 769 nm (corresponds to monomeric bchl a), were resolved in contrast to spectra of alkaline - treated chlorosomes that showed a single peak at 663 nm in the near - infrared region. five proteins (three major and two minor) were detected in untreated osc. trichoides chlorosomes showed two major bands with molecular masses around 11 and 18 kda and a strong broad band 5.7 kda (figure 7(a), lane 2). two minor proteins with masses of 9.5 and 21 kda were also observed. these five proteins could be visualized by cbb (figure 7(a), lane 2) and silver staining (data not shown). figure 7(a) (lane 3) demonstrates that alkaline treatment selectively removed 5.7 kda protein, while the other four proteins remained largely unaffected. aurantiacus chlorosomes and its changes after alkaline treatment are shown in figure 7(b). aurantiacus chlorosomes (lane 2) contain three major proteins with molecular masses 5.7 ; 11 and 18 kda, according to literature data, designated as csma, csmm, and csmn proteins, respectively [2426 ]. figure 7(b) (lane 3) shows that alkaline treatment of cfx.aurantiacus chlorosomes resulted in loss of csma. obviously, protein profiles of untreated and alkaline - treated osc. trichoides and trichoides (family oscillochloridaceae) like thermophile cfx.aurantiacus (family chloroflexaceae) is morphologically filamentous, shows gliding motility, and does not contain fmo protein between the chlorosome baseplate and cytoplasmic membrane of the cell. in view of this, we designated the proteins of osc. aurantiacus chlorosomes : csma (5.7 kda), csmm (11 kda), and csmn (18 kda). it should be noted that the amino acid sequence of the ~6-kda band from ca. (it is related phylogenetically to the chloroflexi group) showed that this protein exhibits substantial sequence similarity (65% identity) to the csma protein from cfx. chlorothrix halophila similarly to oscillochloris trichoides has a combination of features that are present in both phyla of green bacteria : the chlorosome peak is similar to that of chlorobi species, a minimal amount of bchl a is present, and the likely antenna complex is comparable to the b808 - 866 antenna in cfx. aurantiacus. as we reported above, alkaline treatment of osc. trichoides chlorosomes led to a selective removal of bchl a in the baseplate. in this section, we demonstrated that alkaline treatment selectively removed csma protein from osc. thus, a strict correlation between removal of csma protein and removal of bchl a in the baseplate was demonstrated : only this protein was removed from chlorosomes concurrently (figure 7(a)) with the disappearance of bchl a fluorescence, leaving bchl c unchanged spectrally (figures 5(c) and 5(d)). selective bchl a and 5.7 kda protein disappearance should be expected only in case when both of them are located out of the bchl c body, that is, within the baseplate of the chlorosome. thus, the complex study of the structure - function correlations of bchl a and csma protein in osc. trichoides chlorosomes was confirmed (a) by the presence of the band peaking at 820 nm in the fluorescence spectrum of isolated chlorosomes at 77 k (figure 5(b)) ; at room temperature, the corresponding band looks like a shoulder at 805 nm (figure 5(a)) ; (b) by the presence of the band peaking at 769 nm in the absorption spectrum of acetone - methanol extract of chlorosome pigments at room temperature (figure 3(b)). (2) the chlorosome bchl a serves as the direct acceptor of exitation energy from bchl c, which was confirmed (a) by the presence of bchl a band in the fluorescence spectra of isolated chlorosomes both at room temperature and 77 k upon bchl c excitation (figures 5(a) and 5(b)) ; (b) by the bchl a fluorescence excitation spectra that resembled the bchl c near - infrared absorption band both at room temperature and 77 k (figure 4). these data are in full agreement with our recent theoretical calculations that have shown the biological expedience of existence of an intermediate bchl a subantenna with its qy band being within the region of 790800 nm. note that the shoulder at ~805 nm in the fluorescence spectrum of isolated chlorosomes (attributed to bchl a fluorescence, figure 5(a)) is in a good agreement with this estimation of the position of bchl a qy transition that ensures the optimal coupling between chlorosome bchl c b750 subantenna and membrane bchl a b805 - 860 one. (3) upon alkaline treatment, only the 5.7 kda csma protein was removed from the osc. trichoides chlorosomes among five proteins detected by sds - page analysis (figure 7(a)), concomitantly with the disappearance of bchl a fluorescence emission at 820 nm measured at 77 k (figure 5(d)). the absorption bands of bchl c, the main light - harvesting pigment in osc. trichoides chlorosomes, were practically not affected by alkaline treatment (figure 3(a), dotted line). note that ~6 kda csma protein was found earlier in baseplates of chloroflexaceae and chlorobiaceae chlorosomes as a bchl a - binding protein. based on these results, we suggest that : (i) bchl c and bchl a are localized in two different neighboring substructures of osc. trichoides chlorosomes, which is in excellent agreement with the data of electron microscopy (figure 1) ; (ii) bchl a and csma 5.7 kda are localized in one and the same substructure of osc. trichoides chlorosomes, that is, out of the bchl c body and, therefore, in the baseplate of chlorosomes. so, we conclude that the intermediate - energy bchl a subantenna interfacing chlorosome b750 and membrane - bound b805 - 860 light - harvesting antennae is associated with 5,7 kda csma protein and is located within the baseplate in osc. thus, the presented results support our idea that the baseplate bchl a subantenna is a universal interface between the chlorosomal bchl c subantennae and the nearest bchl a ones in all three known families of green photosynthetic anoxygenic bacteria and represents a complex of bchl a with a ~6 kda csma protein. | the baseplate subantenna in chlorosomes of green anoxygenic photosynthetic bacteria, belonging to the families chloroflexaceae and chlorobiaceae, is known to represent a complex of bacteriochlorophyll (bchl) a with the ~6 kda csma proteins. earlier, we showed the existence of a similar bchl a subantenna in chlorosomes of the photosynthetic green bacterium oscillochloris trichoides, member of oscillochloridaceae, the third family of green photosynthetic bacteria. however, this bchl a subantenna was not visually identified in absorption spectra of isolated osc. trichoides chlorosomes in contrast to those of chloroflexaceae and chlorobiaceae. in this work, using room and low - temperature absorbance and fluorescence spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of alkaline - treated and untreated chlorosomes of osc. trichoides, we showed that the baseplate bchl a subantenna does exist in oscillochloridaceae chlorosomes as a complex of bchl a with the 5.7 kda csma protein. the present results support the idea that the baseplate subantenna, representing a complex of bchl a with a ~6 kda csma protein, is a universal interface between the bchl c subantenna of chlorosomes and the nearest light - harvesting bchl a subantenna in all three known families of green anoxygenic photosynthetic bacteria. |
the search for pure salt engaged humanity for millennia and influenced history in profound ways. its preservative property allowed storage of food before refrigeration and had a momentous effect on history enabling transition from a hunter - gatherer to a settled lifestyle and established salt as a major economic commodity. ancient chinese texts describe two different methods to extract salt more than 2000 years ago, mention 40 different types of salt, and describe its uses in food preservation. salt taxes were an important source of revenue for the ancient chinese governments, and later many other governments around the world. roman soldiers were issued their monthly pay as salt money, latin salarium. in ancient libya it led to wars among nations, influenced the establishment of trade routes, such as the ancient roman via salaria, which are the precursors of some of the modern highways in today 's italy. many cities with salt mines derived their names from salt, for example, salzburg, hallstatt, tuzla, and so on. gandhi 's 1930 salt march in defiance of the british salt act was a pivotal moment in the indian independence movement that ended the british rule in india. thus, the recorded history documents that humans knew and valued, even revered salt, attaching sacred attributes to it for nearly 5000 years, because it was needed to preserve food, was extremely scarce, highly coveted, but out of the reach of ordinary people until recently. the cheap purified table salt is a very recent and in many ways an unfortunate addition to human diet. it became widely available for less than two centuries only after modern geology helped identify abundant salt mines throughout the world and after it became possible to produce large quantities efficiently with the aid of modern technology. the evolution of modern humans and our hominid ancestors took place in an environment virtually without any access to salt over a span of two million years. this is in fact true for most land - based animals stretching to several hundreds of millions of years in evolution. for life forms that have originated in briny waters to free themselves from marine environment and transition to land environment, ability to carry thus, in the environment where humans and our distant ancestors evolved (the environment of adaptedness), there was intense and unrelenting selection pressure for genes and mechanisms that could preserve the very small quantities of salt available in natural diet barely 0.25 g salt per day. indeed, all genes identified to date with a link to blood pressure are associated with sodium transport. without an efficient mechanism to preserve salt, its loss in bodily secretions would have fatal consequences as salt is an essential ingredient of our plasma volume. owing to a physiology that has evolved over millions of years, all terrestrial animal life forms are exquisitely well adapted to salt scarcity and can survive without regular access to salt supplements unless faced with excessive salt losses, such as diarrhea or vomiting. all these life forms still exist successfully without access to added salt in their diet. only humans have discovered salt as a dietary additive, and when thrust into salt surfeit conditions, as in the modern diet that contains 1018 g or even more salt per day (50- to 70-fold higher than our natural paleolithic diet), the consequences are high blood pressure, kidney failure, strokes, and heart disease. worldwide, the prevalence of hypertension has now reached 26% in parallel with the increased salt consumption in the modern diet. there is also evidence linking high - salt diet to higher risk of obesity through greater consumption of sugared drinks, stomach cancer, kidney stones, and osteoporosis. high salt intake is clearly linked to increased salt and water retention, increased glomerular filtration, and a blunted pressure natriuresis response this is what evolutionary medicine characterizes as evolutionary mismatch, and hypertension a prototypical maladaptation disorder. observations in contemporary no - salt societies confirm not only the relation between salt and hypertension but also provide evidence of successful adaptation to salt - free environment during the evolution of the human species. for example, the australian aborigines, the african bushmen, or the amazonian yanomami had no access to salt in their diet until recently. their total salt intake was found no more than what they could obtain from natural sources based on the typical hunter when such communities are urbanized and exposed to the salty modern diet, they do suffer from hypertension and its complications, in some cases at disproportionately higher rates than the rest of the population. the extraordinary experiments conducted in 1995 by derek denton in chimpanzees, our closest living evolutionary relatives, showed markedly that the chimpanzees placed on high - salt diet (12 g per day) developed hypertension, which reversed when they resumed their usual low - salt (0.250.5 g per day) diet. numerous animal experiments confirm the role of salt in hypertension and some studies also suggest that excess salt may have harmful effects on cardiovascular health independent of hypertension. for example, one such study showed that in normotensive wistar kyoto rats high salt intake resulted in deposition of fibrous tissue in the heart and kidneys despite only modest rises in their blood pressure, almost to the same extent seen in their hypertensive counterparts, spontaneously hypertensive rats. in the 1940s when there were no drugs available to treat hypertension, walter kempner treated hundreds of severely hypertensive patients on a strict low - salt (0.25 g per day) diet based on rice, fruit juices, and vitamins (kempner 's rice diet) for many weeks, some more than a year making this one of the longest salt restriction studies in the medical literature. meticulous records kept by dr kempner document that the diet markedly improved blood pressure, reversed heart enlargement, improved kidney function, and also reversed the hypertensive changes in the retinal vasculature in these patients. more recent studies, although of shorter durations, confirmed that low - salt diet lowers blood pressure both in individuals with normal blood pressure and in patients with hypertension. perhaps, the best known is the dietary approaches to stop hypertension (dash) diet trial. this 12-week controlled trial showed that reducing dietary intake of salt from the normal ' (8 g per day) to intermediate (6 g), and low (4 g) lowered blood pressure among both hypertensive and normotensive individuals. the recent cochrane review and a comprehensive meta - analysis of data on the health effects of salt restriction documents convincingly the beneficial effects of salt in non - acutely ill adults, and also highlights the efficacy and safety of very - low salt diet (figure 1). this systematic review conducted by scientist from the world health organization 's nutrition policy and scientific advice unit concluded that, evidence in non - acutely ill adults shows that reduced sodium intake reduces blood pressure and has no adverse effect on blood lipids, catecholamine levels, or renal function. ' lower sodium intake was also associated with a reduced risk of stroke and fatal coronary heart disease in adults. in this study, the totality of the evidence suggested that most people would benefit from reducing sodium intake. consensus has now emerged among most hypertension experts that the beneficial effect of salt reduction starts at daily intake levels of 5 g or less, and the relatively high potassium content of low - salt diets may have additional beneficial effects on blood pressure. evidence also suggests that for maximum benefit, salt reduction might need to be started early in life, and some of the cardiovascular effects of sustained high salt intake may be irreversible. why then there is still a controversy and caution that reducing dietary intake may have unforeseen health hazards, and additional evidence is sought ? the findings in some observational studies that show adverse cardiovascular outcomes in very low salt intake groups have been highlighted as an argument against recommending reduced salt intake to very low levels. for example, the report by stolarz - skrzypek. attracted much attention for their finding of a weak but consistent inverse association between cardiovascular mortality and the 24-h urinary sodium excretion at baseline. ' systolic but not diastolic pressure change in parallel with urinary sodium excretion. ' however, this observational study had too many limitations. first, long - term salt intake was estimated from a single 24-h urine collection for the duration of the study. second, all patients with known hypertension and cardiovascular disease were excluded from the analysis, potentially biasing the conclusions in favor of the high salt intake tertiles, known to be associated with higher incidence of hypertension and cardiovascular disease. and thirdly, the finding of a cohort ingesting unusually low quantities of salt (107 mmol, or 6.3 g salt per 24 h) in a high - salt culture is not sufficiently explained. aldosterone system along with increased sympathetic nervous system activity, which are in fact activated to conserve sodium and prevent further sodium losses. these hormonal mechanisms are the precise tools that have emerged during the evolution of terrestrial life forms under pressure to conserve the miniscule quantities of salt intake that they had access to through their natural diet. this level of salt intake is compatible with normal physiology in animals as well as humans. angiotensin aldosterone system activity or sympathetic activity in the absence of high salt intake do not lead to adverse cardiac effects in healthy individuals, as has been observed in no - salt populations and similar to hypertensive patients on diuretics. the danger is when these hormones can not be shut off in patients who are on high salt intake, or have impaired salt balance because of heart, liver, or kidney disease. the adverse outcomes generally derive from unhealthy individuals who are salt - avid, such as patients with heart failure, who usually have very low sodium concentrations in urine, which are taken erroneously as representative of their salt intake. the argument for caution on lowering salt intake further fails as these patients with impaired salt balance are often treated with natriuretic agents, that is, diuretics, for the explicit purpose of reducing their total body sodium. although not openly stated, what these critics seem to suggest is that lowering total body sodium by using a diuretic drug is safer than reducing their salt intake ! worse outcomes in salt - avid populations can not be explained by low dietary intake of salt, but are much more likely related to the seriousness of their pathophysiology that makes them salt - avid. is particularly valuable, because they restricted their analysis to high - quality studies that did not include sick patients, and showed that lowering salt intake below 1200 mg per day is beneficial and safe (figure 1). of note, the characterization of 1200 mg sodium (3 g salt) intake as very low is arbitrary and relative to the current conditions. until approximately 150200 years ago, the majority of the world 's population did not have access to even this much salt in their diet. in no - salt populations, the average salt intake is barely 250500 mg per day, and such populations can survive in good cardiovascular health without hypertension, and also without hypotension or hypovolemia. this is because the human kidney can conserve sodium very efficiently and the average urine sodium in such populations is usually very low (12 mmol per day). the relation between salt and hypertension should no longer be disputed. our understanding of renal and cardiovascular physiology and its evolution in a salt - free environment are also sufficient evidence. world governments faced with the economic burden caused by the ravages of hypertension and the associated cardiovascular morbidity have now started to take action. a worldwide campaign to reduce dietary salt intake to 5 finland and england have already reduced the amount of salt being consumed by a combined policy of getting the food industry to decrease the amount of salt added to foods, labeling the sodium content on food products, and increasing public awareness of the harmful effects of salt on health. a global approach is required to extend these measures to developing countries where approximately 80% of the world 's hypertension - related disease burden exists. experience has shown that even a modest reduction in salt intake can result in major improvements in public health and lead to cost reduction in health - care expenditures. a worldwide coalition, world action on salt and health (wash), has been launched recently (http://www.worldactiononsalt.c om), encouraging other countries and health - care professionals interested in hypertension, kidney, and heart diseases to join in this effort. millions of years of the successful existence of ours and related species in a salt - free environment is sufficient proof that a low - salt diet is man 's original and natural diet, is compatible with normal physiology, and is safe. the modern man while well adapted to low - salt diet is poorly equipped to cope with the salt surfeit imposed on him in recent times. the unnaturally high salt intake, an artifact of recent times, contributes to hypertension and to the increased cardiovascular morbidity and mortality caused by hypertension. reducing daily salt intake to approximately 5 g per day (2 g sodium) or less helps lower blood pressure significantly and reduces the complications of hypertension. this is essentially a cost - free intervention that can help cut health - care expenditure and save millions of lives worldwide. | more than a quarter of human populations now suffer from hypertension paralleling the marked increase in the dietary intake of salt during the recent several decades. despite overwhelming experimental and epidemiological evidence, some still debate the relation between salt and hypertension. pointing to some conflicting data in a few flawed studies, they argue that policy interventions to reduce the dietary intake of salt are premature and maybe unsafe without further studies. a brief review of data relating salt intake to hypertension, along with an overview of the history of the introduction of salt to human diet on an historic and evolutionary time scale, should help dispel doubts on the effectiveness and safety of low - salt diet. the recorded history confirms how rare and inaccessible salt has been until recent times. like all other terrestrial life forms, humans evolved in a salt - free environment under intense evolutionary pressure for the selection of salt - conserving genes. hypertension is a prototypical evolutionary maladaptation disorder of the modern man a species exquisitely well adapted to low salt conditions suddenly confronted with salt excess. the world health organization and many governments have finally taken action to reduce dietary intake of salt, which already has started to reduce the burden of hypertension and the associated cardiovascular morbidity and mortality. this brief review is to broadly look at the evidence linking salt to hypertension from a historic and evolutionary perspective as well as touching upon some of the epidemiological and experimental data. |
glass ionomer cements have wide spread clinical indications, being used as bases, liners, luting agents, and temporary and restorative dental materials. the reasons for such a useful clinical versatility are related to the constant fluoride release, adhesion to dental tissues and base metals, biocompatibility and low coefficient of thermal expansion. resin - modified glass ionomer cements (rmgics) extended these clinical applications since they present better mechanical properties due to the polymeric nature24, overcoming some shortcomings of conventional gics, such as surface crazing during dehydration, brittleness and low fracture strength. conventional gics set by mans of an acid - base reaction in which a polyacid attacks powder particles, forming a mass with some interstitial spaces. according to nicholson (1998), gics consist of interpenetrating networks of inorganic and organic components forming a matrix in which particles of unreacted glass are embedded. in addition to this ionic cure, rmgics have also a resinous polymer - based reaction due to the presence of visible light - polymerizable components. both reactions are processed at the same time ; however, light polymerization results in a rapid initial set while the acid - base reaction, which proceeds normally from the point of mixing, slows down or becomes virtually inhibited by network formation after irradiation. several aspects have been studied in order to improve the mechanical and physical properties of gics, such as composition, viscoelastic properties, fracture toughness, powder particle size reduction radiodensity, strength and powder / liquid (p / l) ratio. composition seems to be the most important factor which affects the radiopacity of dental materials. in addition, the material thickness, x - ray beam angulation, methodology, type of x - ray film, age of developing and fixing solutions, and alteration in p / l ratio can also have an influence. the first radiopaque gics were cermet - containing or metal reinforced cements, in which metals were responsible by high radiodensity levels. after that, the addition of radiopaque fillers resulted on sufficiently radiopaque gics, which could be detected against a background of enamel and dentin, facilitating the evaluation of many clinical situations. the diametral tensile strength (dts) is one of the most common and useful mechanical properties of gics, being extensively found in many studies. depending on the clinical application, gics should be strong enough to resist stresses generated within it when loaded with occlusal forces, as it is truth for luting materials. the type of material, filler size and proportion, addition of metals, composition and p / l ratio are factors which affect strength of gics. mechanically, rmgics generally exhibit substantial plastic deformation in compression and conventional gics display brittle fracture. clinicians tend to make reductions in glass ionomer p / l ratios since some materials are difficult to mix and flow into small cavities, grooves or pits. in general, changing the p / l ratio decreases the physical and mechanical properties of conventional gics and rmgics depending on the reduction of powder per volume. p / l ratios, which could virtually lessen the longevity of clinical procedures. then, the aim of this study was to determine the influence of a 50% reduction in p / l ratio on the dts and radiodensity of different conventional gics and rmgics. five gics (dfl, rio de janeiro, brazil) with different clinical indications were employed in this study. material applications, commercial names, batch numbers and composition are listed in figure 1. two tests were performed in order to analyze the effect of reducing the p / l ratio of different gics : radiodensity and dts. groups were formed according to 2 experimental factors under study : materials, with 5 levels (figure 1) and p / l ratio, with 2 levels, the manufacturer 's recommended p / l ratio and a 50% reduced p / l ratio. samples were fabricated at room temperature according to each experimental method, as described below. chemical cured materials were allowed to set during the period recommended by each manufacturer. light - cured materials were photoactivated for 40 s with a halogen curing unit (xl3000 ; 3 m espe, st. restorative materials used in the study hema, 2-hydroxyethylmethacrylate five 1-mm - thick ring - shaped standard samples were fabricated for each experimental group by mixing the materials according to the manufacturers ' instructions and inserting them in a 1-mm - thick stainless steel mold with diameter of 4.0 mm. after removal of the samples from the mold, the thickness was checked with a digital caliper in order to fit 1.0 mm (0.1 mm). an aluminum stepwedge, ranging from 1.0 mm to 9.0 mm in thickness, served as a control. the samples were positioned onto a phosphor plate and radiographic exposure was performed for 0.2 s at 70 kv and 10 ma, with a source - tosample distance of 40 cm using an x - ray machine (ge 1000 ; general electric, milwaukee, wi, usa). the radiographs were transferred from the phosphor plate to the computer via a digora scanner (digora optime, soredex, helsinki, finland). the radiodensity (in pixels) of the samples was determined with the resident software provided by the manufacturer. the digora system has a windows - based software, digora for windows 2.5 rev 0 (soredex, helsinki, finland), which is capable to measure density curves of digital radiographies obtained by x - ray impregnation on the image phosphor plate. the radiodensity of each radiographed structure or material was obtained by clicking with the software cursor right above the digital image. each digital image had it radiodensity measured immediately after scanning, without any modification in contrast or brightness. this software shows data concerning the highest and the lowest radiodensity of the sample, and an average value, which was considered to be the sample 's initial radiodensity. since each sample was subjected to three exposures the sample 's final radiodensity was considered to be the mean of those values. five cylindrical specimens of each experimental group (n=5) were prepared for the dts test according to ada specification no. three minutes after filling the molds with mixed cements, samples were kept in 100% humidity and 37c for 1 h, followed by gentle removal from the mold and immersion in distilled water for 23 h prior to testing. a compressive load was applied on the diametral surface of the samples to obtain the dts at a crosshead speed of 0.5 mm/ min in a universal testing machine (instron 4411 ; instron testing instruments, canton, ma, usa). for observations of materials characteristics after complete setting, sem analysis was accomplished in samples from ortho glass lc and vitro cem experimental groups. fractured samples were sputter - coated with gold (med 010 ; balzers union, balzers, liechtenstein) and observed with a scanning electron microscope (dsm 940a ; zeiss, oberkoshen, germany). statistical analysis was performed in accordance with the results of shapiro - wilk test of normal distribution, with parametric or non - parametric tests, for each variable. one - way anova followed by tukey 's honestly significant difference (hsd) test was employed for dts and the kruskal - wallis and dunn 's method for radiodensity, using the spss 12.0 for windows statistical software (spss inc., two - way anova (52) followed by tukey 's hsd test, with a general linear model procedure, was also used to analyze the interaction between materials and p / l ratio. for paired comparisons, independent samples t - test was used for dts and mann - whitney test for radiodensity. the aluminum step was compared to each group by anova and dunnett 's 2-sided test. five 1-mm - thick ring - shaped standard samples were fabricated for each experimental group by mixing the materials according to the manufacturers ' instructions and inserting them in a 1-mm - thick stainless steel mold with diameter of 4.0 mm. after removal of the samples from the mold, the thickness was checked with a digital caliper in order to fit 1.0 mm (0.1 mm). an aluminum stepwedge, ranging from 1.0 mm to 9.0 mm in thickness, served as a control. the samples were positioned onto a phosphor plate and radiographic exposure was performed for 0.2 s at 70 kv and 10 ma, with a source - tosample distance of 40 cm using an x - ray machine (ge 1000 ; general electric, milwaukee, wi, usa). the radiographs were transferred from the phosphor plate to the computer via a digora scanner (digora optime, soredex, helsinki, finland). the radiodensity (in pixels) of the samples was determined with the resident software provided by the manufacturer. the digora system has a windows - based software, digora for windows 2.5 rev 0 (soredex, helsinki, finland), which is capable to measure density curves of digital radiographies obtained by x - ray impregnation on the image phosphor plate. the radiodensity of each radiographed structure or material was obtained by clicking with the software cursor right above the digital image. each digital image had it radiodensity measured immediately after scanning, without any modification in contrast or brightness. this software shows data concerning the highest and the lowest radiodensity of the sample, and an average value, which was considered to be the sample 's initial radiodensity. since each sample was subjected to three exposures the sample 's final radiodensity was considered to be the mean of those values. five cylindrical specimens of each experimental group (n=5) were prepared for the dts test according to ada specification no. 27. a 4.0 mm long and 8.0 mm diameter aluminum mold was used. three minutes after filling the molds with mixed cements, samples were kept in 100% humidity and 37c for 1 h, followed by gentle removal from the mold and immersion in distilled water for 23 h prior to testing. a compressive load was applied on the diametral surface of the samples to obtain the dts at a crosshead speed of 0.5 mm/ min in a universal testing machine (instron 4411 ; instron testing instruments, canton, ma, usa). for observations of materials characteristics after complete setting, sem analysis was accomplished in samples from ortho glass lc and vitro cem experimental groups. fractured samples were sputter - coated with gold (med 010 ; balzers union, balzers, liechtenstein) and observed with a scanning electron microscope (dsm 940a ; zeiss, oberkoshen, germany). statistical analysis was performed in accordance with the results of shapiro - wilk test of normal distribution, with parametric or non - parametric tests, for each variable. one - way anova followed by tukey 's honestly significant difference (hsd) test was employed for dts and the kruskal - wallis and dunn 's method for radiodensity, using the spss 12.0 for windows statistical software (spss inc., two - way anova (52) followed by tukey 's hsd test, with a general linear model procedure, was also used to analyze the interaction between materials and p / l ratio. for paired comparisons, independent samples t - test was used for dts and mann - whitney test for radiodensity. the aluminum step was compared to each group by anova and dunnett 's 2-sided test. tables 1 and 2 show the results of dts test (in mpa) and radiodensity measurements (in pixels), respectively, together with the statistical analysis. radiodensity means and standard deviations are presented only to facilitate the understanding. since data were not normally distributed, the sum of the ranks (non - parametric analysis) is also provided. there was a significant interaction (p=0.001) for the factors under study (materials vs. p / l ratio). diametral tensile strength (dts) means and standard deviations (in mpa ; n=5), and results of statistical analysis of groups by anova, tukey 's hsd and t - test (a=0.05) different uppercase letters indicate significant differences within the same p / l ratio groups (vertical comparison only - p0.05). a1-a12 : 1.0 - 12.0 mm thick aluminum digital radiograph of the studied materials and aluminum step wedge. 1 : vitro molar - manufacturer s recommended p / l ratio ; 2 : vitro molar 50% reduced p / l ratio ; 3 : vitro cem manufacturer s recommended p / l ratio ; 4 : vitro cem 50% reduced p / l ratio ; 5 : vitro fil manufacturer s recommended p / l ratio ; 6 : vitro fil 50% reduced p / l ratio ; 7 : ortho glass lc manufacturer s recommended p / l ratio ; 8 : ortho glass lc 0.5p / l ; 9:vitro fil lc manufacturer s recommended p / l ratio ; 10 : vitro fil lc 50% reduced p / l ratio ; a1-a12 : 1.0 - 12.0 mm thick aluminum figures 4 and 5 are sem images of ortho glass lc at the manufacturer 's recommended figures 6 and 7 of vitro cem at the manufacturer 's recommended p / l ratio and 50% reduced p / l ratio, respectively. for the rmgics, reduction in liquid at 50% p / l resulted in poor interaction between matrix and powder particles with clear signs of crack development. for the conventional gics, reduction in liquid at 50% p / l ratio resulted in a greater presence of salt matrix and its close interaction with powder particles without the development of cracks, which could be seen at the manufacturer 's recommended scanning electron microscopy of ortho glass lc at manufacturer s recommended p / l ratio showing a uniform interaction between resin matrix, salt matrix and unreacted powder particles, without clear signals of crack propagation. note that the boundaries of powder particles can not be clearly seen, which mean that the acid base reaction progressed in a free condition scanning electron microscopy ortho glass lc at 50% reduced p / l ratio showing a greater volume of matrix around powder particles ; particle s boundaries can be clearly seen, which mean that the acid - base reaction was partially inhibited by the greater presence of resin. note the presence of crack propagation around the powder particle, which is related to a lower interaction between matrix - powder scanning electron microscopy of vitro cem at manufacturer s recommended p / l ratio showing the presence of big powder particles surrounded by salt matrix. note the presence of bubbles inside the mixture (white arrows), and cracks walking around unreacted powder particles scanning electron microscopy of vitro cem at 50% reduced p / l ratio showing the presence of small unreacted powder particles embedded by a great amount of salt matrix. note the presence of bubbles (white arrows) and a close interaction between particles and the matrix several factors can have an influence on conventional gic and rmgic properties, but the p / l ratio is the one that relies on the responsibility of clinicians. the hypothesis driven on this study was confirmed with the observed alterations in dts and radiodensity of gics and rmgics due to changes in p / l ratio. the assumption that radiodensity depends more on the composition of dental materials than the type of material was confirmed and the dts results are in general agreement with other studies, who reported that the rmgics exhibited higher dts than gics. composition, setting reactions, materials ' maturation and microstructures seemed to be the principal reasons for these observations. materials tested with the same thickness mostly vary in radiodensity by the influence of composition. x - ray beam interactions with the matter are always directly proportional to either the atomic number of the absorber or to its electric density ; then depending on the atomic composition and density of each atom in the matter a radiographic image will be differently influenced. the addition of chemical elements with high atomic numbers such as zinc, strontium, zirconium, barium and lanthanum result in more radiopaque materials.. vitro molar and vitro fil, which present barium/ feo and strontium / feo, respectively, in their composition, were the most radiopaque materials, when mixed in the manufacturer 's recommended p / l ratio. however, even with the presence of strontium / feo in the other materials, their radiodensity were similarly lower than the previous mentioned ones, which mean these components are present in relatively lower quantities. with the reduced p / l ratio, vitro fil and vitro molar still presented the highest radiodensity levels, but vitro cem was similar to vitro molar and the rmgics were similar to vitro cem. vitro molar is a restorative gic and is more viscous than vitro cem. the higher viscosity and the apparent presence of more quantities of radiopaque fillers would render higher radiodensity, but with the reduction in p / l ratio, these differences disappeared. since organic components do not seem to offer radiopaque characteristics to dental materials, the observed lower radiodensity for rmgics at 50% reduced p / l ratio is a direct cause of the increase in liquid per volume within each material, the reduction in p / l ratio significantly reduced the radiodensity of all materials in this study (table 2), and this also may be a direct result of the increase in liquid per volume. since all materials employed on the present study, irrespective of the p / l ratio being similar or more radiopaque than a2 or a3 aluminum steps (figure 2), they could eventually be detected against an enamel background (good clinical property). generally, chain displacement can occur at sufficiently high stress in deformable polymers, than in brittle polymeric and ceramic materials, which undergo crack propagation at high stress. (2003) showed that when the p / l ratio is decreased and the matrix volume increased as a consequence, the characteristics of the resin matrix are significantly emphasized for the rmgics. it is expected that greater presence of resin in rmgics, by reduction of p / l ratio, would render a material with more viscoelastic behavior, greater strain capacity and possibly greater resistance to stress development by load application. however, even if a more viscoelastic behavior is expected, the tendency for generating materials which withstand less load application is real because in the cement mixtures, the volume fraction of the matrix, which has weak mechanical strength, increases at lower p / l ratios. irrespective of the p / l ratio, this study showed that rmgics ' strength was always higher than that of conventional gics (table 1), possibly due to the greater expected viscoelastic behavior of rmgics and the higher cohesive strength of resin matrix versus salt matrix. on the other hand, dts was reduced only for rmgics when 50% reduced p / l ratio was employed (table 1). when comparing rmgics and gics, yamazaki,. (2006) showed they possess similar viscoelastic behavior, irrespective of the polymeric character of rmgics. then, it is possible that resin and salt matrixes act similarly, in these materials, with a higher probability of enabling plastic deformation for the former. however, mitsuhashi,. (2003) found that the fracture toughness of the rmgics is not greatly influenced by the p / l ratio, as it is for gics. only at high reductions in p / l ratio, the fracture toughness started to decrease for rmgics. with lower fracture toughness materials became more brittle, reducing plastic behavior and also the resistance against crack propagation. thus, differently from gics, a significant alteration in rmgics properties is only expected with high reduction of powder amount, because the acid - base reaction of the powder particle and liquid is critical for many physical properties of hardened materials. the sem analysis showed an integrated microstructure for rmgics with manufacturer 's recommended p / l ratio (fig 3), but the same was not true for 50% reduced p / l ratio, which showed a greater presence of matrix per volume, unreacted particles and crack development, recognized as a signal of the jeopardized interaction between matrix and powder particles (figure 5). the high reduction in p / l ratio, as performed in the present study, resulted in significantly lower dts values just for rmgics, and not for conventional gics (table 1). in spite of that, for all materials, a decrease in dts occurred with the reduction in p / l ratio. one is that limitations of the experimental design did not allow differences between manufacturer 's recommended p / l ratio and 50% reduced p the other possibility is closely related to the setting reactions of each type of material. for light - cured gics, the maturation of cure by chemical setting has an important influence on the physical properties of the hardened materials because an increase in the overall properties is observed with material 's maturation. more integrated microstructures with better glass particle - polymer matrix bonding, results in higher values of dts, which could be seen in manufacturer 's recommended p / l ratio (figure 2). (2004) showed that the use of silanated glass in polyacid - modified resin composites results in a decrease in flexural strength because not all particles seem to participate of the setting reaction. this leads to a less cohesive matrix, and the same situation is expected on rmgics, which leads to the conclusion that there is an important role of the salt part of the matrix in the overall strength of these materials. it is speculated, from the dts results of the present study that a high reduction in p / l ratio leads to a more fluid mass, where the completion of the chemical reaction is reduced through the polymerization of the resinous part, as previously stated by yelamanchili and darvel (2008). even if the chemical reaction is generally able to keep going after light polymerization, it seems a greater volume of resin by reduction in p / l ratio, entraps polyacid molecules, and the so important chemical reaction reduces in effectiveness. according to peutzfeldt,. (1997) hema molecule crosslinking keeps the carboxylate groups of different polyacid chains too far apart to be crosslinked via ca as will normally happen without resin. since chemical and light irradiated network formation compete, but with 50% reduced p / l ratio the chemical reaction is slowed down, strength of final product will be negatively influenced. in addition, the higher proportion of the resinous part does not assure better mechanical properties because light irradiation results in a temperature rise, potentially permitting a higher value of the " auto - limiting " glass - transition temperature, with a consequent negative effect of degree of conversion. figure 5 shows an apparent ineffective interaction between particles and matrix as a result of the " hema blocking effect ". the assumption that both the salt matrix and the resinous matrix have a determinant relationship in the overall strength of rmgics was confirmed in this study, but needs more specific studies to be proved. the reduction in p / l ratio for gics did not result in significantly lower dts, which mean that the polyacid is able to react with normally unreacted glass particles and the greater presence of salt matrix does not significantly reduces strength. sem analysis showed a lower presence of salt matrix in manufacturer 's recommended p / l ratio (figure 6) and clear signs of cracks around unreacted powder particles, which mean a high tendency for brittle characteristics in these ionomers. at 50% reduced p / l ratio (figure 7) a greater presence of salt matrix can be seen, with smaller particles and no signals of cracks. these observations led to the assumption that even if a better interaction between matrix and powder particles is seen when more liquid is added to the mixture, gics ' dts can be considered more stable when alterations in p / l ratio are performed, since different morphological structures are formed and can resist differently to load application. however, the reduction in absolute dts means for vitro molar and vitro fil should be taken into consideration, meaning that more salt matrix can possibly result in poor mechanical properties with time. the use of the manufacturers ' recommended p / l ratio is always advisable since properties will be preserved. alterations in the p / l ratio should be avoided since a decrease in radiodensity and dts was seen ; 2. rmgics had a significant decrease in dts, but all materials were affected in their radiodensity due to alterations in p / l ratio ; 3. the dts of rmgics seems to be highly influenced by structural organization (organic molecules and inorganic particles), while radiodensity is affected by chemical composition. authors are grateful to dfl for full donation of the materials used in this study, and to capes - brazil (coordenadoria de aperfeioamento de pessoal de nvel superior) for rodrigo b. fonseca phd program support. | objectiveto determine the influence of p / l ratio on the radiodensity and diametral tensile strength (dts) of glass ionomer cements. material and methodsthere were 2 factors under study : p / l ratio (manufacturer 's recommended p / l ratio and a 50% reduced p / l ratio), and materials (vitro molar, vitro fil, vitro cem conventional gics and vitro fil lc, ortho glass lc rmgics). five 1-mm - thick samples of each material - p / l ratio were produced for radiodensity evaluation. samples were x - ray exposed onto digora phosphor plate and radiodensity was obtained using the software digora for windows 2.5 rev 0. for dts, five (4.0x8.0 mm) cylinder samples of each material were tested (0.5 mm / min). data were subjected to one- and two - way anova (5x2) followed by tukey 's hsd test, or kruskal - wallis and dunn 's method. for paired comparisons, t - test or mann - whitney test were used (a=0.05).resultsthere was a significant interaction (p=0.001) for the studied factors (materials vs. p / l ratio). reduced p / l ratio resulted in significantly lower dts for the rmgics, but radiodensity was affected for all materials (p<0.05). conclusionsreduced p / l ratio affected properties of the tested glass ionomer cements. rmgics were more susceptible to lower values of dts, but radiodensity decreased for all materials following p / l ratio reduction. |
the imaging was performed using a 1300 nm sd - oct system shown in fig. s1. a superluminescent diode (sld) with a bandwidth of 105 nm was used as the light source, giving an axial resolution of 9 m. an identical pair of achromatic doublets with focal lengths of 30 mm was used to image the fiber core to the sample, resulting in a numerical aperture (na) of 0.1. the rayleigh range was calculated to be 50 m (using 4.7 m as the radius of the beam at the beam waist) while the imaging depth from the spectrometer (bayspec, inc.) the beam was raster scanned along the fast - scanning and slow - scanning axes over the sample. customized driving waveforms (85% linear, 15% fly - back) for the x - y galvanometer pair (scanlab, scancube 7) were generated by a daq board (national instruments, ni - pcie-6353), which also generated a control signal to synchronize the scanning with the acquisition from the ingaas line - scan camera (goodrich, su - ldh2). the camera was operated at the maximum a - scan rate of 91.912 khz and was interfaced through an image acquisition board (national instruments, ni - pcie-1427). the software for data acquisition and graphical user interface was developed in labview and the data was processed in real - time through dynamic link library (dll) function calls implemented in c (microsoft visual studio 2008 environment). a computer with an intel core i7 processor (975 @ 3.3 ghz, 12 gb ddr3 ram) was used for running the system and the compute unified device architecture (cuda) extension v4.1 from nvidia was used for gpu kernel calls on the nvidia geforce gtx 580 gpu. the ex vivo samples were placed on a three - axis stage separate from the beam collimator and scanning galvos while the in vivo data was taken by gently pressing the tissue of interest onto a custom mount attached to the scancube and beam collimator. by attaching a coverslip mount directly to the lens tube containing the objective lens, which was attached to the scanning optics and fiber collimator, the phase stability requirements for isam acquired volumes consisted of 1024 pixels per a - scan and 810 a - scans along each transverse dimension resulting in an acquisition time of approximately 8.5 seconds. a multi - streamed gpu program was developed for real - time 3-d isam (see supplementary fig. the streams executed concurrently, performing two orthogonal 2-d isam reconstructions where one stream processed the fast - axis frames while the other stream processed the orthogonal dimension of the previously acquired, 2-d isam processed data. this decomposition required only a single volume buffer in gpu memory at any given time because each previously acquired slow - axis frame can be replaced by the currently acquired fast - axis frame. all the processing on the gpu was done with single precision floating point operations and gamma correction was applied on the processed data for display purposes. in this manuscript, the term real - time means that data acquisition was never paused for processing, and that data is continuously being updated for the user, albeit, the data being displayed is at a small latency of one volume. (8.5 seconds for the dimensions mentioned in experimental setup). if we were to allow the camera to pause between volume acquisitions to complete the isam reconstruction, the latency would be reduced even further (to 1.5 seconds for the same volume). see supplementary methods for further details. to remove any bias in displaying comparisons between oct and isam, all comparisons were viewed on the same histogram scale using the same upper and lower saturation values. in real time, our system has the ability to both process and display cross - sectional and user - selected en face planes in the 2-d and 3-d isam reconstructions. in post - processing for further visualization, the calculated energy in each isam tomogram (en face plane by en face plane) was normalized to that of the oct tomogram. after power - normalization, the noise floors in the oct and the isam tomograms were in excellent agreement. to further visualize complete oct and isam datasets, the volume was first segmented into two regions, the stratum corneum / stratum lucidum (containing the sweat - ducts) and the dermis, then different color maps were used for each. image metrics such as anisotropy, signal - to - noise ratio, and contrast were computed to quantitatively evaluate the improvement in reconstruction quality. the anisotropy image quality metric was originally devised to be sensitive to additive gaussian noise in incoherent imaging systems. further testing on various tissue phantoms and tissue data (data not shown) confirmed that this metric is reliable for noise present in oct data as well. signal - to - noise and contrast were computed by using the 20% (noise) and 90% (signal) quantiles of the intensity histograms. the imaging was performed using a 1300 nm sd - oct system shown in fig. s1. a superluminescent diode (sld) with a bandwidth of 105 nm was used as the light source, giving an axial resolution of 9 m. an identical pair of achromatic doublets with focal lengths of 30 mm was used to image the fiber core to the sample, resulting in a numerical aperture (na) of 0.1. the rayleigh range was calculated to be 50 m (using 4.7 m as the radius of the beam at the beam waist) while the imaging depth from the spectrometer (bayspec, inc.) the beam was raster scanned along the fast - scanning and slow - scanning axes over the sample. customized driving waveforms (85% linear, 15% fly - back) for the x - y galvanometer pair (scanlab, scancube 7) were generated by a daq board (national instruments, ni - pcie-6353), which also generated a control signal to synchronize the scanning with the acquisition from the ingaas line - scan camera (goodrich, su - ldh2). the camera was operated at the maximum a - scan rate of 91.912 khz and was interfaced through an image acquisition board (national instruments, ni - pcie-1427). the software for data acquisition and graphical user interface was developed in labview and the data was processed in real - time through dynamic link library (dll) function calls implemented in c (microsoft visual studio 2008 environment). a computer with an intel core i7 processor (975 @ 3.3 ghz, 12 gb ddr3 ram) was used for running the system and the compute unified device architecture (cuda) extension v4.1 from nvidia was used for gpu kernel calls on the nvidia geforce gtx 580 gpu. the ex vivo samples were placed on a three - axis stage separate from the beam collimator and scanning galvos while the in vivo data was taken by gently pressing the tissue of interest onto a custom mount attached to the scancube and beam collimator. by attaching a coverslip mount directly to the lens tube containing the objective lens, which was attached to the scanning optics and fiber collimator, the phase stability requirements for isam acquired volumes consisted of 1024 pixels per a - scan and 810 a - scans along each transverse dimension resulting in an acquisition time of approximately 8.5 seconds. a multi - streamed gpu program was developed for real - time 3-d isam (see supplementary fig. the streams executed concurrently, performing two orthogonal 2-d isam reconstructions where one stream processed the fast - axis frames while the other stream processed the orthogonal dimension of the previously acquired, 2-d isam processed data. this decomposition required only a single volume buffer in gpu memory at any given time because each previously acquired slow - axis frame can be replaced by the currently acquired fast - axis frame. all the processing on the gpu was done with single precision floating point operations and gamma correction was applied on the processed data for display purposes. in this manuscript, the term real - time means that data acquisition was never paused for processing, and that data is continuously being updated for the user, albeit, the data being displayed is at a small latency of one volume. (8.5 seconds for the dimensions mentioned in experimental setup). if we were to allow the camera to pause between volume acquisitions to complete the isam reconstruction, the latency would be reduced even further (to 1.5 seconds for the same volume). to remove any bias in displaying comparisons between oct and isam, all comparisons were viewed on the same histogram scale using the same upper and lower saturation values. in real time, our system has the ability to both process and display cross - sectional and user - selected en face planes in the 2-d and 3-d isam reconstructions. in post - processing for further visualization, the calculated energy in each isam tomogram (en face plane by en face plane) was normalized to that of the oct tomogram. after power - normalization, the noise floors in the oct and the isam tomograms were in excellent agreement. to further visualize complete oct and isam datasets, the tomograms were depth - normalized to a common histogram scale. the volume was first segmented into two regions, the stratum corneum / stratum lucidum (containing the sweat - ducts) and the dermis, then different color maps were used for each. image metrics such as anisotropy, signal - to - noise ratio, and contrast were computed to quantitatively evaluate the improvement in reconstruction quality. the anisotropy image quality metric was originally devised to be sensitive to additive gaussian noise in incoherent imaging systems. further testing on various tissue phantoms and tissue data (data not shown) confirmed that this metric is reliable for noise present in oct data as well. signal - to - noise and contrast were computed by using the 20% (noise) and 90% (signal) quantiles of the intensity histograms. | high - resolution real - time tomography of scattering tissues is important for many areas of medicine and biology16. however, the compromise between transverse resolution and depth - of - field in addition to low sensitivity deep in tissue continue to impede progress towards cellular - level volumetric tomography. computed imaging has the potential to solve these long - standing limitations. interferometric synthetic aperture microscopy (isam)79 is a computed imaging technique enabling high - resolution volumetric tomography with spatially invariant resolution. however, its potential for clinical diagnostics remains largely untapped since full volume reconstructions required lengthy postprocessing, and the phase - stability requirements have been difficult to satisfy in vivo. here we demonstrate how 3-d fourier - domain resampling, in combination with high - speed optical coherence tomography (oct), can achieve high - resolution in vivo tomography. enhanced depth sensitivity was achieved over a depth - of - field extended in real time by more than an order of magnitude. this work lays the foundation for high - speed volumetric cellular - level tomography. |
this ethical principle is vital to the nurse - patient relationship and reveals a thought of reverence towards patients as human beings and towards patients ' rights [2, 3 ]. indeed, patients ' needs or desires are recognized as a key impulsion for advocacy in nursing. therefore, it is defined as a crucial section of nurses ' attempts to encourage and protect health and interests of patients by supplying information and assisting clients in their decisions, cooperating to patients ' self - determination, autonomy, or empowerment, pleading the reason of client, defending the client from pointless worries, revealing information about misbehavior that imperils the welfare of others, and respecting patients values and beliefs, together with educating and interceding. curtin and gadow stated that advocacy is the elemental foundation for nursing and exclusively illustrates the essence of nursing practice [10, 11 ]. maybe the clearest definition of advocacy in nursing can be found in gadow 's explanation. advocacy not only safeguards but positively contributes to the exercise of self - determination [11, page 53 ]. kohnke recommended that advocacy is a type of caring and kindness in nursing practice and that it is a learned skill that nurses would develop through different experiences, especially if advocacy is esteemed as worthwhile. a great deal of the argument of patient advocacy is originated from the identification and valuing of patient rights and the task of nurses as advocates for the benefits and rights of people [12, 13 ]. these definitions emphasize the nurse 's role as a faithful observer of the patient 's circumstance [4, 14 ], signifying an ethical commitment to make certain quality of care. it is necessary that nurses comprehend and show their scope of practice and specify their professional autonomy in the health care team, in the organizations, and especially in the community. if nursing professionals want to make their clients powerful, they must empower themselves in the first place and develop the ability to make choices, such that the choice or decision on how to deal with patient advocacy would lead to suitable perception and attitudes towards patient advocacy. if the knowledge about patient advocacy is adequate, there would be correct perception leading to positive attitude towards patient advocacy and vice versa. perception is the ability to see, hear, or understand a certain event and apply assumptions about the world 's arrangement to integrate sensory information. an attitude refers to feelings, beliefs, and positive and negative reactions of an individual towards an event, phenomenon, object, or person to arise through the internalization of cognitive structures. the literature contained convincing arguments in favor of nurses ' assumption regarding the role of patient advocate. nurses ' intrinsic characteristics are cited as critical components in nurses ' ability to act as nursing advocates [20, 21 ]. it seems that nurses ' characteristics such as self - concept, personal values, confidence as nurses, and personal beliefs play a main role in nurses ' advocacy actions. generally, only a few quantitative studies have evaluated nursing advocacy, and the majority have been qualitative. in iran, negarandeh. have reported one qualitative grounded theory - type study to inquire into the meaning of patient advocacy from iranian nurses ' perspective. according to the investigators, no research with quantitative method, to measure nurses ' attitudes and perception relationship, so, this descriptive - analytic study aimed to examine nurses ' attitudes and perception towards patient advocacy in an iranian health care context with a quantitative method ; this may help to clarify the meaning of this complex issue. the proposal of this study was approved in kerman razi school of nursing and midwifery. the research approval and ethic committees of kerman university of medical sciences approved this study, and the permission to conduct this study was also obtained from the presidents of four educational hospitals in kerman city. moreover, a descriptive - analytic design was applied, and this study was conducted in those four educational hospitals. informed consent was obtained from all participants, and they were free to decline to participate or to consent but later withdraw their consent without prejudicing their position in the organization. a comprehensive three - part questionnaire was applied to describe the population and collect information regarding nurses ' attitudes and perception towards nursing advocacy. part i, created by us, consisted of one page with seven demographic variables including the hospital name, the ward, age, sex, marital status, nursing work experience, and history of education regarding patient right 's workshop. in part ii, we designed a questionnaire using selective parts of attitude measuring instruments in nursing advocacy that were formerly developed in two other studies by barrett - sheridan and hanks. the participants in this study were persian nurses, so those questionnaires were translated into persian using translation and back - translation techniques by two specialists, and cross - cultured adaptation was also conducted. at first, the questionnaire was designed with twenty - three questions, and then the number of questions was reduced to nineteen, after factor analysis. the questions were scored from 1 to 5 using a five - point likert scale (1 = strongly disagree to 5 = strongly agree). the data was assessed as factorable using kaiser meyer olkin (kmo) test of sampling adequacy,.85, and bartlett 's test of sphericity, p value = 0.00, for factor analysis. so, factor analysis was conducted using principal components analysis (pca) with varimax rotation scheme. the two factors were labeled as factor 1 for cognitive (believe) aspect of attitude (9 items) and factor 2 for behavior (efficacy) aspect of attitude (10 items). part iii, the nineteen - item self - administered perception questionnaire, was built by means of diversity studies. the respondents were required to choose their answers from a set of preprovided answers (yes, no, and do not know). the average score for each question was identified in attitude questionnaire with a sorting index of (1 - 2) negative attitude, (2 - 3) relatively negative attitude, (3) neutral attitude, (3 - 4) relatively positive attitude, and (4 - 5) positive attitude and in perception questionnaire with a sorting index of (00.25) weak, (0.250.5) relatively weak perception, (0.50.75) relatively suitable perception, and (0.751) suitable perception. an expert panel on nursing ethical issues have reviewed the content of the scales from ethical and cultural aspects of nursing advocacy and agreed on a reasonable content validity. to reassess the reliability of the scales, cronbach alpha was calculated to evaluate internal consistency among items. firstly, the alpha coefficient for attitude scale was 0.75, and after factor analysis, this was 0.77, and for questions in factor 1 of attitude scale, it was 0.88, and factor 2 was 0.77. the written information about the aims of this study was given to the participants in the form of a letter ; the questionnaires were handed out by the author to 385 nurses who were selected by quota sampling in four kerman educational hospitals. three hundred seventy - four sets of questionnaires were distributed, with a dropout of eleven. in whole, the descriptive statistics of the samples and measures included frequencies, means, and reliability. one - way analysis of variance (anova) was applied to examine the relationships between the mean level of attitude, perception, and demographic variables. pearson correlation analysis was used to show any significant correlation between nurses ' attitude and perception. a descriptive analysis of the demographic information (table 1) showed that the participants ' age ranged from 22 to 55 years with a mean age of 30 years and were mostly females (93%), married (63%), with the bachelor degree of science in nursing (98%), with six - month to ten - year experience of working in hospitals (70%). however 54% of the participants were general ward 's nurses, and only 37.2% claimed that they were educated patient rights. the descriptive analysis (table 2) indicated a fairly positive attitude (mean = 3.79) and perception (mean = 0.783) towards nursing advocacy amongst the participants. it also indicated that the participants had more positive attitude (4.27) toward cognitive aspects compared to behavioral aspect (3.24). the comparison of the mean scores of the nurses ' attitude and perception toward nursing advocacy and demographic characteristics of the nurses (table 1) revealed that the mean score of attitude and perception amongst the male participants with the masters degree in nursing, and those who had already participated in patient right 's workshop and nurses who were working in psychotic wards, was more than others. in addition, there was a significant difference between the mean scores of attitude (p value = 0.00) and perception (p value = 0.009) of nurses who were working in different hospital. pearson correlation analysis (table 3) showed a significant and positive correlation between nurses ' attitude and perception (r =.351) toward nursing advocacy and also between cognitive (believe) aspects of attitude (r =.497) and behavior (efficacy) aspects of attitude (r =.024) with nurses ' perception toward nursing advocacy. based on these results, it seems that the correlation of nurses ' perception with the cognitive aspect of attitude is more positive than the behavioral aspect. according to the figures presented in table 4, gender and marital status were positively correlated with attitudes, and such a correlation was more positive amongst male and single participants. it should be noted that the correlation between the participants ' attitude and perception and their educational level and the background experience of nursing was also positive, and such a correlation regarding the bachelor 's degree and experience of nursing between twenty to thirty years was more positive when compared to others. the nurses ' perception and attitudes amongst those working in psychiatric hospital and psychiatric wards were positively correlated with each other, and this correlation among participating nurses in patient right 's workshop was positive too. in general, according to the results of this study, improving nurses ' perception would lead to a promotion in their attitude toward nursing advocacy. the results of this study indicated that nurses ' attitudes and perception toward patient advocacy were fairly positive. these results supported the results of two other studies by barrett - sheridan who had found positive nurses ' attitude toward nursing advocacy and by thacker who had stated that most participants agreed with perceptual behavior of nursing advocacy. furthermore, other findings have shown that nurses ' attitudes were positively correlated to their perception (r =.351) toward nursing advocacy. since perception is the ability to see, hear, or understand a certain event and awareness to improve upon such power of perception and attitudes are acquisitive responses that allude to feelings, values, and reactions of an individual towards an occurrence phenomena, if nurses ' knowledge toward nursing advocacy is not acceptable enough, it will be perceived wrongly or negatively, and this can lead to a negative or neutral attitude towards nursing advocacy. if the knowledge about nursing advocacy is sufficient and understandable, correct or right perception would be gained, and this would lead to a positive attitude towards nursing advocacy. the authors of this study concluded that the nurses who were working in psychiatric wards of psychiatric hospitals had more positive attitude and perception compared to others ; it seems that these nurses ' knowledge and perception of nursing advocacy is more than others, and they put this knowledge into practice, indicating their positive attitude. according to boutell and bozett, mental health nurses were more likely to evaluate patient 's spiritual needs because they had more time and were accustomed to consult patients. so, there is a need to conduct more studies in vaster areas and to increase the sample size for improving generalization. it is better to carry out other studies with a larger sample size in more cities. the present study with a focus on iranian nurses ' attitudes and perception towards the responsibilities of patient advocacy explains how nurses ' positive perception would influence on increasing nurses ' positive attitude. it goes without saying that the iranian nurses have been advocated for patients since many years ago, without having nursing advocacy as a professional role in iran, advocacy educational programs, and supports from their employers for doing this role. furthermore, educating nursing advocacy is necessary in the iranian nursing student curriculum, and it should be continued for nurses to improve the quality of this role. more studies will brightly develop the identification of advocate role and support changes needed in the workplace setting to promote advocacy action for patients ' sake. | patient advocacy is an inherent component of professional nursing ethics ; in other words, nurses ' enough knowledge would be essential to gain a positive attitude towards nursing advocacy. using a descriptive - analytic design, this study aimed to assess the correlation between nurses ' perception and attitudes towards patient advocacy, amongst 385 nurses in kerman, iran ; hence, a three - part questionnaire was applied : part i, a demographic data sheet, part ii, attitude measuring instrument, and part iii, perception measuring instrument in nursing advocacy. the results implied that fairly positive attitudes and perception were found amongst the participants, and nurses ' attitudes, in general, were positively correlated to their perception toward nursing advocacy. this means that with an improvement in perception, the attitude would also improve. in addition to our findings, it seems that these nurses needed more advocacy educational programs and support from responsible employers. |
the term odontogenic keratocyst (okc) was used for the first time by philipsen in 1956 in defining an odontogenic cyst with parakeratinized epithelial surface. despite its bland histology, okc shows more aggressive behavior and higher expression rate which make it different from other odontogenic cysts that produce keratin. it has been a long time since many researchers supported that the cyst has a neoplastic nature due to its biological behaviors and based on a number of molecular and genetic evidences, they found. this is a reason why, in its recent classification, who has called this lesion a benign neoplasm entitled keratocystic odontogenic tumor (kcot), because the epithelial surface of okc shows a higher mitotic activity than other odontogenic cysts. okc may be found at any age, but the outbreak of this type of cyst is more common in the second and third decades of life and also it is common more in males than females. it happens in the mandible in 60% to 80% of the cases and it has special tendency toward mandibular angle and ascending ramus. radiographically, okc appears in the form of unilocular or multilocular radiolucency with specific borders and usually with sclerotic smooth margins. the histopathologic characteristics of okc are pathognomonic and include uniform thickness, rete ridgeless stratified squamous epithelium, corrugated superficial parakeratinization, basal cell hyperchromatism, reverse polarization, and palisading arrangement. in the past, however, it was revealed that the orthokeratinized type not only lacks the typical characteristics of the parakeratinized type and composed of orthokeratinized stratified squamous epithelium with a prominent granular layer, but also shows different biological or clinical behaviors in the way that its recurrence is very much lower than the parakeratinized type. this is the reason why nowadays the orthokeratinized type is considered a different cyst with different histopathological and clinical characteristics. therefore, in this study, we were going to compare the okcs and oocs through investigating the extent of p53 protein expression which is one of the tumor suppressors and tgf - alpha expression which is a member of the growth factors family. note that both markers can be found in the head and neck cancers higher than normal tissues. by doing so a total of 30 cases including those of 15 okc and 15 ooc were used in this study. 3 - 4 m sections from paraffin - embedded specimens were mounted on poly - l - lysine - coated glass slides. after deparaffinization and rehydration with five descending alcohol, the sections were incubated in citrate buffer in a microwave oven for 15 min for antigen retrieval and incubated in 0.5% h2o2 in methanol for 10 min to block endogenous peroxidase activity and then rinsed with phosphate - buffered saline (pbs). specimens were incubated for 1 h with the lyophilized monoclonal anti - p53 (ncl - p53-do1, novacastra, germany) at a dilution of 1:50 and the lyophilized monoclonal anti - tgf - alpha (ncl - tgf r1, novacastra, germany) at a dilution of 1:100. immunocomplexes were subsequently treated with post - primary block and then detected by novolink polymer (novacastra, germany) for 30 min, both incubated for 30 min at room temperature. after rinsing with pbs, sections were finally counterstained with hematoxylin, cleared and mounted with pv mount, and slides were blindly viewed independently by two oral pathologists by light microscopy (olympus bx41tf, tokyo, japan). the percentage of positive epithelial cells in ten high - power fields of a microscope was determined and with regard to cytoplasmic positivity for tgf - alpha and nuclear positivity for p53 antigen, classified at a scale 1 - 4 : (+ 1) 025% positive cells ; (+ 2) 26 - 50% positive cells ; (+ 3) 51 - 75% positive cells ; (+ 4) 76100% positive cells. also intensity of staining with p53 and tgf - alpha antigen was evaluated on the following method : (0) when cells had not been staining and (+ 1), (+ 2), (+ 3), (+ 4) for very low, low, moderate, and high staining. finally the sid (staining intensity distribution) score was calculated by multiplication of these two scores for each specimen. the data were analyzed by means of statistical software spss 10 with the mann whitney and wilcoxon statistical tests at a significant level of 0.05 for comparison of data between p53 protein and tgf - alpha. the specimens were examined at 400 magnification in a light microscope. the percentage of positive epithelial cells in ten high - power fields of a microscope was determined and with regard to cytoplasmic positivity for tgf - alpha and nuclear positivity for p53 antigen, classified at a scale 1 - 4 : (+ 1) 025% positive cells ; (+ 2) 26 - 50% positive cells ; (+ 3) 51 - 75% positive cells ; (+ 4) 76100% positive cells. also intensity of staining with p53 and tgf - alpha antigen was evaluated on the following method : (0) when cells had not been staining and (+ 1), (+ 2), (+ 3), (+ 4) for very low, low, moderate, and high staining. finally the sid (staining intensity distribution) score was calculated by multiplication of these two scores for each specimen. the data were analyzed by means of statistical software spss 10 with the mann whitney and wilcoxon statistical tests at a significant level of 0.05 for comparison of data between p53 protein and tgf - alpha. the expression with p53 protein leads to definite bright brown staining in the nucleus of the epithelial cells. according to wilcoxon 's statistical test, the expression in basal and parabasal layers of okc for p53 protein was not statistically significant (p value = 0.356) but was statistically significant for tgf - alpha (p value = 0.041). the expression in the basal and parabasal layers of ooc for p53 protein was not statistically significant but was near to significant (p value = 0.053) and also was statistically significant for tgf - alpha (p value = 0.012). according to mann the average expression for p53 protein in the basal layer in okc was higher than ooc but this difference was not statistically significant (p value = 0.076) [table 1 ] [figure 1a b ]. p53 protein expression in okc and ooc (a) p53 protein expression in the basal and parabasal layers of okc (400) ; (b) p53 protein expression in the basal and parabasal layers of ooc (400) the average expression for p53 protein in the parabasal layer in okc was higher than ooc and this difference was statistically significant (p value = 0.003) [table 1 ] [figure 1a b ]. the expression for tgf - alpha was localized to the cytoplasm. according to mann the average expression for tgf - alpha in the basal layer in okc was higher than ooc but this difference was not statistically significant (p value = 0.284) [table 2 ] [figure 2a b ]. tgf - alpha expression in okc and ooc (a) tgf - alpha expression in the basal and parabasal layers of okc (400) ; (b) tgf - alpha expression in the basal and parabasal layers of ooc (400) the average expression for tgf - alpha in the parabasal layer in okc was higher than ooc and this difference was statistically significant (p value = 0.015) [table 2 ] [figure 2a b ]. figure 3 compares the expression of p53 protein and tgf - alpha in okc and ooc. p53 protein and tgf - alpha expression in the basal and parabasal layers of okc and ooc the growth mechanism and biologic behavior of okc is different from the more prevalent radicular and dentigerous cysts, in which, contrary to other cysts, the main factor in the growth of okc is proliferation of epithelial lining of the cyst under the induced effect of ectomesenchyme. etiology and pathogenesis of ooc are not clear. some of the researchers proposed that ooc be considered as metapelastic orthokeratinized dentigerous cysts because of the common relationship between occ and the unerupted teeth, and its less aggressive biological behavior compared to okc. the findings of the present study indicate that the average of cell expression of p53 protein in the parabasal layer of okc is more than that of ooc. such findings support li which indicates that the number of positive p53 protein cells is the highest in okc and it is mainly in the parabasal layer. slootweg believes that the increase in proliferation is not necessarily related to the mutation of the p53 gene. this phenomenon can not be determined through immunohistochemical studies, because stabilization of p53 protein can occur due to the increase in protein production or protection of p53 protein against damage by bonding to viral or other cell proteins. lo muzio investigated the expression of p63 that is from the p53 family in several cysts. it was indicated that okcs, especially the parakeratinized ones, had the highest number of positive p63 cells and the higher dissemination of p63 in parakeratinized okcs in comparison with orthokeratinized types can account for the clinical and pathological characteristics of okc and ooc. also the expression of p63 in the orthokeratinized type was limited to basal and parabasal layers and only in 50% of the cases, the cells of the middle layer were stained, while the cells had expression in all layers (basal, parabasal, and surface basal) in the parakeratinized type. this finding is in accordance with those of the present study which indicate that okc has significantly higher expression in the parabasal layer for p53 protein in comparison with ooc. findings of the present study also accord with those of dong in which the markers ki67 and p63 were significantly in lower amount in orthokeratinized type than in okc. also, in orthokeratinized type, p63 is observed merely in the basal layer and some parts of the suprabasal layer, while in parakeratinized type, the expression was observed in all layers except for the superficial parakeratinized layer. in contrast, moghaddam. compared ameloblastoma with okc, and found that only 5.12% of okcs had positive areas for p53 protein. accordingly, it was concluded that among okcs, some were p53 positive and some were p53 negative. it was also probable that because the nature of the lesion was benign, okc was not positive for p53 protein. p53 markers between okcs and oocs demonstrated that the expression of okc for ki67 marker was significantly higher than ooc and deduced that is a reason for the aggressive behavior of okc and its tendency to recur more. they attributed this finding, in contrast with the findings of the present study, to the neoplastic changes in the epithelial lining of ooc. the possible reason for such a contrast is the use of two different techniques which may cause different levels of accuracy. another point is that the difference found in baghaei. was not statistically significant and therefore not enough for rejecting the obtained findings of this study. another finding of the present study was the presence of a higher average of expression for tgf - alpha in the basal layer of okc than in ooc. while this difference is not statistically significant, the difference in the parabasal layer is statistically significant. li indicates that the epithelial tissues of all types of odontogenic cysts have tgf - alpha gene manifestation despite the fact that the stainability of various cysts is different and its amount is higher in okcs in comparison to radicular cysts. this feature accords with higher layers of egf - r gene expression and with ki67 nuclear antigene in okc epithelium. this demonstrates that these cysts have a unique growth characteristic which is not observed in other odontogenic cysts. it has been proved that the simultaneous gene expression of tgf - alpha and tgf - r in neoplastic cells results in the growth feature of these cells compared to the normal cells. the strong reaction of tgf - alpha in accordance with high levels of gene expression of tgf - r in okc demonstrates that tgf - alpha can act as a growth factor to stimulate cell proliferation and differentiation in this cyst type. 's study demonstrated that tgf- alpha was stained + 2 and + 3 and in a patchy form for okc in all cases, while the expression for most of the ameloblastomas was high. it is reasoned that this protein is secreted as endogenic by tumor cells and this can reinforce proliferation of ameloblastoma in affected area. the last finding of this study indicates that the expression of tgf - alpha and p53 protein is higher in the basal layer of both okc and ooc than in their parabasal layer. this relation is statistically significant for tgf - alpha and not significant for p53 protein. in previous studies conducted on ki67 protein, the proposed reason was that parabasal layer cells are in a stage of cell cycle which has higher expression of ki67. one possible justification for this finding is that basal layer cells are mostly in the form of stem cells with slow cell cycle and long the g1phase. our hypothesis was that the probability of tgf - alpha and p53 expressions in the parabasal layer will be higher than in the basal layer. this may be due to be expression of p53 protein and tgf - alpha is not directly related to mitosis. therefore, it is not possible to generalize the findings for ki67 to p53 protein and tgf - alpha. considering the higher expression of p53 protein and tgf - alpha in okcs in comparison with ooc, the possibility of carcinomatous changes of okc is, at least theoretically, higher than that of ooc cysts. however, the finding of long - term follow - ups of okc does not confirm this hypothesis and it is rare if any. | background : odontogenic keratocyst (okc) is an aggressive cyst and its recurrence is higher than other odontogenic cysts, orthokeratinized odontogenic cyst (ooc) is a cyst with moderate biological behavior in comparison with okc, but with the probability of carcinomatous changes. the present study aims to evaluate the quantity and intensity of the expression of p53 protein and transforming growth factor alpha (tgf - alpha) in okc and ooc in order to compare the biologic behavior of these two cysts.materials and methods : this is a cross - sectional study. the samples include 30 cysts (15 okc and 15 ooc), all stained immunohistochemically for p53 protein and tgf - alpha by the novolinke polymer method. then, all the cases were examined with an optical microscope with 400 magnification and the stained cells were counted in the basal and parabasal layers. finally the results were analyzed by the mann and wilcoxon tests (p value < 0.05).results : the difference between the expression of p53 protein in the basal layer in okc and ooc was not statistically significant (p value = 0.076). the difference between the expression of p53 protein in the parabasal layer in okc and ooc was statistically significant (p value = 0.003) ; moreover, the difference between the expression of tgf - alpha in the basal layer in okc and ooc was not statistically significant (p value = 0.284). the difference between the expression of tgf - alpha in the parabasal layer in okc and ooc was statistically significant (p value = 0.015).conclusion : since there was a higher expression of p53 protein and tgf - alpha in okc compared to those in ooc, the probability of carcinomatous changes was at least theoretically higher in okc than in ooc. |
long - term outcome of steroid - sensitive nephrotic syndrome (ssns) is usually considered to be good. relapses of ssns become less frequent towards puberty, and eventually permanent remission is achieved. recent reports have shown that almost one - third of children with ssns suffer a relapse during adulthood [1, 2 ]. the clinical course in adult patients with childhood - onset ssns is unclear. four patients with childhood - onset ssns had a relapse after 30 years of age (table 1). nephrotic syndrome (ns) was diagnosed in patients who had heavy proteinuria (more than 40 mg / m / h) and hypoalbuminemia (serum albumin < 2.5 g / dl). patients who responded during 8 weeks of prednisolone (psl) treatment were defined as ssns. relapse was defined as a reappearance of proteinuria (2 + or greater by dipstick for 3 consecutive days). frequent relapses were defined as two or more relapses within the first 6 months after initial response or four or more relapses during any 12-month period. all four patients were treated with psl (60 mg / m / day) until remission was achieved. psl was then tapered using alternate - day doses over a six - week period. the psl was tapered more slowly and the dose of psl was determined individually according to the threshold at which the relapse occurred. the standard deviation (sd) in patient height was calculated using the mean japanese adult height of 170.8 5.8 cm for males and 158.1 5.3 cm for females.table 1profiles of 4 children with relapse of nephrotic syndrome after 30 years of agecase 1case 2case 3case 4sexmalemalefemalefemaleage at onset6 years2 years2 years10 yearsnumber of relapses35535144frequent relapser++++renal biopsy findingfsgstreatmentpsl, cy, mz, cyapsl, cy, mzpsl, cy, cyapsl, cyaage at last relapse39 years35 years34 years33 yearsage at last follow - up41 years36 years35 years33 years fsgs focal segmental glomerulosclerosis, cy cyclophosphamide, mz mizoribine, cya cyclosporine profiles of 4 children with relapse of nephrotic syndrome after 30 years of age fsgs focal segmental glomerulosclerosis, cy cyclophosphamide, mz mizoribine, cya cyclosporine their ages at the onset of ns ranged between 2 and 10 years. neither the number of relapses of ns was between 35 and 53, and frequently relapsing ns was noted in all patients. they were treated with psl combined with cyclophosphamide (cy) in 3 patients (cases 13), mizoribine (mz) in 2 (cases 1 and 2), and cyclosporine (cya) in 3 (cases 1, 3, and 4). cy at a dose of 2 mg / kg for 812 weeks, mz at a dose of 100 mg / m, and cya at a dose to target the trough level of 100 ng / ml were used. the total dose (mg / kg) of cy was 430 in case 1, 275 in case 2, and 90 in case 3. after 20 years of age, the frequency of relapses gradually decreased (table 2). at the last relapse, proteinuria disappeared promptly after the start of psl (table 3). case 1 was obese (body mass index of 30) and was treated with antihypertensive drugs. at the last follow - up, all 4 patients continued to receive psl, had normal renal function, and were in complete remission from ns, when they were between 33 and 41 years of age (table 4). they were not admitted for relapses during adulthood, except for 1 admission for the 33rd relapse of case 1. three patients (cases 13) were working full - time. case 4 was a housewife with two children. there were no signs nor symptoms of insufficiency fracture, aseptic necrosis of femoral capital epiphyses, glucose intolerance, cataract, or malignant diseases in any of the 4 patients.table 2number of relapses per year in each decade of age09 years1019 years2029 years3039 yearscase 11.71.80.90.2case 23.61.70.60.3case 31.42.31.11.0case 42.02.01.5 table 3dose of prednisolone and response to treatment with prednisolone at the onset of nephrotic syndrome and at last relapseat onsetat last relapseprednisolone (mg / m)time to induce remission prednisolone (mg / m)time to induce remission case 145 mg / day (46.9)6 days20 mg / day (9.7)10 dayscase 230 mg / day (55.6)8 days40 mg / day (22.7)9 dayscase 330 mg / day (51.7)7 days5 mg / day (3.6)<4 weeks case 460 mg / day (54.5)9 days45 mg / day (30.4)7 days time from the start of treatment with prednisolone proteinuria was not tested until 4 weeks after treatment with prednisolone table 4clinical findings at last follow - upcase 1case 2case 3case 4height (standard deviation)0.70.81.61.5body mass index (kg / m)30241922blood pressure (mmhg)132/76130/7690/40120/70antihypertensive agentsarb etc.serum creatinine (mg / dl)0.70.70.60.5egfr (ml / min/1.73 m)1009999113proteinuriatreatment prednisolone (mg / day)2.515210 cyclosporine (mg / day)200 arb angiotensin - receptor blocker, egfr estimated glomerular filtration rate number of relapses per year in each decade of age dose of prednisolone and response to treatment with prednisolone at the onset of nephrotic syndrome and at last relapse time from the start of treatment with prednisolone proteinuria was not tested until 4 weeks after treatment with prednisolone clinical findings at last follow - up arb angiotensin - receptor blocker, egfr estimated glomerular filtration rate cy was used at the age of 7 and 10 years, and mz between the age of 19 and 20 years. he had a 33rd relapse at the age of 29 years, and renal biopsy showed focal segmental glomerulosclerosis. an angiotensin - converting enzyme inhibitor was added after the complete remission of ns. at his 34th relapse at 34 years old, he went into complete remission within 2 weeks after the treatment with psl, and psl was tapered for 3 years. at his 35th relapse at 39 years old, cya was started, and angiotensin - converting enzyme inhibitor was changed to angiotensin - receptor blocker and calcium channel blocker. he was treated with cy at 8 and 15 years of age and mz for 1 year at 13 years of age. he developed his 52nd relapse at 31 years of age and was treated with psl for 8 months., his 53rd relapse occurred, and psl was on a tapering dose at the last follow - up. cy, followed by cya for 1 year, was used for growth impairment due to continuous treatment with psl at 510 mg / day at 13 years of age. after 30 years of age, 6 relapses occurred when psl was tapered to 1 mg / day. at her 51st relapse in 34 years old, she was treated with psl 5 mg / day, and her proteinuria disappeared within 1 month. she was on psl 2 mg / day at the last follow - up. she had 4 relapses during treatment with cya, and cya was discontinued at her first pregnancy. she had relapses during pregnancy at 26 and 31 years of age and received psl treatment. her 42nd to 44th relapses occurred between 32 and 33 years of age, and psl treatment was restarted. relapses of ssns become less frequent towards puberty, and eventually permanent remission is achieved. studies from the 1980s reported that no more than 10 % of children had additional relapses in adulthood. more recent reports indicated a relapse rate after 18 years of age of between 27 and 42 % [1, 2 ]. most patients who have relapsed during adulthood developed ns at a young age and were frequent relapsers during childhood [2, 9, 10 ]. between 1972 and 2013, 148 patients were diagnosed with ssns before 15 years of age at toho university omori hospital and sakura hospital. of the 34 patients who were followed after 20 years of age, 12 had at least one relapse after 20 years. the mean onset age for the 12 patients was 6.8 3.6 (2.212.7) years, and 11 of them were frequent relapsers. of the 5 patients who relapsed after 20 years of age and were followed after 30 years of age, 4 patients had one or more relapses after 30 years. to our knowledge, only a few patients who relapsed after 30 years of age have been reported. relapses in a 33-year - old patient and a 39-year - old patient were reported [9, 11 ]. reported 5 patients whose onsets of frequently relapsing minimal change nephrotic syndrome (mcns) ranged between 1 and 3 years of age, and who had relapses between 32 and 42 years of age. patients with adult - onset mcns who have transient hypertension and impaired renal function during the nephrotic phase are not rare, and therapeutic response is slower than in children. according to a report by the international study of kidney disease in children, 8590 % of children with ns achieved complete remission within 4 weeks and 9095 % within 8 weeks after the start of steroid treatment. in adult patients with mcns, in some studies of adult patients given psl, remission occurred in 5060 % of patients after 8 weeks of treatment, and in 7075 % after 16 weeks. in the 4 patients presented here, the time period from the start of treatment with psl to the induction of complete remission was similar to that of childhood ns. frequency of relapse decreased with age, and renal function was normal after 30 years of age. the prognosis was similar to the children with ssns, despite their prolonged course of ns. there does not seem to be any proven way to predict the individual relapse courses of patients with ssns at onset. evaluating the number of relapses during a long - term course, as shown in table 2 of this report, may predict the frequency of relapse during adulthood in some patients. both increased duration and the cochrane database of systematic reviews suggested that duration of psl therapy was more important than the dose of psl. in children with ssns treated with psl for 7 months, frequency of relapse over the course of 2 years significantly decreased compared with that for ssns children treated with psl for 2 months. at relapse in adult patients case 1 presented here suffered a relapse at 39 years of age, and a relapse in a 42-year - old patient has also been reported. although the long - term outcome of ssns is usually considered to be favorable, the chronic course and prolonged treatments involved affect the quality of life of children and also that of their families. social performance and quality of life seemed to be relatively good in the 4 patients reported here. pediatricians should be aware that some children with ssns may require long - term treatment even after they have entered adulthood. | some children with steroid - sensitive nephrotic syndrome (ssns) have been reported to suffer relapses in adulthood, but the clinical course of such adults is unclear. four children with ssns suffered relapses after 30 years of age. those 4 patients developed frequently relapsing nephrotic syndrome (ns) between 2 and 10 years of age. they were treated with prednisolone (psl) combined with cyclophosphamide in 3 patients, mizoribine in 2, and cyclosporine in 1 during childhood, and with cyclosporine in 2 during adulthood. after 20 years of age, the frequency of relapses gradually decreased. the last relapse occurred between 33 and 39 years of age, and proteinuria disappeared within 1 month after the start of treatment with psl. at the last follow - up, all 4 patients continued to receive psl, had normal renal function, and were in complete remission of ns when they were between 33 and 41 years of age. although the long - term outcome of ssns is usually considered to be favorable, pediatricians should be aware that some children with ssns may require long - term treatment during adulthood. |
shock wave lithotripsy (swl) is the preferred treatment modality for most renal stones 2 cm in maximum length. in addition, swl has high patient and practitioners acceptance owing to its convenience and non - invasiveness. in spite of its safety, the high rate of residual fragments (rfs) of the lower caliceal stones (lcss) after swl has been the cause of concern. it has been shown that these rfs can grow and may act as a nidus for further stone formation. there is no standard auxiliary procedure to facilitate the clearance of lcs fragments after swl. as a result, alternative invasive treatment modalities with a high clearance rate, such as percutaneous nephrolithotripsy (pcnl) or ureterorenoscopy (urs), are used instead of swl for lcss. as the gravity - dependent position of the lower calyces is the main factor inhibiting stone clearance after swl for lcs, adjuvant procedures such as intraoperative inversion, urinary diuresis and mechanical percussion are used to facilitate stone clearance. the aim of the current study was to evaluate the combination of two adjuvant procedures, namely intraoperative forced diuresis and inversion, as a method for enhancing clearance of lcs fragments after swl. after institutional ethics board approval, this randomized controlled study was conducted between august 2010 and january 2014. patients 18 years old with symptomatic, single lcs of 520 mm maximum diameter who agreed to participate in the study and provided an informed written consent were included. all patients underwent (a) detailed history, (b) clinical examination, (c) laboratory investigations including routine urinalysis, complete blood cell count, serum creatinine, liver function tests, fasting blood glucose, serum electrolytes and coagulation profile, (d) renal ultrasonography, (e) plain abdominal x - ray for kidneys, ureter and urinary bladder (kub) and (f) non - contrast computed tomography (ncct) supplemented by contrast study if indicated. the stone to skin distance (ssd) and stone attenuation value were also determined on ncct as described elsewhere. patients with morbid obesity, musclo - skeletal disorder that impede positioning, severe cardio - vascular or neurological diseases, previous failed swl, urinary tract infection, elevated serum creatinine, uncontrolled coagulation disorders, abnormal renal anatomy (as horseshoe kidney, duplex kidney or bifid pelvis), severe hydronephrosis, obstructed calyx or urinary tract obstruction at any level in the ipsilateral renal unit and those with medical problems that contradicted overhydration, forced diuresis or inversion therapy, as well as patients requiring a pre - treatment auxiliary procedures such as jj ureteral stent insertion or percutaneous nephrostomy drainage were excluded from the study. patients who met the inclusion criteria were randomly assigned to two treatment groups. to distribute patients efficiently between groups, patients were assigned to one of the two groups across two pre - determined stone size groupings at a cutoff 10 mm. the first group (study group, sg) underwent swl with the patient in the trendelenburg position with an angle of 30 degree combined with intravenous hydration with 1000 ml normal saline solution and 20 mg furosemide that were started 30 min before and continued through the procedure ; hence, swl was performed during the diuresis phase. an electromagnetic shockwave generator was used and all patients were treated with the same machine (dornier lithotripter sii, dornier medtech, wessling, germany). all patients were treated on an outpatient basis without anesthesia. only intravenous sedoanalgesia (meperidine hydrochloride 1 mg / kg, to a maximum dose of 100 mg) was given 10 min before starting the swl session. with the patients in a supine position, stone localization was performed using ultrasound and/or fluoroscopy guidance with exposure when needed. the shock wave frequency was 6090/min and number of shocks per session was 3500 or until the stone was completely fragmented. at the end of the procedure, patients were discharged home on antibiotic and analgesic and encouraged to drink plenty of fluids during the post - swl period. the patients were followed - up and re - swl was carried out for a maximum of three sessions depending on the response. patients were evaluated 4, 8, 12 and 24 weeks after the last swl session by medical history taking, physical examination, urinalysis, serum creatinine, x - ray kub and renal ultrasonography. at the follow - up week 4, patients with no signs of stone fragmentation were discharged from the treatment regimen and shifted to another treatment modality. at the end of follow - up, patients with large residual fragment (rf) (4 mm) were advised to continue swl for up to two sessions while patients with small rf (10 mm) were found to have significantly higher sfr at the end of the study (p = 0.013). within both groups, stones 10 mm had significantly higher sfr, shorter time to stone clearance, lower re - treatment rate and fewer number of swl sessions and shock waves than larger stones (p 1000 hu. we found that stones with a higher attenuation value (> 500 hu) had significantly higher sfr in sg than cg. within both groups, stones with lower attenuation values had significantly higher sfr, shorter time to stone clearance, lower re - treatment rate and a fewer number of swl sessions and shock waves (p 10 mm) and stones with higher attenuation value (> 500 hu) in patients treated with simultaneous inversion / diuresis than those treated with standard swl. in clinical practice, this indicates the higher benefit of adjuvant procedures even if it is applied to larger and harder stones. all reported complications were mild and comparable to that previously reported. despite being a prospective study the main limitation is using two measures simultaneously to facilitate stone clearance ; therefore, it is unclear which measure had greater contribution. however, as these measures are simple and non - invasive, it can be combined together without harm to achieve the ultimate goal of stone clearance. also, our patients were followed by x - ray kub and renal ultrasonography, which may have limited the ability to detect residual fragments. further three - arm studies and follow - up of patients with low radiation ncct may be warranted to document a greater depth of precise information about the beneficial effect of the two adjuvant procedures separately. swl with forced diuresis and inversion seems to be an effective minimally invasive option to improve the clearance of lcs fragments. this therapy might be a valuable alternative to the more invasive treatment modalities such as pcnl or urs for lower caliceal stones less than 20 mm. | objective : to improve the clearance of lower caliceal stones (lcss) after shock wave lithotripsy (swl) using a combination of intra - operative forced diuresis and inversion therapy.materials and methods : one hundred and fifty - seven consecutive patients with symptomatic, single lcss of 520 mm size were prospectively randomized into two groups. the first (study group, sg) underwent swl at the time of the maximum diuresis with the patient in the trendelenburg position with an angle of 30 degree, while the second group (control group, cg) underwent standard swl. after the last swl session, patients were followed - up regularly using plain abdominal x - ray and renal ultrasound. the primary endpoint of the study was the stone - free rate (sfr) at 12 weeks.results:a total of 141 patients completed the study treatment protocol and follow - up : 69 patients in sg and 72 patients in cg. both groups were comparable in baseline data. sg showed significantly higher sfr at all follow - up time points. at week 12, 78.3% of sg were rendered stone free, whereas only 61.1% were stone free in cg (p = 0.030). also, there was a significantly higher sfr for larger stones (> 10 mm) and stones with higher attenuation value (> 500 hounsfield units) in sg than cg. mild non - significant complications were reported in both groups.conclusion:swl with intraoperative forced diuresis and inversion seems to be an effective measure with minimal extra cost to improve lcs clearance post - swl. |
previous studies from our laboratory, examining the neural basis of morphine - induced inhibition of maternal behavior, brought up the suggestion of a rather unsuspected and integrative role of the periaqueductal gray (pag) in influencing the selection of adaptive behavioral responses [1, 2 ]. examining the neural basis underlying maternal behavior inhibition by low doses of morphine in morphine - experienced dams, we found that morphine treatment induces a behavioral switch from maternal to predatory behavior. hence, morphine - challenged dams, tested in an environment containing both pups and roaches (which served as prey), clearly preferred hunting instead of nursing. we have further shown that, under physiological conditions, there is a natural endogenous opioid tone that may be able to stimulate hunting in lactating dams. the results of behavioral, neuronal immediate early gene activation, and lesion experiments indicate that a particular site in the pag, at the level of the oculomotor nucleus, located in the outer half of the lateral column, and referred to as the rostrolateral pag (rlpag), should be responsible for this switching from maternal behavior to prey hunting in morphine - treated dams. first, we showed that the rlpag upregulates fos expression in lactating rats acutely challenged with morphine, similar to what had been found for animals performing insect hunting. next, by testing morphine - treated dams in an environment containing pups and roaches, we were able to show that lesions of the rlpag, but not other parts of the pag, impaired predatory hunting and restored the maternal response. these findings support the idea that this opioid sensitive pag site is critical for influencing the motivation drive to hunt and forage ; and should be a nodal part of a neural circuit involved in the decision - making process between hunting, foraging, and other behavioral responses. to start unraveling this circuit, in the present study, we performed a comprehensive investigation on the rlpag afferent connections, using the fluorogold as retrograde tracer. a number of retrograde tract - tracing studies have investigated the afferent sources of inputs to the pag, but they used a much less sensitive retrograde tracer (i.e., the retrograde transport of the horseradish peroxidase) and were based on large injection sites encompassing different pag functional domains. the retrograde tract - tracing method using fluorogold as a tracer, and revealed by immunohistochemical procedures, is one of the most sensitive retrograde tract - tracing tools available, and yields relatively small injection sites, a feature particularly suitable for investigating the afferent connections of relatively small sites, such as the rlpag, in the present case. overall, the present results support the idea that the rlpag combines a unique set of inputs rendering this region particularly suitable for influencing the decision - making process between hunting, foraging, and other behavioral responses. subjects were adult female wistar rats (n = 18) weighing 190220 g and approximately 90 days of age at the beginning of the experiments. food and water were available ad libitum to the animals in light - controlled (06:00 am to 06:00 pm) and temperature - controlled (2325c) rooms. conditions of animal housing and all experimental procedures were conducted under institutional guidelines of the committee on animals of the (colgio brasileiro de experimentao animal, brazil) and the committee on the care and use of laboratory animal resources, national research council. animals were anesthetized with a mixture of ketamine (vetaset ; fort dodge laboratory, campinas, brazil) and xylazine (rompum, 1:2 v / v ; 1 ml / kg body weight ; bayer ; sao paulo, brazil), and unilateral iontophoretic deposits of a 2% solution of fluorogold (fluorochrome inc., colo, usa) were placed stereotaxically into the rlpag (2.9 mm rostral to the interaural line, 0.65 mm from the midline, and 4.5 mm ventral to the surface of the brain). deposits were made over 5 minutes through a glass micropipette (tip diameter, 25 m) by applying a + 3 a current, pulsed at 7-second intervals, with a constant - current source (midgard electronics, wood dale, ill, usa, model cs3). after a survival time of 712 days, the animals were deeply anesthetized with sodium pentobarbital (65 mg / kg, ip) and perfused transcardially with a solution of 4.0% paraformaldehyde in 0.1 m phosphate buffer, ph 7.4 ; the brains were removed and left overnight in a solution of 20% sucrose in 0.1 m phosphate buffer at 4c. the brains were then frozen, and five series of 30-m - thick sections were cut on a sliding microtome in the transverse (frontal) plane and collected from the caudal medulla through the rostral tip of the prefrontal cortex. one complete series was processed for immunohistochemistry with an antiserum directed against fluorogold (chemicon international, calif, usa) at a dilution of 1:5000. the antigen - antibody complex was localized with a variation of the avidin - biotin complex system (abc), with a commercially available kit (abc elite kit, vector laboratories, calif, usa). the sections were mounted on gelatin - coated slides and then treated with osmium tetroxide to enhance visibility of the reaction product. an adjacent series was always stained with thionin to serve as reference for cytoarchitecture. fluorogold deposits in the injection sites, and the distribution of retrogradely labeled neurons, were plotted with the aid of a camera lucida onto maps prepared from adjacent thionin - stained sections. the distribution of retrograde labeling was transferred onto a reference atlas of the rat brain. the figures were prepared using adobe photoshop (v.5.5 ; adobe systems, san jose, calif, usa) for photomicrographs and adobe illustrator (v.10, adobe systems) for drawings. the distribution of neurons projecting to the rlpag region was examined by using fluorogold. in five experiments, the deposit of the tracer appeared to be confined almost entirely to the rlpag (located in the outer half of the lateral column at the levels of the oculomotor nucleus). the appearance of a representative fluorogold injection site in the rlpag is illustrated in figure 1, and the distribution of retrogradely labeled neurons from this experiment is illustrated schematically in figure 2. the results of this experiment are described in detail, because the injection site was virtually confined to the rlpag. furthermore, in this experiment, the pattern of retrograde labeling was representative of that one seen in each of the other experiments with deposits centered in the rlpag. the following is a summary of regions that appear to send fibers to the rlpag. retrogradely labeled neurons were found in the isocortex, amygdala and in the septal region. no retrogradely labeled cells were found in the hippoccampal formation. in the isocortex, a large number of retrogradely labeled neurons was observed in the prelimbic, infralimbic, anterior cingulate, and secondary motor areas (figures 2(a), 2(b), 2(c) and 3(a), 3(b)), in addition to substantial labeling in the gustatory and visceral areas (figures 2(b), 2(g), 2(h), and 2(i)). a few retrogradely labeled neurons were found in the posterior part of agranular insular area (figure 3(c)) and primary motor, perirhinal, and ectorhinal areas. all of the cortical labeled cells were pyramidal neurons of the layer v. in the lateral septal nucleus, a relatively sparse number of marked cells were observed in the rostral part of the nucleus, distributed mainly through the dorsal region of the ventrolateral zone (figures 2(b), 2(c)). in the septal region, we have also observed a substantial number of retrogradely marked neurons in the posterior division of the bed nuclei of the stria terminalis, particularly in the interfascicular nucleus, and also, to a lesser degree, in the transverse nucleus (figures 2(e), 2(f)). in the amygdala, a large number of retrogradely labeled cells were found to be restricted to the medial part of the central amygdalar nucleus (figures 2(g), 2(h), 2(i), and 4(a)). thalamusat the thalamus, a substantial retrograde labeling was found in the ventral part of the zona incerta (figures 2(h), 2(i), 2(j)). no marked cells were observed in the dorsal thalamus. at the thalamus, a substantial retrograde labeling was found in the ventral part of the zona incerta (figures 2(h), 2(i), 2(j)). hypothalamusin thepreoptic region, a substantial retrograde labeling was found in the median preoptic nucleus (figures 2(c), 2(d), and 4(b)), in addition to a sparse number of marked cells in the anteroventral preoptic nucleus (figures 2(c) and 2(d)).at the anterior hypothalamic levels, the anterior part of the anterior hypothalamic nucleus presented a dense cluster of retrogradely labeled cells, which were distributed in the region that seems to overlap, at least partially, with a territory known to contain a large number of neurons expressing enkephalin (figures 2(e), 2(f), 2(g), and 4(c)). moreover, at these levels, a substantial retrograde labeling was also found in the lateral hypothalamic area immediately dorsal to the optic tract and the supraoptic nucleus, which seems to correspond to a region densely targeted by the lateral component of retinohypothalamic tract (figures 2(f), 2(g), and 4(c)).at tuberal levels, a dense number of retrogradely labeled cells was found in the anterior part of the ventromedial nucleus, in addition to a moderate number of marked cells in the retrochiasmatic area, the ventrolateral and central parts of the ventromedial nucleus, the tuberal nucleus, and rostral parts of the posterior hypothalamic nucleus (figures 2(g), 2(h), 2(i), 2(j), and 4(d)). furthermore, at these levels, we have found a large number of retrogradely labeled cells in the lateral hypothalamic area, distributed in the dorsal, suprafornical, justadorsomedial, and justaventromedial areas (figures 2(g), 2(h), 2(i), and 2(j)).at the mammilary levels, a large number of labeled neurons were found in the dorsal premammilary nucleus, mostly distributed in the dorsal part of the nucleus (figure 2(k)). finally, at these levels, we have found moderate retrograde labeling in the parasubthalamic nucleus, in addition to sparse labeling in the subfornical region of the lateral hypothalamic area (figures 2(j) and 2(k)). in thepreoptic region, a substantial retrograde labeling was found in the median preoptic nucleus (figures 2(c), 2(d), and 4(b)), in addition to a sparse number of marked cells in the anteroventral preoptic nucleus (figures 2(c) and 2(d)). at the anterior hypothalamic levels, the anterior part of the anterior hypothalamic nucleus presented a dense cluster of retrogradely labeled cells, which were distributed in the region that seems to overlap, at least partially, with a territory known to contain a large number of neurons expressing enkephalin (figures 2(e), 2(f), 2(g), and 4(c)). moreover, at these levels, a substantial retrograde labeling was also found in the lateral hypothalamic area immediately dorsal to the optic tract and the supraoptic nucleus, which seems to correspond to a region densely targeted by the lateral component of retinohypothalamic tract (figures 2(f), 2(g), and 4(c)). at tuberal levels, a dense number of retrogradely labeled cells was found in the anterior part of the ventromedial nucleus, in addition to a moderate number of marked cells in the retrochiasmatic area, the ventrolateral and central parts of the ventromedial nucleus, the tuberal nucleus, and rostral parts of the posterior hypothalamic nucleus (figures 2(g), 2(h), 2(i), 2(j), and 4(d)). furthermore, at these levels, we have found a large number of retrogradely labeled cells in the lateral hypothalamic area, distributed in the dorsal, suprafornical, justadorsomedial, and justaventromedial areas (figures 2(g), 2(h), 2(i), and 2(j)). at the mammilary levels, a large number of labeled neurons were found in the dorsal premammilary nucleus, mostly distributed in the dorsal part of the nucleus (figure 2(k)). finally, at these levels, we have found moderate retrograde labeling in the parasubthalamic nucleus, in addition to sparse labeling in the subfornical region of the lateral hypothalamic area (figures 2(j) and 2(k)). brainstemat mesodiencephalic levels, a large number of marked cells were found in the precommissural nucleus (figure 2(l)). in the midbrain, at the injection site level, substantial retrograde labeling was found in the lateral part of the intermediate layer of the superior colliculus (figure 2(m)). additionally, at this level, a moderate number of retrogradely labeled neurons were also found in the pag, which appeared to be distributed within the dorsomedial part (figure 2(m)). proceeding caudally, at the intermediate rostrocaudal levels of the dorsal raphe nucleus, a moderate number of marked cells was found in the ventrolateral part of the pag (figure 2(n)).at rostral pontine levels, a few labeled cells were found in the laterodorsal tegmental nucleus, as well as in the central lateral, dorsal lateral, and ventral lateral parts of the parabrachial nucleus (figure 2(o)).it should be noted that, in the experiments with fluorogold deposits centered in the rlpag, retrograde labeling was mostly ipsilateral. however, some of the main sources of projections to this area, including the prefrontal cortex, retrochiasmatic area, anterior hypothalamic nucleus, ventromedial hypothalamic nucleus, lateral hypothalamic area, zona incerta, precommissural nucleus, and the pag, also displayed conspicuous retrograde labeling contralateral to the injection site. at mesodiencephalic levels, a large number of marked cells were found in the precommissural nucleus (figure 2(l)). in the midbrain, at the injection site level, substantial retrograde labeling was found in the lateral part of the intermediate layer of the superior colliculus (figure 2(m)). additionally, at this level, a moderate number of retrogradely labeled neurons were also found in the pag, which appeared to be distributed within the dorsomedial part (figure 2(m)). proceeding caudally, at the intermediate rostrocaudal levels of the dorsal raphe nucleus, a moderate number of marked cells was found in the ventrolateral part of the pag (figure 2(n)). at rostral pontine levels, a few labeled cells were found in the laterodorsal tegmental nucleus, as well as in the central lateral, dorsal lateral, and ventral lateral parts of the parabrachial nucleus (figure 2(o)). it should be noted that, in the experiments with fluorogold deposits centered in the rlpag, retrograde labeling was mostly ipsilateral. however, some of the main sources of projections to this area, including the prefrontal cortex, retrochiasmatic area, anterior hypothalamic nucleus, ventromedial hypothalamic nucleus, lateral hypothalamic area, zona incerta, precommissural nucleus, and the pag, also displayed conspicuous retrograde labeling contralateral to the injection site. the results of the present retrograde axonal tract - tracing study suggest that the rlpag receive inputs from several widely distributed areas in the forebrain and, to a lesser extent, from the brainstem, as well. in addition, clear telencephalic inputs appear to arise from the gustatory, visceral, and perirhinal cortical areas, as well as from the medial part of the central amygdalar nucleus and the interfascicular nucleus of the bed nuclei of the stria terminalis. in the diencephalon, massive inputs to the rlpag arise from several hypothalamic sites, including the median preoptic nucleus, anterior hypothalamic nucleus, retrochiasmatic area, anterior, and ventrolateral parts of the ventromedial nucleus, dorsal premammillary nucleus (pmd), and several districts of the lateral hypothalamic area. in contrast, the rlpag appears to receive inputs from considerably fewer brainstem sites, where significant retrograde labeling was found in other parts of the pag (i.e., the dorsomedial and ventrolateral parts) and in the intermediate layers of the lateral part of the superior colliculus. in agreement with previous anterograde tract - tracing studies, medial prefrontal cortical areas, including the infralimbic, prelimbic, anterior cingulated, and secondary motor areas, appear to represent one of the most important afferent sources of projections to the rlpag [1120 ]. in line with previous anterograde findings, we have also found that the rlpag receives projections from visceral, gustatory, and perirhinal cortical areas [1214, 17 ]. in the septal region, a substantial number of retrograde labeled cells were found in the interfascicular nucleus of the bst, a finding likewise supported by previous phal studies. the present results also revealed that the medial part of the central nucleus of the amygdala is an important source of telencephalic inputs to the rlpag. this finding has also been confirmed by means of phal anterograde tract - tracing method, which showed that the medial part of the central nucleus of the amygdala provides a substantial terminal field to the lateral pag. the present findings also revealed that the rlpag is targeted by several hypothalamic districts. at preopotic levels, the rlpag seems to receive substantial inputs from the median preoptic nucleus, in addition to somewhat sparse inputs from the anteroventral preoptic nucleus. both of these projections have been confirmed by previous phal studies. at anterior hypothalamic levels, a prominent group of retrogradely labeled cells was found in the anterior part of the anterior hypothalamic nucleus, in a region containing a characteristic cluster of enkephalinergic cells. we were able to confirm this projection by means of phal deposits placed in the anterior part of the anterior hypothalamic nucleus, which yielded a distinct terminal field in the rlpag [s. r. mota - ortiz and n. s. canteras, personal observation ]. in addition, at these levels, we have found that the rlpag receives inputs from the retrochiasmatic area and a lateral hypothalamic region immediately adjacent to the optic tract and the supraoptic nucleus, which receives direct projections from the lateral part of the retinohypothalamic tract and corresponds to the so - called retinoceptive region of the lateral hypothalamic area. both the projections from the retrochiasmatic area and from the retinoceptive region of the lateral hypothalamic area to the rlpag have been previously confirmed through phal experiments [24, n. s. canteras, personal observation ]. at tuberal levels, in agreement with the results of our retrograde transport experiments, previous evidence based on phal anterograde tract - tracing indicates that the rlpag receives dense projections from the anterior part of the ventromedial nucleus and the posterior hypothalamic nucleus, in addition to somewhat weaker inputs from the ventrolateral part of the ventromedial nucleus and the adjacent tuberal nucleus [25, 26 ]. in addition, at these levels, we found that the rlpag receives substantial inputs from the dorsal, suprafornical, justadorsomedial, and justaventromedial areas of the lateral hypothalamus, as well as immediately adjacent parts of the zona incerta. these projections have been confirmed in phal studies [27, m. goto, personal observation ]. notably, this region of the lateral hypothalamus overlaps with the region of cells expressing melanin - concentrating hormone (mch) and hypocretin / orexin. moreover, in agreement with previous phal studies, we have also found, in the lateral hypothalamic area, that the parasubthalamic nucleus represents another source of inputs to the rlpag. finally, at mammilary levels, we found a distinct projection mostly from the dorsal part of the pmd. the pmd represents one of the main hypothalamic sources of inputs to the pag, and previous phal studies have shown that the dorsal and ventral parts of the nucleus provide a differential pattern of projection to the pag, and confirmed that the dorsal part of the pmd provides a strikingly dense projection to the rlpag. the present results also revealed that a small number of brainstem sites appear to innervate the rlpag. evident retrograde labeling was found in the precommissural nucleus, the dorsomedial and ventrolateral parts of the pag, and the lateral part of the intermediate layer of the superior colliculus. all of these projections have been confirmed through phal anterograde tract - tracing method [31, s. r. mota - ortiz and n. s. canteras, personal observation ] in addition, we have also found that the rlpag receives relatively sparse projections from the laterodorsal tegmental nucleus and the lateral part of the parabrachial nucleus. unfortunately, to our knowledge, these projections remain yet to be demonstrated with anterograde tracer studies. as mentioned above, previous retrograde tract - tracing studies have investigated the afferent sources of inputs to the pag, but they were based on large injection sites, providing just an overall picture of the afferent inputs to the pag without differentiating among the particular pag domains. by determining the specific set of afferent inputs to the rlpag, we have provided important information to distinguish this pag site from adjacent different functional domains, such as the immediately adjacent dorsolateral pag (dlpag). in contrast to the rlpag, the dlpag upregulates fos expression in response to both actual and contextual predatory threats [3234 ], and seems to be responsible for organizing the expression of unconditioned and conditioned antipredatory responses. as for the rlpag, the dlpag also receives substantial inputs from the anterior cingulate, prelimbic, and infralimbic areas [11, 12, 19, 20 ], but in sharp contrast to what has been found for the rlpag, the visceral area appears to project only sparsely to the dlpag. additionally, in contrast to the rlpag, the dlpag does not seem to receive substantial inputs from the amygdala, and seems to be densely innervated by medial hypothalamic sites involved in processing predatory cues, including the posterior part of the anterior hypothalamic nucleus, the dorsomedial part of the ventromedial nucleus and the dorsal premammillary nucleus [25, 35, 36 ]. notably, the projection from the dorsal premammillary nucleus to the dlpag arises chiefly from the ventrolateral part of the nucleus, which is the hypothalamic site most responsive to the predator or its cues. in the zona incerta, differently from what we have just found for the rlpag, the dlpag seems to receive dense projections from the rostral zona incerta, also referred to as the incertohypothalamic area. the superior colliculus also has a differential pattern of projection to the pag, where the lateral part of the intermediate layer projects to the rlpag, as presently shown, and the medial part of the intermediate and deep layers target the dorsal pag. notably, the medial part of the intermediate and deep layers of the superior colliculus respond to visual - threatening stimuli, such as suddenly expanding shadows in the upper visual field (which look like an approaching predator), and, via a projection to the dlpag, may exert a marked influence on the control of defensive responses. the evidence just discussed supports the idea that different functional pag domains are likely to have particular sets of afferent inputs, and next, we will provide a discussion on how the diverse inputs to the rlpag may be related to its function on hunting and foraging behavior. as commented in the introduction, the rlpag is an opioid sensitive site, which is critical for influencing the motivation drive to hunt and forage, and is likely to be part of a neural circuit involved in the decision - making process between hunting, foraging, and other behavioral responses. the present findings help to reveal this complex network, and reinforce the rlpag 's nodal role in integrating a wealth of different kinds of information likely to influence foraging or hunting activity. one of the main findings of the present investigation is that the rlpag appears to be particularly driven by inputs from medial prefrontal cortical areas. in the present context, it is noteworthy that, in the prefrontal cortex, the anterior cingulate, the prelimbic, and the infralimbic cortices have been associated with diverse emotional, cognitive, and mnemonic functions that underlie attentional and decision - making processes [39, 40 ]. in addition to the prefrontal cortex, the rlpag also seems to be influenced by the septohippocampal system via a projection from the enkephalinergic region of the anterior hypothalamic nucleus. of particular relevance in the present account, the septohippocampal system has been shown to have a role in prioritizing the temporal order of motivated responses, which, in the present case, seems to utilize an enkephalinergic pathway. here, it is noteworthy to recall that the opioidergic influence on the rlpag has been shown to control the selection of adaptive behavioral responses, switching from maternal to hunting behavior. as commented in the introduction, the rlpag upregulated fos expression in animals performing insect hunting, and in the present study, we have shown that the rlpag integrates inputs from a number of neural sites related to the circuitry underlying predatory hunting. thus, we have presently found that the medial part of the central amygdalar nucleus provides a significant projection to the rlpag. the medial part of the central amygdalar nucleus is part of a distinct amygdalar circuit comprising ; the anterior part of the cortical nucleus, the anterior part of the basomedial nucleus and the posterior part of the basolateral nucleus, which has been shown to be mobilized during insect predation and seems to be particularly involved in processing visceral, gustatory and olfactory information [4144 ]. the central nucleus the main output way station of this amygdalar circuit has been shown to be involved in controlling feeding behavior [45, 46 ]. in particular, the nucleus appears to integrate food hedonic values [47, 48 ] and to influence searching and consumption of palatable food through a pathway involving opioidergic neurotransmission [48, 49 ]. in the context of feeding behavior, it is also particularly relevant to point out that, according to the present findings, the rlpag is also in a position to integrate visceral and gustatory information from the visceral and gustatory cortical areas, and to a lesser degree, from the parabrachial area. the rlpag also seems to be innervated by lateral hypothalamic regions activated during predatory hunting, namely, the parasubthalamic nucleus and the region containing cells expressing mch and hypocretin / orexin. the parasubthalamic nucleus is also targeted by the medial part of the central amygdalar nucleus, and provides inputs to hindbrain control regions involved in modulating digestive and metabolic responses occurring in both cephalic and consummatory phases of feeding behavior. the lateral hypothalamic region containing cells expressing mch and hypocretin / orexin appears to be involved in controlling arousal and exploratory activity related to feeding behavior [27, 51, 52 ]. in fact, this lateral hypothalamic region may also represent an important interface for the hypothalamic periventricular sites involved in the control of homeostatic feeding. moreover, according to the present findings, the rlpag also receives direct inputs from the retrochiasmatic region, which has also been included in the hypothalamic circuit controlling feeding. we have presently shown that the rlpag is also targeted by the lateral part of the intermediate layer of the superior colliculus, which we have also previously shown to be mobilized during insect hunting. in fact, cells in this collicular region seem to respond to moving objects in the temporal visual field, and we have found that animals bearing lesions in this collicular region, besides failing to orient themselves toward the moving prey, were also clearly less motivated to pursue the roaches [i. c. furigo and s. r. mota - ortiz, personal observation ] perhaps reflecting the functional role of the superior colliculus rlpag pathway. the rlpag is also significantly targeted by hypothalamic districts that do not seem to be directly involved in controlling feeding or hunting behavior, namely, the median preoptic nucleus, the retinoceptive region of the lateral hypothalamic, area and the dorsal premammillary nucleus. the median preoptic nucleus is classically known to respond to plasma osmolarity and influence drinking behavior, and this pathway to the rlpag may be thought of as having some role in the search of water supplies. conversely, the projection from the retinoceptive region of the lateral hypothalamic area may bring the information regarding the environmental luminescence, which may be thought of as having a direct impact on foraging activity [n. s. canteras, personal observation ]. finally, the dorsal part of the dorsal premammillary nucleus provides an important projection to the rlpag. unfortunately, to the best of our knowledge, the potential roles of this path remain yet to be determined. here, it is important to consider that functional studies using electrical or chemical stimulation have suggested a role for the lateral pag in defensive responses. in the rat, chemical stimulation with kainic acid (ka) in the region of the rlpag produced strong backward defense, a kind of response that would be expected when the animal is exposed to a predator. however, this finding is hard to conciliate with the fact that rlpag does not seem to be activated in response to predator exposure, as would be expected. in reality, the effects of ka injection in this region should be taken very cautiously since the immediately adjacent dlpag presents a much heavier binding to ka when compared to the rlpag. actually, the dlpag appears as the pag site presenting the largest activation to predator exposure, and, as previously discussed, its hodological pattern is fully compatible with its role in antipredatory defense. in addition, the concept of a distinct functional column for the entire lateral pag should also be revised. in fact, the role here assigned to the rlpag does not seem to apply to the caudal half of the lateral pag, since lesions encroaching upon this latter pag region do not seem to affect the motivational drive to hunt [2, m. h. sukikara and s. r. mota - ortiz, personal observation ]. conversely, there is a prominent fos upregulation in the caudal, but not in the rostral, lateral pag in response to predator exposure [32, 33 ]. overall, the present findings support the idea that the rlpag has a central role in a complex network controlling the decision - making process between hunting, foraging, and other behavioral responses. on one hand, the rlpag appears to be importantly driven by medial prefrontal cortical areas involved in controlling attentional and decision - making processes. on the other hand, the rlpag receives a wealth of information from different neural sites related to feeding, drinking, or hunting behavioral responses. therefore, this unique combination of afferent connections puts the rlpag in a privileged position to influence the motivation drive to choose whether hunting and foraging are the most appropriate adaptive responses. | previous studies have shown that a particular site in the periaqueductal gray (pag), the rostrolateral pag, influences the motivation drive to forage or hunt. to have a deeper understanding on the putative paths involved in the decision - making process between foraging, hunting, and other behavioral responses, in the present investigation, we carried out a systematic analysis of the neural inputs to the rostrolateral pag (rlpag), using fluorogold as a retrograde tracer. according to the present findings, the rlpag appears to be importantly driven by medial prefrontal cortical areas involved in controlling attention - related and decision - making processes. moreover, the rlpag also receives a wealth of information from different amygdalar, hypothalamic, and brainstem sites related to feeding, drinking, or hunting behavioral responses. therefore, this unique combination of afferent connections puts the rlpag in a privileged position to influence the motivation drive to choose whether hunting and foraging would be the most appropriate adaptive responses. |
the mortality rate associated with cns aspergillosis is reported to be as high as 90%, especially in the immunocompromised patients. indications for surgical intervention are usually decompression of symptomatic lesions causing focal neurological deficits and for tissue diagnosis. our patient is a 35-year - old lady who presented with fever and paraparesis of one week duration. she had backache without radicular symptoms, cough and occasional hemoptysis for five months prior to admission (day-150). a tissue biopsy five years prior confirmed pulmonary sarcoidosis for which she has been on chronic oral steroids. on examination, she was febrile with normal vital signs. neurological exam revealed mrc grade 3 paraparesis and a sensory level corresponding to t6 spinal level. acid fast bacilli were detected in sputum examination and chest x - ray was normal. x - ray of the thoracolumbar spine showed prevertebral and paravertebral opacity with mild loss of t8 vertebral height and no evidence of disc space collapse or other bony destruction (fig. mri of thoracolumbar spine showed abnormal marrow signals in the mid - thoracic vertebrae with a partial collapse of t8 and a large epidural abscess from t5-t9, all suggestive of spinal tuberculosis (fig. a diagnosis of pulmonary tuberculosis with probable associated tuberculous spondylitis was made and the patient was started on the 4-drug antitubercular regimen (inh 300 mg, rifampicin 600 mg, ethambutol 800 mg, pyrazinamide 1500 mg) that was later modified, in view of drug - induced hepatotoxicity to streptomycin(750 mg), ethambutol(800 mg) & ofloxacin(400 mg). however, in spite of the antitubercular treatment, her weakness worsened to complete paraplegia by the fourth week (day+28). she then underwent t5-t8 laminectomy, debridement of epidural granulation tissue and posterior stabilization from t2-t12. spinal tuberculosis was ruled out by negative multiplex tb pcr and bd bactec culture of the epidural tissue. histopathology of the excised epidural tissue revealed suppurative granuloma with septate hyphae indicative of aspergillus species [fig. multiplex pcr and fungal culture [fig. 2c and d ] also confirmed aspergillus fumigatus. treatment with intravenous voriconazole, loading dose of 400 mgs twice daily followed by 200 mgs twice daily was initiated (day+30). her steroid was tapered to the lowest possible maintenance dose to avoid clinical symptoms of adrenal insufficiency. after initial defervescence, the fever recurred after three weeks (day+51) of intravenous voriconazole. by the fourth postoperative week, she developed seizures, altered sensorium and persistent fever. she was electively intubated for airway protection and nonenhanced head ct brain showed well defined hypodense lesions in the left parietal and bilateral frontal lobes [fig. csf analysis showed glucose of 31.6 mg / dl (corresponding blood glucose 120 mg / dl), 12 cells per high power field with 30% polymorphonuclear and 70% mononuclear. mri brain showed multiple t2flair hyperintense, well - defined focal lesions with dense diffusion restriction and ring enhancement pattern on contrast administration located at the grey - white junction of the left parietal region, left frontal and right frontal lobes (fig. csf bacterial and afb cultures were negative ; whereas, csf multiplex pcr was positive for aspergillus fumigates and negative for tuberculosis and bacterial panel. as there was evidence of progression of infection to the brain on systemic voriconazole therapy, our patient was started on lipid complex amphotericin b (day+58). she was initially started at 5 mg / kg / day, subsequently increased to 10 mg / kg / day as fever and altered sensorium persisted. in the interim, she developed signs of adrenal insufficiency requiring escalation of her steroid dosage. after defervescence her amphotericin b dose was reduced to 5 mg / kg / day. the modified antitubercular regimen, started for her sputum afb culture positivity, was continued. following defervescence and control of seizures, she improved neurologically and was transitioned to long - term rehabilitation. follow - up mri brain and spine showed no new cerebral or spinal lesions. with clinical and radiological improvement, antifungal therapy with intravenous lipid complex amphotericin was continued for a total of six months with close monitoring. meanwhile her sputum became afb - negative and antitubercular treatment was continued to complete a nine month course. in the interim, steroids were gradually tapered and discontinued. mri brain and spine at 1 year showed complete resolution of her lesions (fig. 4). at two year follow - up, she remains afebrile with a normal sensorium and is seizure - free. the spinal infection has resolved radiologically and her myelopathy is clinically improving to the extent that she has mrc grade 3 power in the lower limbs, and is able to stand with support. invasive aspergillosis is a disseminated fungal infection associated with a high mortality despite treatment. a systematic review of the literature of 1223 cases of invasive aspergillosis reported case fatality rates of 99% for cerebral aspergillosis, 86% for pulmonary, and 66% for paranasal sinus infection. mortality rates remained high despite improvements in diagnosis and newer, safer forms of antifungal treatment. voriconazole is the recommended therapy for cns aspergillosis ; with a favourable outcome of up to 35% when coupled with surgery. as this case depicts, differentiation between spinal aspergillosis and tuberculosis may be impossible on clinical and radiological grounds alone,, especially in an immunocompromised individual. however, the distinction between the two is important, as delay in diagnosis contributes to the high morbidity and mortality of invasive aspergillosis. in our case, the clinical presentation and mri findings in the setting of sputum afb positivity was highly suggestive of spinal tuberculosis. spinal tuberculosis most commonly leads to disc space collapse and paradiscal involvement of the vertebral body ; whereas in invasive aspergillosis, the lesion expands circumferentially and destroys all the surrounding spinal structures, including vertebral bodies, discs, and neural arches, as well as all the contiguous structures, like ribs, thoracic wall, lungs, etc.. the pathophysiology of the cerebral aspergillus infection implicates an infective vasculopathy - mediated septic infarction or hemorrhage, causing infectious cerebritis that evolves into an abscess. its anatomic distribution is mainly in the corticomedullary junction, thalami, basal nuclei or the corpus callosum. the apparent affinity of cns aspergillosis for perforating artery distributions is most likely due to the invasive character of aspergillus spp. culture of clinical specimens to isolate the etiologic fungal agent remains the gold standard for diagnosis. given the limitations of conventional methods of diagnosis for invasive fungal infections, a negative result on direct or pathologic smears and cultures does not rule out infection. the detection of fungal cell wall markers in serum has been reported using galactomannan (gm),, beta - d - glucan (bdg) and mannan enzyme immune assays,,. galactomannan is relatively specific for aspergillus species, and can be detected in body fluid specimens with enzyme immunoassay. the polymerase chain reaction (pcr) assay serves as a powerful non - culture method for the diagnosis of systemic fungal infection in high - risk patients. the sensitivity of csf pcr in detecting cns aspergillosis is 100% as compared to galactomannan 80%. molecular methods yielding results within 6 h have now revolutionized the diagnosis of fungal infections, allowing for diagnosis and therapy during the incubation period and early stage of infection. despite the increasing burden of opportunistic fungal infections, early accurate diagnosis of fungal infections remains a challenge. voriconazole is the recommended therapy for cns aspergillosis, with favourable response of up to 35% when coupled with surgery. azole resistance in a. fumigatus has been associated with mutations in the cyp51a gene, the target for antifungal azoles. in our patient, though we were unable to monitor therapeutic drug levels of voricanozole, we believe that coadministration of rifampicin would have contributed to clinical voricanozole failure. for patients intolerant of or refractory to voriconazole, a formulation of amphotericin b is an appropriate alternative. though amphotericin b deoxycholate has historically been used in the treatment of invasive aspergillosis, the lesser nephrotoxicity makes liposomal amphotericin b preferable. intrathecal administration of amphotericin b does not allow penetration beyond the pia - mater. instead, high - dose systemic amphotericin b is recommended to achieve higher parenchymal concentration. in our patient we used lipid emulsion amphotericin with great success. because of continued high rates of mortality despite the use of newer antifungals, surgical resection of large infective foci is an important adjunct to improve outcome. surgical debridement and stabilization of the spine was necessary in our patient once she developed acute compressive myelopathy. once our patient developed multiple cerebral abscesses, stereotactic aspiration, though considered, was not required since she responded favorably to medical management alone. though duration of therapy for aspergillosis has not been optimally defined, amphotericin b or voriconazole for a total duration of 8 12 weeks is recommended. to date, clear - cut guidelines are lacking due to the rarity of these infections. infectious diseases society of america (idsa) guidelines advise treatment of invasive aspergillosis until resolution or stabilization of all clinical and radiographic manifestation. our case also highlights the possibility of voriconazole resistance, as she developed fever with new symptomatic cerebral lesions while on therapy for spinal aspergillosis requiring treatment with lipid complex amphotericin b. azole resistance in a. fumigates is an emerging problem and may develop during azole therapy. withdrawal of corticosteroids or reduction of dosage is often critical for successful outcome in invasive aspergillosis. failure to reduce an immunosuppressive dosage of systemic corticosteroids usually results in relentless invasive fungal infection. for patients with successfully treated invasive aspergillosis who require continued immunosuppression, antifungal prophylaxis may prevent recurrent infection from residual foci of infection. this case highlights the efficacy of aggressive medical and surgical co - management for invasive fungal disease of the cns. though voriconazole is the treatment of choice for invasive aspergillosis, resistance is common and can lead to rapid disease progression and mortality. additionally it is critical to monitor therapeutic drug levels, where available, during voricanozole therapy to ensure clinical efficacy and decrease adverse effects. hence close monitoring for clinical and radiological resolution, as well as, alternate drug therapy are strongly advised. transitioning treatment from voriconazole to amphotericin b along with early surgical intervention | 35 yr old steroid dependent lady with pulmonary tb underwent debridement of epidural abscess & posterior stabilization for paraparesis. with histopathology and cultures showing aspergillus fumigatus, voricanozole was started. by the fourth week, she developed persistent fever, and altered mental status. brain mri and csf study including multiplex pcr evaluation confirmed cerebral aspergillosis. voricanozole was changed to intravenous lipid complex amphotericin b to achieve sustained clinical and radiological response after six months of therapy. |
. this could be due to increase in motor vehicle accident12). increase in motor vehicle accident could be due to ignorance of traffic police and poor traffic system, all contribute to the incidence of this dilemma3). at present these injuries require trained and expert orthopedic surgeons who unfortunately are less available in developing country like pakistan34) as surgeons expertise and approach to particular acetabular fracture also influence the outcome56). acetabular fractures are major clinical problems as being the foremost weight bearing joint of the body. lack of advances in radiological assessment and surgeon experience leads to conservative and delayed management of these fractures. letournel7) proposed that surgical treatment of acetabular fractures has got better prognosis than conservative management in patients with acetabular fractures. only a few setups in the country this study aims to evaluate the functional and radiological outcomes of surgical management of acetabular fractures among patients in tertiary care hospital. this was a prospective study conducted at the department of orthopaedic surgery in liaquat national hospital (karachi, pakistan), a tertiary care hospital. the study was approved by the ethics review committee of hospital (0292 - 2016). factors examined include age, gender, mechanism of injury, associated injury, time between injury and surgery (in days), preoperative degree of displacement, fracture pattern and quality of reduction. all the surgeries were performed by a single surgeon who is fellowship trained and had 15 year experience in pelvis and acetabulum surgeries. all patients had definite indications of surgery with more than 2 mm displaced acetabular fractures, articular impaction, unstable and non - concentric reduction, matta roof arc angle less than 45. the selection technique was case series study. only those patients who had completed at least 24 months of follow - up were included in the study. out of 60 patients, 10 patients were lost to follow - up and were not included in the study. the remaining patients attended our outpatient clinic and was evaluated clinically with harris hip score (hhs) and radiologically with matta outcome grading910). to evaluate functional outcome patients were categorized into excellent (hhs, 90 - 100), good (hhs, 80 - 90), fair (hhs, 70 - 80), and poor (hhs, 2 mm displacement, 2 mm displacement) preoperative radiographic evaluation shows fracture displacement of > 2 mm in 15 cases and 2 mm in 35 cases. fracture types were divided into simple and associated fractures which are then further classified into five subcategories as shown in table 1. surgeries were performed using various approaches and decision was mainly based upon fracture pattern and surgeon preference as well. kocher - langenbeck (n=25, 50.0%) approach was the most common surgical approach followed by ilioinguinal (n=16, 320%) and triradiate (n=9, 18.0%) approach. open reduction and internal fixation of fractures were performed using reconstruction plates of 3.5 mm, screws (3.5 or 4.5 mm) or combination of plate and screws. postoperative anteroposterior view of pelvis and judet views (iliac oblique and obturator views) were used to assess the quality of reduction. the patients were categorized into three groups based on matta radiological grading such as anatomical, congruent or incongruent. anatomical if all fracture gaps and steps had been removed intraoperatively and postoperative films shows restoration of all five anatomical lines (ilioinguinal, iliopectineal, dome, posterior wall and anterior wall) with the head centered and parallel beneath the acetabular roof (fig. 1, 2, 3). a congruent reduction was best judged on anteroposterior film which was useful in assessing the hip with reference to contralateral normal hip joint. patients with poor restoration of five anatomical lines with inward subluxation of hip were included in the incongruent group (fig. had already been treated with total hip replacement because of developing secondary osteoarthritis between one and two years after open reduction and internal fixation of acetabulum. the patients were followed up clinically and radiologically at six weeks, three months, six months, one year and two years. patient 's demographics and postoperative outcomes were noted in a predesigned study proforma and the data was analyzed using statistical package for social sciences (spss) version 20.0 (ibm co., armonk, ny, usa). the most common mechanism of injury was motor vehicle accident 37 (74.0%) while in associated injury most common was extremities injury 24 (48.0%). most of the patients 37 (74.0%) were admitted in ward rather than intensive care unit (icu). kocher - langenbeck approach was done in 25 (50.0%) patients. regarding the presenting patterns of fractures, it was observed that simple fractures were seen in 42 patients while associated fractures seen in 8 patients. four of the six patients had neuropraxia pre - surgery and had recovery with complete nerve function while no recovery of nerve function in two cases, both these cases had neglected injuries. infection detected in 4 (8.0%) while avn femoral head was found in 2 (4.0%) of patients. functional outcomes were divided into two group 's ; i.e., acceptable and not acceptable. on the basis of this division, 35 (70.0%) outcomes were acceptable and 15 (30.0%) outcomes were not acceptable. stratification with respect to age, gender, mechanism of injury, associated injury, fracture pattern, time taken between injury and surgery and initial degree of displacement on radiographs was done to observe effect of these modifiers on functional outcome. the results showed that there was significant association of functional outcome with respect to age, mechanism of injury, time between injury and surgery and initial displacement on radiographs as shown in table 3. the patients are divided into three groups such as anatomical, congruent and incongruent group. on the basis of this division, 39 (78.0%) outcomes are in anatomical group, 5 (10.0%) are in congruent group and 6 (12.0%) are in incongruent group. stratification with respect to age, gender, mechanism of injury, associated injury, fracture pattern, time between injury and surgery and displacement on radiographs was done to observe effect of these modifiers on radiological outcome. the results showed that there is significant association of age, mechanism of injury, associated injury, fracture pattern, time between injury and surgery and initial displacement on radiological outcome as shown in table 4. the most common mechanism of injury was motor vehicle accident 37 (74.0%) while in associated injury most common was extremities injury 24 (48.0%). most of the patients 37 (74.0%) were admitted in ward rather than intensive care unit (icu). kocher - langenbeck approach was done in 25 (50.0%) patients. regarding the presenting patterns of fractures, it was observed that simple fractures were seen in 42 patients while associated fractures seen in 8 patients. four of the six patients had neuropraxia pre - surgery and had recovery with complete nerve function while no recovery of nerve function in two cases, both these cases had neglected injuries. infection detected in 4 (8.0%) while avn femoral head was found in 2 (4.0%) of patients. functional outcomes were divided into two group 's ; i.e., acceptable and not acceptable. on the basis of this division, 35 (70.0%) outcomes were acceptable and 15 (30.0%) outcomes were not acceptable. stratification with respect to age, gender, mechanism of injury, associated injury, fracture pattern, time taken between injury and surgery and initial degree of displacement on radiographs was done to observe effect of these modifiers on functional outcome. the results showed that there was significant association of functional outcome with respect to age, mechanism of injury, time between injury and surgery and initial displacement on radiographs as shown in table 3. the patients are divided into three groups such as anatomical, congruent and incongruent group. on the basis of this division, 39 (78.0%) outcomes are in anatomical group, 5 (10.0%) are in congruent group and 6 (12.0%) are in incongruent group. stratification with respect to age, gender, mechanism of injury, associated injury, fracture pattern, time between injury and surgery and displacement on radiographs was done to observe effect of these modifiers on radiological outcome. the results showed that there is significant association of age, mechanism of injury, associated injury, fracture pattern, time between injury and surgery and initial displacement on radiological outcome as shown in table 4. fractures of the acetabulum though being un - common are of great clinical importance as being the most important weight bearing joint of the lower extremity. these fractures are mainly caused by high velocity motor vehicle accidents due to rise in incidence of motor vehicle accident. the incidence of these fractures is rising in developing countries in contrast to the united states and western europe where the incidence is stable11). these fractures need well - planned management treatment guidelines and expertise. in countries like pakistan where adequate facilities are scarce and lack of well trained experts in the field, majority of the fractures tend to get managed conservatively. this leads to complications such as deep vein thrombosis, joint stiffness, hypostatic pneumonia12). our setup is one of the only few which have the capacity to deal with these types of fractures in the region. mean time duration between injury and surgery in our study being 5.003.59 days is comparatively lower than study done from the neighboring country showing the mean injury surgery duration of 21 days13). male gender being more common could be attributed due to the reason that males in this society are more involved in travelling purposes for various reasons14). our study also shows that patients having any associated injuries most commonly in the extremities. sciatic nerve injury can be caused by acetabular fractures and the surgical interventions associated with fracture management. all the patients who presented to our setup had traumatic nerve injuries and none of the patients had iatrogenic injuries. in a study done by lehman.17), the incidence of nerve injuries was found to be 4% on admission whereas in our study it was found to be 12%. this could be due to the reason that our study is limited to 50 cases and hence the true incidence of these nerve injuries might not be correctly calculated in countries like pakistan where majority of patients do not come to hospital17). the radiological outcome correlate significantly with the functional outcome and quality of reduction of fracture was the single most important predictor of clinical function, radiological grade and development of arthritis. this is slightly higher but still comparable to a findings mentioned by shrestha.11) of mean hhs of 78 ; and 74 is a study done by gupta). our study reports surgical site infection as 8.0%. in a study done by suzuki.18), the surgical site infection was 5.2% superficial and deep infections combined, which is less than our study. in our study, all the patients had superficial surgical site infections with culture being positive for drug sensitive staphylococcus aureus. these were treated with antibiotics and repeated dressings and the infections were recovered. following patients for up to 24 months, two (4.0%) patients developed osteoarthritis and subsequently underwent total hip arthroplasty. both of these patients were elderly with mean age of 55 years and had initial displacement of > 2 mm on initial radiographs. our study reported that initial displacement of > 2 mm had significant effect on both functional and radiological outcome. meena.19) also demonstrates poor results with initial displacement of > 2 mm. this shows that initial displacement of > 2 mm is one of the prognostic factor for the development of osteoarthritis. literature shows conversion of open reduction and internal fixation (orif) to total hip arthroplasty in 28% of the patients among elderly population with mean age of 60 years. this is because of increased fragility of bones of elderly patients. in our study, the elderly study population comprised of 10% ; five of the patient population and three of these patients had conversion of orif to arthroplasty among elderly which is 40% among elderly being higher than the findings of study done by o'toole). previous study concludes that delay in surgical reconstruction of acetabular fractures has worse effect on outcome2122). we evaluate the effect of delay in surgical reconstruction on outcome by dividing patients into two groups. patients who were operated within a week had significantly better results than patients operated after one week. letournel7) in his study concludes that outcome is worse if acetabular reconstruction were undertaken beyond three weeks. fracture pattern had significant effect on radiological outcome but not on functional outcome in our study. kreder.23) found that functional outcome was significantly worse in patients with radiological arthritis and in those with associated posterior wall and posterior column fractures. we use the letournel and judet system to classify acetabular fractures that does n't consider fracture comminution and displacement. this classification can guide surgeons about surgical approach but ca n't useful to assess functional outcome. moed.22) concludes that associated injury have significant negative effect on functional outcome. study results indicated that mechanism of injury, time between injury and surgery, initial degree of displacement and quality of reduction had significant effect on functional as well as radiological outcome. with the availability of good imaging facilities, surgeon experience, better instrumentation along with good perioperative care, we believe that the surgical fixation of displaced acetabular fractures would yield better results. | purposeacetabular fractures are mainly caused by trauma and the incidence is rising in developing countries. initially these fractures were managed conservatively, due to lack of specialized and dedicated acetabulum surgery centres. our aim is to study the radiological and functional outcomes of surgical management of acetabular fractures in tertiary care hospital.materials and methodstotal 50 patients were enrolled. the patients with acetabular fractures were enrolled between the years 2012 to 2014. patients were evaluated clinically with harris hip score (hhs) and radiologically with matta outcome grading. the factors examined include age, gender, fracture pattern, time between injury and surgery, initial displacement and quality of reduction on the final outcome.resultsthere were 34 males and 16 females. mean age was 44.2011.65 years while mean duration of stay was 9.282.36 days. duration of follow - up was 24 months. most common mechanism of injury was motor vehicle accident (n=37, 74.0%). open reduction and internal fixation of fractures were performed using reconstruction plates. mean hhs at 24 months was 82.368.55. the clinical outcome was acceptable (excellent or good) in 35 (70.0%) cases and not acceptable (fair or poor) in 15 (30.0%) cases. the radiological outcome was anatomical in 39 (78.0%) cases, congruent in 5 (10.0%) cases, incongruent in 6 (12.0%) cases.conclusionstudy results indicated that mechanism of injury, time between injury and surgery, initial degree of displacement and quality of reduction had significant effect on functional as well as radiological outcome. |
hrpe1 smo - egfp cells were serum starved 24 - 72 h prior to electrophysiological recording in order to slow cell growth and induce ciliogenesis. primary cells cultured from the arl13b - egfp mouse tissues (mef and mrpe) were cultured for less than two passages before they were patch clamped. data were collected using an axopatch 200b patch clamp amplifier, digidata 1440a, and pclamp 10 software. for temperature - controlled experiments, the perfusate was heated using a warner tc-344b heater controller and in - line heater / cooler while bath temperature was monitored using a thermistor placed in close proximity to the recording electrode. for pressure clamp experiments, membrane pressure was applied using a hspc-1 high - speed pressure system (ala scientific) controlled by pclamp software. whole - cell and excised inside - out patch currents were digitized at 25 khz and low pass filtered at 10 khz. the permeability of monovalent cations relative to that of na was estimated from the shift in reversal potential on replacing external na bath solution (150 mm x, 10 mm hepes, 0.5 mm cacl2, ph 7.4), where x was nacl, names, kcl, bacl2, cacl2, or nmdg. permeability ratios were calculated as : where [x]out is defined as the extracellular concentration of the given ion, p is defined as the permeability of the ion indicated by the subscript, f is faraday 's constant, r is the gas constant, t is absolute temperature and vrev is the reversal potential for the relevant ion. the number of channels per cilia was calculated as n = i / po(i), where n = number of channels ; i is the whole - cilium current at + 100 mv ; po is the open probability (calculated at + 100 mv from 1 s - long traces), and i is the single channel amplitude at + 100 mv. based in the slope of the single channel records from rpe cilia, the channel conductance, = 80 3 ps inward and = 96 3 ps outward. rpe cilia channel mean open time at -100 mv and 100 mv were 0.22 0.02 and 14 3 ms, respectively. for simplification we represent the cilia as a cylinder and thus its surface area is given by a = 2r(r+h) where h is the height (5 m on average) and radius (r) varies from 0.15 to 0.25 m as measured in electron micrographs. assuming r = 0.2 m, then the surface area is 6.3 m and capacitance (cm, assuming 1 f / cm) = 0.063 pf (for comparison, surface areas of typical cells are 2000 m with cm = 20 pf). current density measured in the detached cilia patch and the hrpe cell body was 1730 98 pa / pf and 31 4 pa / pf respectively. the equivalent circuit of the whole - cilia recording assumes that the resistance between cilia and cell is sufficiently high to neglect the contributions from the cell, since cilia ripped from the cell and sealed over show essentially the same result as cilia attached to the cell. hpkd2, hpkd2-l1 and hpkd2-l2 cdnas were obtained from open biosystems and a hemagglutinin (ha) tag was added to the n - terminus using pcr. the resulting cdnas were subcloned into an ires enhanced green fluorescence protein (egfp)-containing vector. hpkd1-l1 cdna was synthesized (bio basic, amherst ny) with a flag - tag at the c - terminus. hpkd1l1, hpkd1 and hpkd1-l2 were subcloned into an ires red fluorescence protein (rfp)-containing vector. transfected cells cultured at 37c and plated onto glass coverslips and were recorded 24 - 48 h later. for the co - transfected cells in figure 4, whole - cell patches for recordings from the cell body were obtained using electrodes of 1.5 - 2.5 m and excised inside - out patches were obtained using pipettes of 5 - 7 m resistances. smo - egfp expressing hrpe1 cells were transfected with 50 pm of sirna and 5 l rnaimax (life technologies) in a 3.5 cm dish. 60 h after transfection, cells were serum - starved and cilia recordings performed 48 h after serum starvation. hrpe1 cells in a 3.5 cm dish were washed 1 with pbs and lysed in 1 ml trizol for rna extraction according to the manufacturer 's instructions. gene specific products were amplified by pcr and gene expression was visualized by agarose gel electrophoresis. hek 293 t cells were transfected in a 10 cm dish with the indicated combinations of pkd1-l1 flag and pkd2-l1-ha using lipofectamine according to the manufacturer 's instructions (life technologies). 24 h after transfection cells were lysed in 2 ml ripa buffer (20 mm tris - hcl ; ph 7.5, 150 mm nacl, 1 mm edta, 1% np-40 and 1% sodium deoxycholate and protease inhibitors added (complete, roche). after centrifugation at 15,000 g for 10 min at 40c, 1.5 ml of the supernatant was added to 30 l anti - flag m2 agarose (sigma - aldrich) and incubated overnight at 40c. agarose beads were spun down at 1000 g for 5 min and washed 5 with ripa buffer. the agarose beads were resuspended in lds samples buffer containing 2% -mercaptoethanol, heated at 75c for 5 min, and proteins were separated on a 4%-12% bistris gel. after transfer to pvdf membrane, blots were probed with anti - flag (sigma aldrich) or anti - ha (roche) antibodies. hrpe1 smo - egfp cells were serum starved 24 - 72 h prior to electrophysiological recording in order to slow cell growth and induce ciliogenesis. primary cells cultured from the arl13b - egfp mouse tissues (mef and mrpe) were cultured for less than two passages before they were patch clamped. data were collected using an axopatch 200b patch clamp amplifier, digidata 1440a, and pclamp 10 software. for temperature - controlled experiments, the perfusate was heated using a warner tc-344b heater controller and in - line heater / cooler while bath temperature was monitored using a thermistor placed in close proximity to the recording electrode. for pressure clamp experiments, membrane pressure was applied using a hspc-1 high - speed pressure system (ala scientific) controlled by pclamp software. whole - cell and excised inside - out patch currents were digitized at 25 khz and low pass filtered at 10 khz. the permeability of monovalent cations relative to that of na was estimated from the shift in reversal potential on replacing external na bath solution (150 mm x, 10 mm hepes, 0.5 mm cacl2, ph 7.4), where x was nacl, names, kcl, bacl2, cacl2, or nmdg. permeability ratios were calculated as : where [x]out is defined as the extracellular concentration of the given ion, p is defined as the permeability of the ion indicated by the subscript, f is faraday 's constant, r is the gas constant, t is absolute temperature and vrev is the reversal potential for the relevant ion. the number of channels per cilia was calculated as n = i / po(i), where n = number of channels ; i is the whole - cilium current at + 100 mv ; po is the open probability (calculated at + 100 mv from 1 s - long traces), and i is the single channel amplitude at + 100 mv. based in the slope of the single channel records from rpe cilia, the channel conductance, = 80 3 ps inward and = 96 3 ps outward. rpe cilia channel mean open time at -100 mv and 100 mv were 0.22 0.02 and 14 3 ms, respectively. for simplification we represent the cilia as a cylinder and thus its surface area is given by a = 2r(r+h) where h is the height (5 m on average) and radius (r) varies from 0.15 to 0.25 m as measured in electron micrographs. assuming r = 0.2 m, then the surface area is 6.3 m and capacitance (cm, assuming 1 f / cm) = 0.063 pf (for comparison, surface areas of typical cells are 2000 m with cm = 20 pf). current density measured in the detached cilia patch and the hrpe cell body was 1730 98 pa / pf and 31 4 pa / pf respectively. the equivalent circuit of the whole - cilia recording assumes that the resistance between cilia and cell is sufficiently high to neglect the contributions from the cell, since cilia ripped from the cell and sealed over show essentially the same result as cilia attached to the cell. the number of channels per cilia was calculated as n = i / po(i), where n = number of channels ; i is the whole - cilium current at + 100 mv ; po is the open probability (calculated at + 100 mv from 1 s - long traces), and i is the single channel amplitude at + 100 mv. based in the slope of the single channel records from rpe cilia, the channel conductance, = 80 3 ps inward and = 96 3 ps outward. rpe cilia channel mean open time at -100 mv and 100 mv were 0.22 0.02 and 14 3 ms, respectively. for simplification we represent the cilia as a cylinder and thus its surface area is given by a = 2r(r+h) where h is the height (5 m on average) and radius (r) varies from 0.15 to 0.25 m as measured in electron micrographs. assuming r = 0.2 m, then the surface area is 6.3 m and capacitance (cm, assuming 1 f / cm) = 0.063 pf (for comparison, surface areas of typical cells are 2000 m with cm = 20 pf). current density measured in the detached cilia patch and the hrpe cell body was 1730 98 pa / pf and 31 4 pa / pf respectively. the equivalent circuit of the whole - cilia recording assumes that the resistance between cilia and cell is sufficiently high to neglect the contributions from the cell, since cilia ripped from the cell and sealed over show essentially the same result as cilia attached to the cell. hpkd2, hpkd2-l1 and hpkd2-l2 cdnas were obtained from open biosystems and a hemagglutinin (ha) tag was added to the n - terminus using pcr. the resulting cdnas were subcloned into an ires enhanced green fluorescence protein (egfp)-containing vector. hpkd1-l1 cdna was synthesized (bio basic, amherst ny) with a flag - tag at the c - terminus. hpkd1l1, hpkd1 and hpkd1-l2 were subcloned into an ires red fluorescence protein (rfp)-containing vector. transfected cells cultured at 37c and plated onto glass coverslips and were recorded 24 - 48 h later. for the co - transfected cells in figure 4, whole - cell patches for recordings from the cell body were obtained using electrodes of 1.5 - 2.5 m and excised inside - out patches were obtained using pipettes of 5 - 7 m resistances. smo - egfp expressing hrpe1 cells were transfected with 50 pm of sirna and 5 l rnaimax (life technologies) in a 3.5 cm dish. 60 h after transfection, cells were serum - starved and cilia recordings performed 48 h after serum starvation. hrpe1 cells in a 3.5 cm dish were washed 1 with pbs and lysed in 1 ml trizol for rna extraction according to the manufacturer 's instructions. gene specific products were amplified by pcr and gene expression was visualized by agarose gel electrophoresis. hek 293 t cells were transfected in a 10 cm dish with the indicated combinations of pkd1-l1 flag and pkd2-l1-ha using lipofectamine according to the manufacturer 's instructions (life technologies). 24 h after transfection cells were lysed in 2 ml ripa buffer (20 mm tris - hcl ; ph 7.5, 150 mm nacl, 1 mm edta, 1% np-40 and 1% sodium deoxycholate and protease inhibitors added (complete, roche). after centrifugation at 15,000 g for 10 min at 40c, 1.5 ml of the supernatant was added to 30 l anti - flag m2 agarose (sigma - aldrich) and incubated overnight at 40c. agarose beads were spun down at 1000 g for 5 min and washed 5 with ripa buffer. the agarose beads were resuspended in lds samples buffer containing 2% -mercaptoethanol, heated at 75c for 5 min, and proteins were separated on a 4%-12% bistris gel. after transfer to pvdf membrane, blots were probed with anti - flag (sigma aldrich) or anti - ha (roche) antibodies. hpkd2, hpkd2-l1 and hpkd2-l2 cdnas were obtained from open biosystems and a hemagglutinin (ha) tag was added to the n - terminus using pcr. the resulting cdnas were subcloned into an ires enhanced green fluorescence protein (egfp)-containing vector. hpkd1-l1 cdna was synthesized (bio basic, amherst ny) with a flag - tag at the c - terminus. hpkd1l1, hpkd1 and hpkd1-l2 were subcloned into an ires red fluorescence protein (rfp)-containing vector. transfected cells cultured at 37c and plated onto glass coverslips and were recorded 24 - 48 h later. for the co - transfected cells in figure 4, whole - cell patches for recordings from the cell body were obtained using electrodes of 1.5 - 2.5 m and excised inside - out patches were obtained using pipettes of 5 - 7 m resistances. smo - egfp expressing hrpe1 cells were transfected with 50 pm of sirna and 5 l rnaimax (life technologies) in a 3.5 cm dish. 60 h after transfection, cells were serum - starved and cilia recordings performed 48 h after serum starvation. hrpe1 cells in a 3.5 cm dish were washed 1 with pbs and lysed in 1 ml trizol for rna extraction according to the manufacturer 's instructions. gene specific products were amplified by pcr and gene expression was visualized by agarose gel electrophoresis. hek 293 t cells were transfected in a 10 cm dish with the indicated combinations of pkd1-l1 flag and pkd2-l1-ha using lipofectamine according to the manufacturer 's instructions (life technologies). 24 h after transfection cells were lysed in 2 ml ripa buffer (20 mm tris - hcl ; ph 7.5, 150 mm nacl, 1 mm edta, 1% np-40 and 1% sodium deoxycholate and protease inhibitors added (complete, roche). after centrifugation at 15,000 g for 10 min at 40c, 1.5 ml of the supernatant was added to 30 l anti - flag m2 agarose (sigma - aldrich) and incubated overnight at 40c. agarose beads were spun down at 1000 g for 5 min and washed 5 with ripa buffer. the agarose beads were resuspended in lds samples buffer containing 2% -mercaptoethanol, heated at 75c for 5 min, and proteins were separated on a 4%-12% bistris gel. after transfer to pvdf membrane, blots were probed with anti - flag (sigma aldrich) or anti - ha (roche) antibodies. supplemental video 1 : obtaining a whole - cilium patch using confocal microscopy an hrpe cell cilia expressing smo - gcamp3 is patch clamped by applying the glass pipette to the tip of the cilia and establishing a giga - seal by gentle suction (frames 4 - 13). intentional movement of the pipette pulls the attached cilium (frames 14 - 18). the ciliary membrane was held at -60 mv and ruptured, establishing a whole - cilium patch configuration in which the entire cilia membrane is voltage - clamped and the inside of the cilia perfused by the patch pipette. dic (left), fluorescence (right), and merged (center) images were acquired in an inverted confocal microscope using a 60x objective. break - in to the whole - cilium is accompanied by an increase in gcamp fluorescence (frames 22 - 29). supplemental video 2 : obtaining an excised - cilia patch from an rpe smo - egfp cell. rpe cells expressing smo - gfp enable visualization of the primary cilium under confocal microscopy. once the cilium was patched in the whole - cilium configuration, the cilia - pipette connection is demonstrated by intentional movement of the pipette from side to side (frames 1 - 2). part of the cilia was then detached from the cell by lifting the electrode (frames 3 - 4). the focus plane was raised and lowered (gap in fluorescence), demonstrating that the cilium has separated from the cell body (frames 4 - 9). | summarya primary cilium is a solitary slender non - motile protuberance of structured microtubules (9 + 0) enclosed by plasma membrane1. housing components of the cell division apparatus between cell divisions, they also serve as specialized compartments for calcium signaling2 and hedgehog (hh) signaling pathways3. specialized sensory cilia such as retinal photoreceptors and olfactory cilia employ diverse ion channels4 - 7. an ion current has been measured from primary cilia of kidney cells8 but the responsible genes have not been identified. the polycystin proteins (pc, pkd), identified in linkage studies of polycystic kidney disease9, are candidate channels divided into two structural classes : 11-transmembrane (tm) proteins (pkd1, pkd1-l1 and pkd1-l2) remarkable for a large extracellular n - terminus of putative cell adhesion domains and a gpcr proteolytic site, and the 6-tm channel proteins (pkd2, pkd2-l1, pkd2-l2 ; trpps). evidence suggests that the pkd1s associate with the pkd2s via coiled - coil domains10 - 12. here, we employ a transgenic mouse in which only cilia express a fluorophore and employ it to directly record from primary cilia and demonstrate that pkd1-l1 and pkd2-l1 form ion channels at high densities in several cell types. in conjunction with the companion manuscript2, we show that the pkd1-l1/pkd2-l1 heteromeric channel establishes the cilia as a unique calcium compartment within cells that modulates established hedgehog pathways. |
calcium channel blockers (ccb) constitute the leading form of cardiovascular drug overdose and have been implicated in up to 48% of deaths resulting from such overdose. the difficulty arises because patients severely poisoned with ccbs can develop profound refractory bradycardia, hypotension and either cardiogenic or noncardiogenic pulmonary edema. this case was reported to raise awareness about the severe complications of amlodipine poisoning, as well as the appropriate therapeutic approach to these complications among practicing medical professionals. the case we present here is about an 18-year - old girl who was referred to our hospital from a local health center with shock, following the consumption of 17 tablets containing a fixed dose combination of amlodipine 5 mg and atenolol 50 mg. gastric lavage had been performed at the local center prior to referral, approximately 2 h after the consumption of the tablets. she developed hypotension 2 h later and was transferred to our hospital 7 h after the time of ingestion. on questioning, she admitted to having ingested the tablets with suicidal intent. she denied concomitant consumption of alcohol or any other drugs. at presentation, she was confirmed to be hypotensive, with cold peripheries, a thready pulse at 130/min and a blood pressure of 70/50 mm hg in the supine position. hourly urine output was initially below 0.5 ml / kg body weight but improved rapidly after infusion of intravenous (iv) fluids and vasopressors. pulse oximetry showed mild hypoxia (spo2 : 88% on room air), which improved with oxygen supplementation. routine laboratory tests showed mild renal failure (serum urea : 49 mg / dl, creatinine : 1.9 mg / dl). other tests including complete blood counts, liver function tests and serum electrolytes were within the normal limits. arterial blood gas analysis showed relative hypoxia with severe metabolic acidosis and respiratory compensation (ph : 7.31, po2 : 86 mm hg, pco2 : 12 mm hg, hco3 : 8.8 mmol / l, fio2 : 0.6). calculated parameters showed high anion gap metabolic acidosis (anion gap : 22), a pao2/fio2 ratio of 143 and an elevated a - a gradient (327 mm hg). when hypotension failed to improve, inotropic support was initiated with an iv infusion of norepinephrine. as hypoxia continued to worsen over the next 24 h, chest x - ray was repeated and revealed bilateral extensive inhomogeneous shadows involving the middle and lower zones of both lung fields ; both upper zones were spared [figure 1 ]. transthoracic echocardiography was performed, and cardiac dysfunction was excluded (ejection fraction 58%, no evidence of diastolic dysfunction). although direct measurement of pulmonary venous pressure could not be done, the overall picture was strongly suggestive of acute respiratory distress syndrome (ards). chest x - ray showing bilateral extensive inhomogeneous shadows involving the middle and lower zones of both lung fields, with sparing of both upper zones oxygen supplementation was provided initially through a venturi mask, and thereafter by means of non - invasive ventilation. iv calcium gluconate (30 ml of 10% calcium gluconate iv stat followed by an infusion of 10% calcium gluconate at 10 ml / h) was administered when the patient developed ards. the patient responded to inotropic support with progressive resolution of hypotension over the next 48 h. ards also improved over the same period of time. eight days after the episode, she had recovered completely from hypotension, hypoxia and acute kidney injury. there are a limited number of case reports describing severe amlodipine intoxication ; understandably therefore, there are no standardized guidelines for managing such cases. gastrointestinal decontamination with activated charcoal constitutes the first line of therapy for patients with ccb overdose ; unfortunately this approach is only beneficial when used within the 1 h of consumption, and could not be employed in our case. other therapeutic strategies that have been used with varying degrees of success include the hyperinsulinemia - euglycemia technique, continuous iv infusion of calcium, and inotropic support with dopamine, norepinephrine, dobutamine and levosimendan. in this case, it was decided to administer calcium infusion when the patient developed ards. this approach contrasts sharply with most case reports, wherein calcium infusion was employed to correct hemodynamic instability. for instance, saravu and balasubramanian described a young male with late onset ards after consuming 400 mg of amlodipine, who received calcium gluconate for hypotension, prior to the onset of ards. described a female with refractory hypotension following consumption of 280 mg of amlodipine, in whom treatment with calcium gluconate infusion yielded only marginal improvement in hypotension. similarly hasson. have reported another young female with refractory hypotension and ards after consuming 280 mg of amlodipine. again, the patient was initiated on iv calcium gluconate primarily for the management of hypotension. yet another case series by ghosh and sircar describes two individuals with refractory hypotension following amlodipine intoxication who had a successful outcome incorporating iv calcium infusion in their management. kute. also described a patient with ards and hypotension who was administered iv calcium only when hypotension failed to respond to conventional inotropes. selective pre - capillary vasodilation producing excessive pulmonary capillary transudation has been suggested as a mechanism for development of ards in patients with ccb overdose. in such a situation, administration of iv calcium raises extracellular calcium concentration, driving calcium intracellularly and thus reversing ccb induced vasodilation. intuitively, such pharmacological antagonism would suggest that calcium gluconate be useful for primary management of ccb induced pulmonary edema. this hypothesis is consistent with the temporal patterns seen in this case, wherein progression of ards was seen in the first 24 h after admission despite correction of hypotension. another notable finding in this case was the development of severe metabolic acidosis, in the absence of lactic acidosis. we suggest that severe metabolic acidosis might be an independent sequel of amlodipine intoxication, with contribution from systemic hypotension and acute kidney injury. interestingly, this patient did not develop any overt signs of beta - blocker toxicity such as conduction blocks or even bradycardia despite the ingestion of 850 mg of atenolol, instead presenting with reflex tachycardia probably secondary to amlodipine induced vasodilation and hypotension. it is possible that the relatively poor bioavailability of oral atenolol along with proven variability of biological response to high doses of atenolol could be responsible for the absence of beta - blocker toxicity in this instance. pertinently, doses of up to 1200 mg / day of atenolol have been reported to be well - tolerated without clinical manifestations. the relatively innocuous adverse effects of amlodipine such as peripheral edema and constipation, have resulted in a general perception that amlodipine is a safe and well - tolerated drug. while, this is certainly true of amlodipine in therapeutic doses, physicians should be aware of the severe organ dysfunction that can arise from overdosage of amlodipine given the popularity of this drug as an antihypertensive. | we report the case of an 18-year - old girl presenting with shock following ingestion of 85 mg of amlodipine and 850 mg of atenolol with suicidal intent. subsequently, the patient developed severe metabolic acidosis, acute kidney injury, and acute respiratory distress syndrome, which were managed conservatively. the patient ultimately made a full recovery. given the popularity of amlodipine and atenolol as antihypertensive drugs in this part of the world, it is likely that more such cases will be encountered in the future. physicians should be aware of the severe complications that can develop with amlodipine overdose. |
scrutiny of pharmaceutical expenditures is increasing as this is the fastest growing cost component in ambulatory care, with pharmaceutical expenditures now typically the largest cost or equal largest component in this sector across europe (ess., 2003 ; godman., 2008a ; simoens, 2008a ; coma., 2009 ; barry., 2010 ; sermet., pharmaceutical expenditure is proportionally higher in middle and lower income countries at between 20 and 60% of total healthcare spending, although from a lower baseline (cameron., 2009). the reasons for increasing expenditures are well known and include demographic changes, the continued launch of new expensive medicines, rising patient expectations and stricter clinical targets (gumbs., 2007 ; garattini., 2008 ; lee and emanuel, 2008 ; barry., 2010). new biological drugs marketed at appreciably higher acquisition costs than previous standards provide additional impetus to this growth in expenditure enhancing the scrutiny (caroll, 2005 ; barrett., 2006 ; lee and emanuel, 2008). this unsustainable growth has resulted in increasing urgency among governments, health authorities and health insurance companies to introduce reforms to improve prescribing efficiency for both new and existing drugs (traulsen and almarsdttir, 2005 ; toth, 2010). supply side reforms for existing drugs include compulsory price cuts, measures to lower generic prices, reference pricing in a class (anatomical therapeutic classification level 4 world health organization [who ], 2009) including voluntary reference pricing, as well as delisting products from the reimbursement list when they are considered no longer to be cost effective versus current standards (godman., 2008a, b, 2009a, b, c, 2010a ; simoens, 2008b ; teixeira and vieira, 2008 ; wettermark., 2008 ; coma., 2009 ; elshaug., 2009 ; mcginn., 2010 ; sermet., demand - side reforms and initiatives for existing drugs include measures to enhance the prescribing and dispensing of generics. this includes academic detailing and prescribing guidance incorporating electronic prescribing support systems, prescribing targets, financial incentives including incentives to enhance substitution in pharmacies, mandatory substitution unless prohibited by government agencies and administrative barriers to relegate the prescribing of patent protected products in a class or related classes to second line (tilson., 2005 ; gumbs., 2009a, b, 2010a ; krska and godman, 2010 ; martikainen., 2010 a number of these strategies are aimed at counter - acting the commercial activities of pharmaceutical companies, who have typically been the principal source of information among physicians for new drugs (jones., 2001 ; this together with the complex nature of prescribing helps explain why pharmaceutical companies in the uk currently invest over 850 mn / year in marketing activities, with similar experiences across europe (beishon. governments, health authorities and health insurance agencies have instigated a range of measures to address physician and patient concerns with the effectiveness and/or side - effects of generics to release valuable resources. this urgency has increased with estimated global sales of products of $ us50 bn to $ us100 bn of products likely to lose their patents between 2008 and 2013 (frank, 2007 ; jack, 2008). the initiatives are similar and include defined criteria for granting substitutability status for generics, publishing lists of substitutable and non - substitutable products, not reimbursing generics where there are concerns with their quality, physician and patient education, encouraging international non - proprietary name (inn) prescribing as well as incentivizing pharmacists to talk with patients when substituting to allay any fears (allenet and barry, 2003 ; valles., 2003 ; kjoenniksen., 2006 ; kopp and vandevelde, 2006 ; godman., 2008a, 2009a ; teixeira and vieira, 2008 ; versantvoort., 2008 these concerns though generally only apply in a limited number of situations (valles., 2003 ; kjoenniksen., 2006 ; heikkil., 2007 ; shrank.,, there should be considerable opportunities for european countries to further enhance their prescribing efficiency without compromising care. this should be welcomed as further reforms are essential to maintain comprehensive and equitable healthcare throughout europe as we are already seeing european countries experiencing difficulties with funding new premium priced ambulatory care drugs even when these are considered cost effective. current activities to help fund new innovative drugs include (cooke., 2005 ; doh, 2006, 2008 ; godman.,, 2010b) : placing them on waiting lists until more funding becomes available, e.g., lithuania funding a limited number through special programs, e.g., therapeutic programs in poland increasing planning activities to pro - actively address potential funding concerns. this includes ascertaining the potential role for new treatments ahead of launch as well as identifying potential areas to release resources, such as current treatments that well soon lose their patent, to fund new innovative treatments at launch, e.g., sweden and uk. subsequently monitoring prescribing of the new products against agreed guidance post launch it is recognized it is difficult for countries to learn from each other in view of different circumstances and starting points, with one approach unlikely to fit all countries. in addition, prescribing behavior is complex (grol and grimshaw, 2003 ; prosser., 2003 ; having said this, there are examples of european countries learning from each other when considering new health reforms (toth, 2010). in addition, the plethora of different measures introduced across europe to enhance prescribing efficiency should stimulate debates within countries on additional reforms and initiatives that could be introduced. coupled with this, cross national comparisons of drug utilization and expenditure also help identify possible additional reforms that countries could introduce through analytical studies linking datasets from different countries and regions and matching changes in utilization and expenditure with health policy initiatives. the objectives of this paper are to assess the influence of the many supply and demand - side reforms and initiatives introduced across europe to enhance prescribing efficiency in ambulatory care once a decision has been made to prescribe a particular class of drug. subsequently utilize the findings to suggest potential future initiatives that countries could consider to further enhance their prescribing efficiency given continued resource pressures. this is a cross national retrospective observational study involving the analysis of reimbursed utilization and expenditure on a yearly basis for the proton pump inhibitors (ppis) and hmg coa reductase inhibitors (statins) among european countries. these were austria (at), croatia (hr), estonia (ee), france (fr), finland (fi), germany (de), italy (it), lithuania (lt), norway (no), portugal (pt), poland (po), republic of ireland (ie), serbia (rs), slovenia (si), spain (es only catalonia), sweden (se), turkey (tr), and the united kingdom (gb - eng england and gb - scot scotland). the countries reflect differences in geography, epidemiology, financing of healthcare, available resources for healthcare as well as different approaches to the pricing of generics, originators, and single sourced products (table 1). they also reflect appreciable differences in the nature and extent of reforms and initiatives introduced to enhance the prescribing of generics. as a result, provide a number of exemplar initiatives and countries (table a1 of appendix). characteristics of the european countries in 2008 (using published definitions for generic pricing). reference pricing : vp, voluntary reference pricing ; ni, not introduced ; rjt, proposed but rejected ; ac, active consideration ; part, partial applying to some product classes but not all. the following definitions have been used to classify the different pricing approaches for generics across europe, which build on previous publications (godman., 2010a, c) : prescriptive pricing (pp) mandated price reductions for generics for reimbursement compared with for instance pre - patent loss prices for the molecule market forces (mf) no prescriptive pricing approaches ; price reductions left to market forces with typically patients paying an additional co - payment for a more expensive product including branded generics than the current referenced priced molecule mixed approaches (ma) typically prescriptive pricing for the first generic or generics ; market forces after that we acknowledge though that in some countries only branded generics are available. however, to reduce confusion only the term generics will be used throughout the paper. only administrative databases were used to ensure standardization across countries. these included (100% coverage of the population unless stated) : at (austria) data warehouse of the federation of austrian social insurance institutions (98% of the population) de (germany) gamsi - database, i.e., the gkv arzneimittel schnell - information, which covers all prescriptions paid by the social health insurance funds (shi approximately 90% of the population) ee (estonia) estonian health insurance fund es (spain only catalonia) dmart (catalan health service) database (all patients in catalonia). data only available from 2003 onwards fi (finland) prescription register of the social insurance institution fr (france) medic'am database (cnam - ts database for salaried personnel covering 75% of the population) gb eng (england) information centre for health and social care gb scot (scotland) prescribing information system (pis) from nhs national services scotland corporate warehouse hr (croatia) croatian institute for health insurance ie (republic of ireland) hse - pcrs (gms population covering approximately 30% of the population with higher morbidity than the general population reflected in consuming approximately 65% of total pharmaceutical expenditure) it (italy) osmed database lt (lithuania) electronic database of the national health insurance fund no (norway) norwegian prescription database (norpd). expenditure data only available from 2004 onwards po (poland) national health fund database pt (portugal) infarmed (nhs) database (approximately 75% of the population) rs (serbia) republic of serbia 's health insurance fund database se (sweden) apoteket ab (national corporation of swedish pharmacies monopoly up to 1 january 2010) si (slovenia) the national institute of public health and health insurance institute prescription database tr (turkey) social security institution (sgk) single national public payer purchasing approximately 95% of pharmaceutical expenditure in turkey as discussed, two classes were chosen for in - depth analysis of ambulatory care prescribing efficiency. these were the ppis anatomical therapeutic chemical (atc) a02bc, and the hmg coa reductase inhibitors (statins) atc group c10aa (who, 2009). these two classes were chosen as (afssaps, 2005 ; merec extra, 2006 ; national institute for health and clinical excellence, 2006 ; wessling and lundin, 2006 ; godman., 2008b, 2009a, b ; eriksson and lundin, 2009 ; martikainen., 2010 ; 2010) : they are both commonly prescribed in ambulatory care they also contain a mixture of generics, originators and single sourced products in a class and many patients, if not all in the case of ppis, can be adequately managed with generic products ppis and statins are typically the subject of country and/or regional initiatives to enhance prescribing efficiency utilization rates for the different molecules in each class were computed using defined daily doses (ddds), with utilization patterns in 2007 generally compared with 2001. these dates were chosen as typically both generic simvastatin and generic omeprazole became available and were reimbursed during this time period among western european countries (table 2). simvastatin was the first major statin to become available as a generic in europe with generally no or limited utilization of lovastatin. both events resulted in demand - side initiatives to try and enhance the prescribing of generics ahead of more expensive patent protected products to improve prescribing efficiency (table a1 of appendix). dates when generic omeprazole and generic simvastatin were first dispensed and reimbursed among european countries. the concepts of atc classification and ddds were developed to facilitate cross country comparisons in drug utilization especially where there are differences in pack sizes and available tablet strengths (bergman. ddds are now internationally accepted for comparing drug utilization patterns across countries (birkett, 2002 ; who, 2003 ; walley., the atc index from 2010 was used in this study in line with who recommendations (rnning., 2000). demand - side measures, i.e., initiatives and reforms to influence subsequent prescribing or dispensing of generics, have been collated under the 4 es, i.e., education, engineering, economics and enforcement. this approach has been used in other settings and successfully adapted to healthcare to provide a concise and easily understandable methodology to compare and contrast the complexity and multiplicity of demand - side measures implemented within and between countries (coma., 2009 ; godman., examples of the 4 es include : educational activities includes development and distribution of prescribing guidance right through to more intensive strategies such as educational outreach visits and benchmarking of physician prescribing habits engineering activities gincludes organizational or managerial interventions such as prescribing targets and compulsory inn prescribing as well as price : volume agreements for single sourced existing products economic interventions includes devolved budgets with penalties, positive and negative financial incentives, as well as differential patient co - payments for more expensive products than the current reference molecule enforcement includes regulations by law such as mandatory generic substitution and prescribing restrictions reimbursed expenditures from 2001 to 2007 were typically captured for each class to assess the influence of recent reforms on overall expenditure from a health authority or health insurance perspective. the only exceptions were austria, germany and norway where there are difficulties with disassociating co - payments from total expenditure. however, this typically represents only a small proportion of overall expenditure in these three countries.. reimbursed expenditures, as opposed to total expenditures, were chosen for the analysis as this is the actual expenditure incurred by health authorities or health insurance agencies reflecting the focus of the paper. reimbursed expenditures in 2007 was subsequently converted to /1000 inhabitants / year to compare expenditures across countries adjusted for population sizes. this was based on established rates for the country ; alternatively an average for the year from national banks (godman and wettermark, 2009a, b). 2007 was chosen for this calculation as this was the latest year for comprehensive data from all countries. again, expenditure/1000 inhabitants / year is the internationally accepted standard approach for comparing expenditures across countries. exchange rates used were 1 = 0.734gb, ltl3.453, 8.219nok, 3.783pln, 79.24rsd and 9.25sek (2007). there has been no allowance for inflation in the analysis in order to directly compare the impact of different policies over time. in addition, health authorities and health insurance agencies typically refer to pre - patent loss prices when establishing reimbursed prices for generics especially for prescriptive pricing or mixed approaches to the pricing of generics (godman. the data sets collected to compare prescribing efficiency for the ppis and statins among the european countries included : total ddds 2001 and 2007 ddds/1000 inhabitants / day (ddds / tid) reimbursed expenditure in 2001 and 2007 /1000 inhabitants / year in 2007 principal reforms to lower the price of generics principal demand - side reforms to enhance the prescribing of generic ppis and statins compared with single sourced products collated under the 4es two principal analyses were undertaken for both the ppis and statins to assess overall efficiency, with criteria subsequently broken down into three categories. the three cut - off points for assessing efficiency were chosen intuitively ; however, tested among the co - authors for internal validity. principal measures used to evaluate changes in prescribing efficiency for both the ppis and statins during the study years as well as categorize countries. generic ppis and statins were often available in central and eastern european countries before 2001 distorting the figures in reality. the republic of ireland will not be included when assessing potential savings (figures 3 and 4) as the gms population has greater morbidity than the general population reflected in appreciably higher utilization of pharmaceuticals. in view of the limited number of peer - reviewed publications documenting current reforms for the pricing of generics, as well as current demand - side reforms and their impact especially for the ppis and statins among the 19 european countries and regions outside of those from a number of the co this method was also chosen to add robustness and standardization to the documentation since many of the co - authors are involved with either implementing or suggesting additional reforms in their country or region. this especially as there have been concerns with the accuracy of some of the health policy information contained within some of the web based publications (blaszczyk., 2007). no attempt has been made to analyze the appropriateness of prescribing of either the ppis or statins. this is due to a lack of access to patient databases to determine the indication and/or doses prescribed. in addition, the main emphasis of this paper is regarding prescribing efficiency once a decision has been made by the physician to prescribe either a ppi or statin., 2009 ; godman., 2009a, b, c ; mcginn., 2010). no impact analyses have been undertaken as typically multiple supply and demand - side initiatives were instigated in each country during the study period and the datasets generally covered the whole population. in addition, the intensity of different initiatives may vary over time and between different regions further hindering the usefulness of such analyses. no regression lines have been added to figures 3 and 4 as each point represents a different country subject to different supply and demand - side reforms (table a1 of appendix). table a1 in the appendix documents the main pricing reforms for generics during the study period among the 19 european countries and regions. table a1 also documents the nature and intensity of the demand - side reforms introduced to enhance prescribing efficiency principally for the ppis and statins collated under the 4 es. any co - payments for the product and/or indication, in addition to the standard co - payment for the package, are also included in table a1. this recognizes that some european countries use this economic approach to influence utilization. figure 1 demonstrated the influence of the various supply and demand - side measures (table a1 of appendix) on ppi prescribing efficiency among the different european countries and regions as measured by the rate of change in utilization (ddds) versus reimbursed expenditure principally between 2001 and 2007. the countries have been broken down by : geography into central and eastern european countries and the remainder, for the reasons discussed in table 3 the different approaches to pricing of generics prescriptive pp, market forces mf, mixed ma those showing considerable efficiency, in addition to general efficiency, i.e., below the line drawn, are highlighted using the definitions in table 3. rate of increase in expenditure (local currency) versus the rate of increase in utilization (ddd based) for the ppis principally from 2007 versus 2001 among european countries (unless stated), with generic pricing approaches divided into three categories. es = catalonia (2007 versus 2003), ee = 2007 versus 2004, hr = 2007 versus 2000, it = 2008 versus 2006, no = 2007 versus 2004, tr = 2009 versus 2007. in both lithuania and poland, there was approximately a twofold difference in the rate of increase in utilization (ddd basis) versus the rate of increase in reimbursed expenditure for the ppis between 2001 and 2007, e.g., in lithuania utilization increased 10.8-fold between 2001 and 2007 and poland over 150-fold between 2002 and 2007. this appreciable increase in utilization following reimbursement, which was considerably greater than seen in the other european countries, led to their exclusion from figure 1. serbia was also excluded from figure 1 with comprehensive data only recently becoming available, and after the availability of generic ppis. the various demand and supply side reforms instigated among the european countries and regions similarly influenced prescribing efficiency for the statins (figure 2). the same categorization for efficiency has been used (table 3), and again countries have been broken down into geography and approaches to the prescribing of generics. percentage change in utilization (ddds) versus the percentage change in reimbursed expenditure (local currency) for the statins principally from 2001 to 2007 among european countries. the countries again divided into former central and eastern european countries (cee) with the approaches to generic pricing divided into three categories. es = catalonia (2007 versus 2003), ee = 2007 versus 2004, hr = 2007 versus 2000, it = 2008 versus 2006, no = 2007 versus 2004, tr = 2009 versus 2007. in poland, there was over a 140-fold increase in statin utilization between 2002 and 2007 following their reimbursement, with overall a 4.5-fold difference between the rate of increase in utilization versus the rate of increase in reimbursed expenditure between 2001 and 2007. again, serbia was excluded from figure 2 with comprehensive data only recently becoming available. the differences seen in prescribing efficiency for the ppis (figure 1) translate into considerable differences in overall expenditure adjusted for the differences in population sizes, i.e., expenditure expressed in /1000 inhabitants / year and utilization by ddds/1000 inhabitants / day (ddd / tid) by 2007 (figure 3). the differences in geography and approaches to the prescribing of generics have again been highlighted. expenditure figures for the ppis will be affected by whether there are high patient co - payment levels (table a1 of appendix). utilization (ddd / tid) and overall expenditure (/1000 inhabitants / year) for ppis among european countries in 2007 (italy 2008, serbia 2008). republic of ireland not included as the gms population has greater morbidity than the general population. the differences seen in the rates of prescribing efficiency for the statins between 2001 and 2007 among european countries (figure 2) are again reflected in considerable differences in overall expenditure in 2007 adjusted for population sizes (figure 4). the differences in geography and approaches to the prescribing of generics have again been highlighted, with overall expenditures again affected by whether there are high co - payment levels for the statins (table a1 of appendix). utilization (ddd / tid) and overall expenditure (/1000 inhabitants / year) for the statins among european countries in 2007 (italy 2008, serbia 2008). republic of ireland not included as the gms population has greater morbidity than the general population. additional reforms are essential across europe to continue funding increased volumes and new drugs without prohibitive increases in either taxes or health insurance premiums. as such, we consider the findings from this study help provide future direction to health authorities and health insurance agencies as they seek to instigate additional measures. general findings from the study include more limited utilization, and hence expenditure, of the ppis and statins among central and eastern european countries compared with western european countries (figures 3 and 4). this is principally due to prescribing restrictions and higher patient co - payments in these countries (table a1 of appendix). this endorses the need to document ongoing reforms when comparing utilization rates across countries otherwise there could be concerns with the accuracy of the data provided. table a1 of appendix also demonstrates that the 4 es provides a methodology for health authorities and health insurance agencies to comprehensively categorize their current demand - side initiatives ready for comparisons. more specific findings include the fact that both supply and demand - side reforms are essential to maximize prescribing efficiency. the findings also demonstrate that the influence of the reforms appears to be additive, with enforcement having appreciable influence on subsequent utilization patterns. prescribing efficiency in norway for the ppis (figure 1) is enhanced by its aggressive prescriptive pricing policy for generics, overcoming to some extent more limited demands side measures for the ppis compared with sweden and the uk (table a1 of appendix). the various pricing policies for generics in austria, france and portugal also helped improve prescribing efficiency with for instance the ppis despite limited demand - side measures in these countries for this class (table a1 of appendix). overall though just concentrating on one aspect of reforms, i.e., either supply or demand - side measures but not both, will not help health authorities or health insurance agencies fully realize potential efficiency gains from the availability of generics. this is illustrated when comparing for instance prescribing efficiency for the statins in sweden and the uk (england and scotland) versus germany (figures 2 and 4). in germany in 2007, there was very limited utilization of atorvastatin following the introduction of reference pricing for the class in 2003 at just 2% of overall statin utilization (godman. this compares with 21 and 33% respectively on a ddd basis for the appreciably more expensive atorvastatin and rosuvastatin in sweden and england in 2007 (godman., 2010c). however, comparative expenditure appears similar or greater in germany due to higher expenditure / ddd for simvastatin (figure 4). there are also differences in prescribing efficiency between croatia and finland even though there are high patient co - payment levels in both countries. prescribing efficiency has been enhanced in finland by active reforms to lower generic prices, e.g., a 3-month 's course of simvastatin during early 2006 was 17 versus 127 in 2002 before the introduction of generic substitution (martikainen., 2010), as well as measures restricting the prescribing of atorvastatin and rosuvastatin (table a1 of appendix). the additive nature of the demand - side measures is illustrated by greater prescribing efficiency in catalonia (spain), sweden and the uk with their multiple and intensive measures based on education, engineering and economic initiatives (table a1 of appendix) compared with france, portugal, the republic of ireland and turkey (figures 1 and 2) due generally to more limited demand - side measures in these countries (table a1 of appendix) ; although this is now changing in france (sermet. these findings concerning the additive influence of demand - side measures endorse the results from previous studies which also showed that multiple interventions appear more successful in changing prescribing behavior than single interventions (bero., 1998 ; barton, 2001 ; grol and grimshaw, 2003 ; prosser and walley, 2005). introducing prior authorization, or other similar enforcement schemes, also enhances prescribing efficiency, e.g., statins in austria, germany and norway (figure 2), coupled with reforms to lower generic prices (table a1 of appendix). this compares with a more limited influence on prescribing efficiency for the ppis in austria with just education and economic measures in the absence of enforcement (figure 1 ; table a1 of appendix). the improved efficiency seen with introducing measures for the statins in these three countries appears similar to the combination of extensive educational, engineering and economic initiatives for the statins in for instance england and sweden (figure 2 ; table a1 of appendix) along with measures to lower generic prices. enforcement can be also additive and introduced at any stage as seen in austria where prescribing restrictions for atorvastatin built on existing educational and economic activities (table a1 of appendix ; godman., 2009c). this was also seen in sweden where recent restrictions on angiotensin receptor blockers have increased their prescribing second line building on existing educational, engineering, and economic initiatives among the counties (wettermark., 2010a). as a result of the differences in the nature and extent of demand - side initiatives across these countries, there are appreciable differences in overall prescribing efficiency between france, ireland and portugal when compared with catalonia (spain), sweden and the uk (figures 1 and 2), and when adjusted for population sizes (figures 3 and 4). reimbursed expenditure is also high in ireland in their selected gms population at over 60,000/1000 inhabitants / year for both the ppis and statins. there have been concerns that extensive demand - side measures including prescribing restrictions can alter the quality of subsequent care (fein, 2010). however, a recent ecological study demonstrated similar surrogate outcomes in patients with hypercholesterolemia whether they were prescribed formulary drugs, i.e., generic simvastatin, or non - formulary drugs including atorvastatin (norman., recent studies conducted in the uk have also shown that patients can be successfully switched from atorvastatin to generic simvastatin without compromising care (usher - smith., conserved resources can be re - directed to fund programs to improve compliance as well as fund increased volumes with the growing incidence of cardiovascular diseases. compliance is a real concern in patients with chronic asymptomatic diseases (cramer., 2008) rather than any minor differences in effectiveness between the statins in clinical trials, which is not seen in practice (usher - smith. this builds on earlier examples generally within healthcare (toth, 2010), with some examples contained in table 4. examples of countries learning from each other (godman., 2008b, 2009a ; wettermark., 2008, 2010a ; barry., 2010 ; martikainen., 2010 ; mcginn., 2010). as discussed, we accept there are limitations with the study design, which are summarized in table 5. however, some of these issues are less important when comparing changes in utilization and/or expenditure as opposed to comparing absolute numbers. as a result, never - the - less, we consider the findings will be of interest to health authorities and health insurance agencies as they plan future supply and demand - side measures to further improve their prescribing efficiency. we also believe the findings will be of interest to pharmaceutical companies as they plan for the future, especially as health authorities and health insurance agencies become increasingly proactive to conserve resources for existing products (moon., 2010). ongoing initiatives to optimize the managed entry of new drugs will be discussed in future papers particularly as they underscore the notion that the funding of new premium priced products is an important challenge in europe (garattini., 2008). the majority of the authors are employed directly by health authorities or health insurance agencies or are advisers to these organizations. no author has any other relevant affiliation or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript, although morten andersen has received teaching grants from the danish association of pharmaceutical industries for providing education on pharmacoepidemiology. | introduction : european countries need to learn from each other to address unsustainable increases in pharmaceutical expenditures. objective : to assess the influence of the many supply and demand - side initiatives introduced across europe to enhance prescribing efficiency in ambulatory care. as a result provide future guidance to countries. methods : cross national retrospective observational study of utilization (ddds defined daily doses) and expenditure (euros and local currency) of proton pump inhibitors (ppis) and statins among 19 european countries and regions principally from 2001 to 2007. demand - side measures categorized under the 4es education engineering, economics, and enforcement. results : instigating supply side initiatives to lower the price of generics combined with demand - side measures to enhance their prescribing is important to maximize prescribing efficiency. just addressing one component will limit potential efficiency gains. the influence of demand - side reforms appears additive, with multiple initiatives typically having a greater influence on increasing prescribing efficiency than single measures apart from potentially enforcement. there are also appreciable differences in expenditure (/1000 inhabitants / year) between countries. countries that have not introduced multiple demand side measures to counteract commercial pressures to enhance the prescribing of generics have seen considerably higher expenditures than those that have instigated a range of measures. conclusions : there are considerable opportunities for european countries to enhance their prescribing efficiency, with countries already learning from each other. the 4e methodology allows european countries to concisely capture the range of current demand - side measures and plan for the future knowing that initiatives can be additive to further enhance their prescribing efficiency. |
developing self - management skills is now seen as a standard component of pulmonary rehabilitation (pr) programs.13 indeed, pr is considered an integral component of managing chronic obstructive pulmonary disease (copd), a progressive disabling respiratory and systemic condition.4 guidelines also recommend that pr should include a detailed assessment, exercise, education, and psychosocial support.1,2 exercise in pr has demonstrated improvements in physical capacity, health - related quality of life, dyspnea, and fatigue.5 recommendations have stipulated at least three exercise sessions weekly, two of which are to be preferably supervised.6 however, the supporting evidence for this degree of supervised exercise is limited7 and was subsequently contradicted by others who provided only once - weekly exercise supervision.811 these later studies question the conventionally recommended degree of exercise supervision. as we will explain in the materials and methods section, our intervention was once - weekly supervised exercise added to a self - management program, which we offered as a combined pr approach. condition - specific education in pr has traditionally been delivered in a didactic format and has not demonstrated additional benefit in terms of physical or exercise capacity.2,1214 consequently, exercise, not education, has become the cornerstone of pr. however, the benefits of the traditional format for pr, especially on physical capacity and exercise behavior, wane over time.15,16 this has led to an emphasis on behavioral strategies in pr as a potential means of maintaining the acute gains,3,16 but this is little tested. indeed, self - management interventions, underpinned by health psychology principles, have been seen as an alternative to the conventional pr approach. such programs are seen particularly as strategies for improving health behaviors, such as regular exercise, and integrating them into daily life. chronic disease self - management is defined as a process that facilitates an individual s confidence and capability to engage in health - promoting behaviors in order to deal with the impact of their condition on all aspects of their health namely, a sense of self, physical, emotional, social, and medical domains so as to maximize function and quality of life.17,18 self - management education or training is recognized as needing to be interactive, to facilitate not only the acquisition of health behavior knowledge but its implementation, by fostering the self - management skills of collaborative goal - setting with associated action plans, problem solving, and decision - making.13 however, with the exception of increased uptake of a symptom - based action plan to manage copd exacerbations (ie, self - management of symptoms),19 and some decrease in hospitalization rates,20 the evidence for the efficacy of holistic copd - specific self - management approaches, while popular and continuing to increase in practice, has been limited.2025 however, in more general chronic disease situations, statistically sustained significant improvements in health care utilization, health status, and health behaviors such as self - reported exercise have been reported for participants who attended the stanford chronic disease self - management program (cdsmp).26 the cdsmp, either provided at a medical center or community - based, is a generic, 6-week, group - based self - management education approach led by two trained leaders.26 such benefits were subsequently supported in a review of self - management approaches27 and have been widely implemented, as indeed is the case in our institution. the cdsmp includes educational information delivered in a lecturette style and supplemented by a companion book. the topics cover generic health information, the basis of which is comparable to that relating to health behaviors in conventional pr (table 1). the interactive format of the cdsmp deliberately fosters self - efficacy (confidence) to manage one s health condition through mastery (practicing skills through setting action plans), vicarious experiences (role modelling and peer support), social persuasion (encouragement via guided feedback), and reinterpretation of symptoms (exploring different explanations of symptoms).28 unlike pr, the cdsmp is not designed to include supervised exercise, yet has been reported to increase self - reported exercise26,29 and decrease dyspnea.29 any added benefit of formally adding supervised exercise to the cdsmp has not been reported, especially in copd. thus, with an increasing worldwide focus on self - management of chronic conditions, the opinion of hospital management at our institution was that the rehabilitation needs of the target population might be most effectively met through the cdsmp rather than conventional pr. a small pilot of the cdsmp for people with copd compared with our traditional pr30 found that the cdsmp alone improved physical capacity by 30 m, measured by the 6-minute walk distance (6mwd).31 however, with supervised exercise considered an essential component of pr and the benefits of such exercise for people with copd established,5 we felt that it was incumbent on us to investigate more fully the likely benefits of the cdsmp approach on exercise capacity, specifically in copd patients, as well as on more subjective endpoints in this context. we wished to establish whether supervised exercise in addition to the cdsmp would have added benefit compared with the cdsmp alone. this would be a step to informing us of whether the cdsmp with supervised exercise, or without, might offer an alternative to more traditional pr. this was a parallel group, randomized clinical trial aiming to investigate both the efficacy of the cdsmp itself in copd and, more particularly, the addition of supervised exercise to the cdsmp on physical capacity measured by the 6mwd. the trial was registered with the australian and new zealand clinical trials registry (actrn12610000781044). in our pilot study already mentioned, which had included a similar population of people with copd, we found the mean baseline 6mwd to be 365 m, with a standard deviation of 74 m.30 the acknowledged minimal clinical important difference (mcid) at that time was reported as 54 m.32 to achieve this difference in exercise capacity, for power of 0.8 and level of significance of 0.05, using a two - sided t - test for our primary outcome of 6mwd, we calculated that the current study would require 31 participants in each arm to allow demonstration of superiority for the active intervention by this amount. allowing for the 25% dropout rate seen in the pilot, we estimated a total of 78 participants needed to be recruited, with 1:1 randomization to the exercise intervention arm of the cdsmp plus a single supervised exercise session versus the cdsmp alone. randomization used a random numbers table with allocation stored in opaque sealed envelopes until completion of baseline data measurement. however, a double - blind clinical trial was not possible, as there was no appropriate dummy for the exercise component, and the whole population attended the cdsmp together. participants were recruited from patients referred by clinicians to pr at the royal hobart hospital, a tertiary, university - affiliated, public hospital. ethical approval was granted by the tasmanian human research ethics committee (h0008105). referring staff were aware that our rehabilitation service was intending to trial the addition of formal exercise to the cdsmp. inclusion criteria included being over age 18 years, agreeable to attend supervised exercise as well as the cdsmp as randomized, a firm diagnosis of copd, and there being at least 2 months since an acute exacerbation. exclusion criteria were cognitive impairment, inability to provide informed consent or complete a self - administered questionnaire, previous cdsmp or pr attendance within the past 2 years, and standard contraindications to exercise.33 the intervention group underwent 6 weeks of a 1-hour, weekly, supervised group exercise session of aerobic and strengthening exercises for upper and lower limbs, individualized for each participant, in the same week as the 6-week cdsmp. we were able to offer only this 1-hour supervised exercise session per week due to operational constraints. however, this approach has been supported by others.8,11 participants were offered a choice of attending the supervised exercise session either in the morning prior to the cdsmp or later in the week. the first exercise session took place in the week of the first cdsmp session. in collaboration with the physiotherapist a physiotherapy assistant trained in exercise supervision and not otherwise involved in the study supervised the actual exercise sessions, with the physiotherapist available for consultation if needed. exercise intensity in the intervention group was determined by using the modified borg rating of perceived exertion (rpe) scale (010 ; 10= maximum).34 a minimum of moderate intensity (rpe =3) exercise was aimed for, according to recommended exercise guidelines,35 and a maximum intensity of strong (rpe = 5). as it was not possible for us to offer separate cdsmp sessions for intervention and control participants, they attended the cdsmp together, each receiving the same encouragement and information about following a home exercise regime. to avoid contamination, controls, receiving the cdsmp alone, were offered supervised exercise on completion of their study participation. the participants attended the 6-week group - based cdsmp, with sessions of 2.5 hours duration offered once per week, facilitated by the respiratory nurse and the physiotherapist the sessions were interactive with lecturettes, problem solving, brain storming, action planning, and reporting - back activities. a comparison of the cdsmp lecturette topics with those previously delivered in pr education is shown in table 1. outcomes were assessed in the week prior to and the week following completion of the interventions. it was deemed more ethical to first determine whether or not a significant and clinically meaningful change resulted for the intervention in the short - term before extending the study to longer - term follow - up. the primary outcome was physical capacity measured by the 6mwd31 and was assessed by an assistant not connected with the trial, affording some degree of objectivity at that point. secondary outcomes, more directed to the likely efficacy outcomes of the cdsmp, are detailed in table 2.31,3541 we selected the short - form 36 questionnaire, version 2 (sf-36) as the quality - of - life measure to account for the impact of comorbidities. it has been deemed as responsive as the copd - specific saint george s respiratory questionnaire42 at detecting even small changes in people with copd.43 data were analyzed on an intention - to - treat basis, using the statistical software package spss (spss, chicago, il, usa), version 15. missing variables were replaced by carrying forward the last item measured, and for missing cases, the baseline data was carried forward to the post - data. the exception was for the champs (community healthy activities model program for seniors) self - report of physical activity, whereby all missing data were scored as zero.39 results are reported as medians with ranges or means with standard deviations, depending on distribution of data points. differences in outcomes were compared using student s t - tests for parametric data and mann whitney u tests for nonparametric data. significance for the primary outcome was set at a p - value < 0.05. due to the large number of secondary outcomes, significance levels for these were calculated using a highly conservative bonferroni correction (p<0.003).44 this was a parallel group, randomized clinical trial aiming to investigate both the efficacy of the cdsmp itself in copd and, more particularly, the addition of supervised exercise to the cdsmp on physical capacity measured by the 6mwd. the trial was registered with the australian and new zealand clinical trials registry (actrn12610000781044). in our pilot study already mentioned, which had included a similar population of people with copd, we found the mean baseline 6mwd to be 365 m, with a standard deviation of 74 m.30 the acknowledged minimal clinical important difference (mcid) at that time was reported as 54 m.32 to achieve this difference in exercise capacity, for power of 0.8 and level of significance of 0.05, using a two - sided t - test for our primary outcome of 6mwd, we calculated that the current study would require 31 participants in each arm to allow demonstration of superiority for the active intervention by this amount. allowing for the 25% dropout rate seen in the pilot, we estimated a total of 78 participants needed to be recruited, with 1:1 randomization to the exercise intervention arm of the cdsmp plus a single supervised exercise session versus the cdsmp alone. randomization used a random numbers table with allocation stored in opaque sealed envelopes until completion of baseline data measurement. however, a double - blind clinical trial was not possible, as there was no appropriate dummy for the exercise component, and the whole population attended the cdsmp together. participants were recruited from patients referred by clinicians to pr at the royal hobart hospital, a tertiary, university - affiliated, public hospital. ethical approval was granted by the tasmanian human research ethics committee (h0008105). referring staff were aware that our rehabilitation service was intending to trial the addition of formal exercise to the cdsmp. inclusion criteria included being over age 18 years, agreeable to attend supervised exercise as well as the cdsmp as randomized, a firm diagnosis of copd, and there being at least 2 months since an acute exacerbation. exclusion criteria were cognitive impairment, inability to provide informed consent or complete a self - administered questionnaire, previous cdsmp or pr attendance within the past 2 years, and standard contraindications to exercise.33 the intervention group underwent 6 weeks of a 1-hour, weekly, supervised group exercise session of aerobic and strengthening exercises for upper and lower limbs, individualized for each participant, in the same week as the 6-week cdsmp. we were able to offer only this 1-hour supervised exercise session per week due to operational constraints. however, this approach has been supported by others.8,11 participants were offered a choice of attending the supervised exercise session either in the morning prior to the cdsmp or later in the week. the first exercise session took place in the week of the first cdsmp session. in collaboration with the physiotherapist a physiotherapy assistant trained in exercise supervision and not otherwise involved in the study supervised the actual exercise sessions, with the physiotherapist available for consultation if needed. exercise intensity in the intervention group was determined by using the modified borg rating of perceived exertion (rpe) scale (010 ; 10= maximum).34 a minimum of moderate intensity (rpe =3) exercise was aimed for, according to recommended exercise guidelines,35 and a maximum intensity of strong (rpe = 5). as it was not possible for us to offer separate cdsmp sessions for intervention and control participants, they attended the cdsmp together, each receiving the same encouragement and information about following a home exercise regime. to avoid contamination, controls, receiving the cdsmp alone, were offered supervised exercise on completion of their study participation. the participants attended the 6-week group - based cdsmp, with sessions of 2.5 hours duration offered once per week, facilitated by the respiratory nurse and the physiotherapist the sessions were interactive with lecturettes, problem solving, brain storming, action planning, and reporting - back activities. a comparison of the cdsmp lecturette topics with those previously delivered in pr education is shown in table 1. outcomes were assessed in the week prior to and the week following completion of the interventions. it was deemed more ethical to first determine whether or not a significant and clinically meaningful change resulted for the intervention in the short - term before extending the study to longer - term follow - up. the primary outcome was physical capacity measured by the 6mwd31 and was assessed by an assistant not connected with the trial, affording some degree of objectivity at that point. secondary outcomes, more directed to the likely efficacy outcomes of the cdsmp, are detailed in table 2.31,3541 we selected the short - form 36 questionnaire, version 2 (sf-36) as the quality - of - life measure to account for the impact of comorbidities. it has been deemed as responsive as the copd - specific saint george s respiratory questionnaire42 at detecting even small changes in people with copd.43 data were analyzed on an intention - to - treat basis, using the statistical software package spss (spss, chicago, il, usa), version 15. missing variables were replaced by carrying forward the last item measured, and for missing cases, the baseline data was carried forward to the post - data. the exception was for the champs (community healthy activities model program for seniors) self - report of physical activity, whereby all missing data were scored as zero.39 results are reported as medians with ranges or means with standard deviations, depending on distribution of data points. differences in outcomes were compared using student s t - tests for parametric data and mann whitney u tests for nonparametric data. significance for the primary outcome was set at a p - value < 0.05. due to the large number of secondary outcomes, significance levels for these were calculated using a highly conservative bonferroni correction (p<0.003).44 there were 316 potential participants referred for pr (figure 1), with similar proportions from the private sector (30%), public inpatient service (32%), and public outpatient clinics (35%). of those not screened, the majority declined an appointment, while many others failed to keep their appointment (figure 1). those referred from the hospital wards were least likely, whereas those referred from the private sector were most likely, to attend a screening appointment (p<0.001). participants attended a median of five sessions of the cdsmp, and where appropriate, of the supervised exercise sessions. there were 15 withdrawals (five intervention and ten controls) due to illness, family issues, or medical appointments. overwhelmed by the process and subsequently withdrew permission for their data to be used ; one could not complete the baseline walking test due to increasing breathlessness, and for two, baseline questionnaires were misplaced. outcome variables at baseline were similar, with no statistically significant differences between groups (table 4). the 15 withdrawals had no baseline differences compared with completers, defined as those attending at least one cdsmp session and both data collections. there were statistically significant increases in 6mwd in both groups, of around 20 m on average (table 5). however, there was no statistically significant difference between the groups. the number of participants in each group who reached the mcid of 54 m was similar, 22% in the cdsmp - plus - exercise group and 23% in the cdsmp - only group. the associations between the changes in 6mwt distance and the selected variables of age, sex, education, breathlessness, exercise duration, and frequency, sf-36 physical and mental component summaries, exercise self - efficacy, and body mass index were weak ; all pearson s correlation coefficients were less than 0.3, and none reached statistical significance. severity and frequency of breathlessness were selected rather than copd grade, as the latter had missing data. the strongest correlations with the change in 6mwd were frequency of moderate exercise (r=0.188, p=0.066), and exercise self - efficacy (r=0.140, p=0.132), although neither were statistically significant. when entered in a multivariable linear regression model, the frequency of moderate exercise (=0.257, p=0.048) and exercise self - efficacy (=0.220, p=0.089) explained only 7.9% of the variance in the change in 6mwd. both groups increased similarly in the frequency of moderate exercise, achieving 3 days per week, and showed small increases in physical function, role physical, and exercise self - efficacy (confidence to exercise), but none of these intragroup changes reached statistical significance. there were no statistically significant differences between the groups in these changes for any secondary outcome measure (table 5). however, only the intervention group had a statistically significant increase, but only of 1 hour per week, in the duration of moderate intensity self - reported exercise (p=0.002). there were 316 potential participants referred for pr (figure 1), with similar proportions from the private sector (30%), public inpatient service (32%), and public outpatient clinics (35%). of those not screened, the majority declined an appointment, while many others failed to keep their appointment (figure 1). those referred from the hospital wards were least likely, whereas those referred from the private sector were most likely, to attend a screening appointment (p<0.001). participants attended a median of five sessions of the cdsmp, and where appropriate, of the supervised exercise sessions. there were 15 withdrawals (five intervention and ten controls) due to illness, family issues, or medical appointments. overwhelmed by the process and subsequently withdrew permission for their data to be used ; one could not complete the baseline walking test due to increasing breathlessness, and for two, baseline questionnaires were misplaced. outcome variables at baseline were similar, with no statistically significant differences between groups (table 4). the 15 withdrawals had no baseline differences compared with completers, defined as those attending at least one cdsmp session and both data collections. there were statistically significant increases in 6mwd in both groups, of around 20 m on average (table 5). however, there was no statistically significant difference between the groups. the number of participants in each group who reached the mcid of 54 m was similar, 22% in the cdsmp - plus - exercise group and 23% in the cdsmp - only group. the associations between the changes in 6mwt distance and the selected variables of age, sex, education, breathlessness, exercise duration, and frequency, sf-36 physical and mental component summaries, exercise self - efficacy, and body mass index were weak ; all pearson s correlation coefficients were less than 0.3, and none reached statistical significance. severity and frequency of breathlessness were selected rather than copd grade, as the latter had missing data. the strongest correlations with the change in 6mwd were frequency of moderate exercise (r=0.188, p=0.066), and exercise self - efficacy (r=0.140, p=0.132), although neither were statistically significant. when entered in a multivariable linear regression model, the frequency of moderate exercise (=0.257, p=0.048) and exercise self - efficacy (=0.220, p=0.089) explained only 7.9% of the variance in the change in 6mwd. both groups increased similarly in the frequency of moderate exercise, achieving 3 days per week, and showed small increases in physical function, role physical, and exercise self - efficacy (confidence to exercise), but none of these intragroup changes reached statistical significance. there were no statistically significant differences between the groups in these changes for any secondary outcome measure (table 5). however, only the intervention group had a statistically significant increase, but only of 1 hour per week, in the duration of moderate intensity self - reported exercise (p=0.002). this is the first study to investigate the effect of the cdsmp itself on physical capacity in copd, and whether limited supervised exercise produces additional benefit. this is important, because current formats of pr with an emphasis on lecture - style education and multiple weekly, health professional - supervised exercise sessions is costly, reaches only up to 50% of those to whom it is offered,3 and has waning effects over time.3 wider - reaching, more cost - effective and sustainable alternatives are needed. we found a statistically significant mean increase in 6mwd for both groups, but no extra benefit for the supervised exercise component, apart from some evidence for an increase in the mean duration of moderate self - reported exercise by 1 hour in the intervention group. there was also an increase in role physical of the sf-36 for both groups, although this lost statistical significance following the bonferroni adjustment. our study is the first on the cdsmp, when used for copd self - management, to report in the literature a statistically significant increase in 6mwd for a self - management intervention alone (our control group). all participants in our study received a behaviorally - based educational and self - management skills training intervention (cdsmp), in contrast to other studies where controls have typically been assigned to usual medical care7,8,20,21 or usual activities only.9 however, the mean increase in 6mwd achieved by both groups was small and might be regarded as of borderline clinical significance. although the direction of change is consistent with an updated systematic review5 and other randomized controlled studies,8,9 it does not approach the previously reported mcid of 54 m (95% confidence interval [ci ] 3771 m).32 on the other hand, our results do approach the more recently reported mcid of 25 m (95% ci 2061 m),45 and others have also reported an mcid of 262 m in people with severe copd following pr.46 such small improvement may have particular relevance for severe copd, and in our study, half of the participants were severely affected in this way. the 20 m change we observed is also within the lower limit of the ci observed by holland.45 other groups have also tried to reduce the pr frequency of weekly exercise component. these studies are relevant to our research, as we could offer only 1 weekly session of supervised exercise, in contrast to current recommendations.1,3 thus, singh compared twice - daily home - based walking recorded in a log and monitored once - weekly over 4 weeks with usual activities and found a significant increase in 6mwd for the intervention group but not the control group (54.226.7 m versus 6.710.3 m, p<0.001). finnerty compared education, plus once - weekly supervised exercise, plus an unsupervised home exercise program of 5 days per week over 6 weeks with usual care and reported a median increase in 6mwd of 51 m (range 2081 m) in the intervention group. these studies contrast with the limited earlier evidence7,47 on which the recommendation of at least twice - weekly supervised exercise sessions is based. furthermore, no significant difference between once weekly or twice weekly exercisers for the incremental shuttle walking test was demonstrated in a recent randomized controlled trial.11 others included a structured home exercise program to supervised exercise, also finding no additional benefit of two supervised sessions to the incremental shuttle walking test distance.10 these later studies of weekly versus twice - weekly supervised exercise point to uncertainty over the optimal frequency and mode of supervision required to increase physical capacity. they suggest that once - weekly exercise supervision with a structured home exercise program may be as good as more intensive regimes. to an extent, that is what we have tested, with negative results. in contrast with our study, others have reported a significant increase in self - reported exercise immediately following the cdsmp alone for people with chronic conditions, including copd.48 one explanation could be that the measure of self - reported exercise we used is more comprehensive than the stanford measure49 used previously. thus, our study is the first cdsmp - related study to focus on copd and the amount of moderate exercise which is required for optimal health benefits.35 disappointingly, our results indicate that the cdsmp alone is of limited benefit for meeting the minimum recommendations of exercising in the community for 30 minutes on each of at least 5 days per week. furthermore, the single supervised exercise session did not add anything in this regard either. while a major strength of our study was its execution in real world clinical practice, utilizing existing resources, this also imposed some limitations. firstly, due to ethical considerations, we were unable to include a second control group who did not receive any rehabilitation - type intervention. secondly, it would have been informative to have a group in a twice - weekly or three - times - weekly supervised exercise schedule to determine whether this more exacting approach to exercise would add to the effects of the cdsmp. while this more intense exercise has its advocates,6 such an approach is highly resource - intensive ; most other similar centers would have been unable to achieve this. therefore, the effect of adding more than one supervised exercise session to the cdsmp is unknown, or indeed whether there are optimal numbers of weekly exercise sessions. however, even if more sessions are better, resource limitation will always be a major factor for generalizability within many health centers. thirdly, participants were recruited from referrals to a hospital - based program that may differ from those who might self - refer to community - based cdsmps. nevertheless, our study reflects the usual practice for australian pr, thus enhancing local generalizability. fourthly, due to resource limitations, we were unable to offer separate sessions for cdsmp - exercise and cdsmp - only groups. while participants were requested not to discuss the exercise experience, vicarious contamination of the control group by the active intervention can not be excluded. fifthly, the leaders in this study were health professionals rather than peer leaders as is typical for other cdsmps. nevertheless, a recently published systematic review concluded that there were few differences between peer - led or health professional - led self - management programs,27 suggesting this was unlikely to be a source of bias. although this did not vary a great deal, it may have yielded information as to a differential effect of the intervention and would be a consideration for future research. finally, the cdsmp does not include a structured home exercise program, since under the license agreement, we were precluded from doing so. this is the first study to investigate the effect of the cdsmp itself on physical capacity in copd, and whether limited supervised exercise produces additional benefit. this is important, because current formats of pr with an emphasis on lecture - style education and multiple weekly, health professional - supervised exercise sessions is costly, reaches only up to 50% of those to whom it is offered,3 and has waning effects over time.3 wider - reaching, more cost - effective and sustainable alternatives are needed. we found a statistically significant mean increase in 6mwd for both groups, but no extra benefit for the supervised exercise component, apart from some evidence for an increase in the mean duration of moderate self - reported exercise by 1 hour in the intervention group. there was also an increase in role physical of the sf-36 for both groups, although this lost statistical significance following the bonferroni adjustment. our study is the first on the cdsmp, when used for copd self - management, to report in the literature a statistically significant increase in 6mwd for a self - management intervention alone (our control group). all participants in our study received a behaviorally - based educational and self - management skills training intervention (cdsmp), in contrast to other studies where controls have typically been assigned to usual medical care7,8,20,21 or usual activities only.9 however, the mean increase in 6mwd achieved by both groups was small and might be regarded as of borderline clinical significance. although the direction of change is consistent with an updated systematic review5 and other randomized controlled studies,8,9 it does not approach the previously reported mcid of 54 m (95% confidence interval [ci ] 3771 m).32 on the other hand, our results do approach the more recently reported mcid of 25 m (95% ci 2061 m),45 and others have also reported an mcid of 262 m in people with severe copd following pr.46 such small improvement may have particular relevance for severe copd, and in our study, half of the participants were severely affected in this way. the 20 m change we observed is also within the lower limit of the ci observed by holland.45 other groups have also tried to reduce the pr frequency of weekly exercise component. these studies are relevant to our research, as we could offer only 1 weekly session of supervised exercise, in contrast to current recommendations.1,3 thus, singh compared twice - daily home - based walking recorded in a log and monitored once - weekly over 4 weeks with usual activities and found a significant increase in 6mwd for the intervention group but not the control group (54.226.7 m versus 6.710.3 m, p<0.001). finnerty compared education, plus once - weekly supervised exercise, plus an unsupervised home exercise program of 5 days per week over 6 weeks with usual care and reported a median increase in 6mwd of 51 m (range 2081 m) in the intervention group. these studies contrast with the limited earlier evidence7,47 on which the recommendation of at least twice - weekly supervised exercise sessions is based. furthermore, no significant difference between once weekly or twice weekly exercisers for the incremental shuttle walking test was demonstrated in a recent randomized controlled trial.11 others included a structured home exercise program to supervised exercise, also finding no additional benefit of two supervised sessions to the incremental shuttle walking test distance.10 these later studies of weekly versus twice - weekly supervised exercise point to uncertainty over the optimal frequency and mode of supervision required to increase physical capacity. they suggest that once - weekly exercise supervision with a structured home exercise program may be as good as more intensive regimes. to an extent, that is what we have tested, with negative results. in contrast with our study, others have reported a significant increase in self - reported exercise immediately following the cdsmp alone for people with chronic conditions, including copd.48 one explanation could be that the measure of self - reported exercise we used is more comprehensive than the stanford measure49 used previously. thus, our study is the first cdsmp - related study to focus on copd and the amount of moderate exercise which is required for optimal health benefits.35 disappointingly, our results indicate that the cdsmp alone is of limited benefit for meeting the minimum recommendations of exercising in the community for 30 minutes on each of at least 5 days per week. furthermore, the single supervised exercise session did not add anything in this regard either. while a major strength of our study was its execution in real world clinical practice, utilizing existing resources, this also imposed some limitations. firstly, due to ethical considerations, we were unable to include a second control group who did not receive any rehabilitation - type intervention. secondly, it would have been informative to have a group in a twice - weekly or three - times - weekly supervised exercise schedule to determine whether this more exacting approach to exercise would add to the effects of the cdsmp. while this more intense exercise has its advocates,6 such an approach is highly resource - intensive ; most other similar centers would have been unable to achieve this. therefore, the effect of adding more than one supervised exercise session to the cdsmp is unknown, or indeed whether there are optimal numbers of weekly exercise sessions. however, even if more sessions are better, resource limitation will always be a major factor for generalizability within many health centers. thirdly, participants were recruited from referrals to a hospital - based program that may differ from those who might self - refer to community - based cdsmps. nevertheless, our study reflects the usual practice for australian pr, thus enhancing local generalizability. fourthly, due to resource limitations, we were unable to offer separate sessions for cdsmp - exercise and cdsmp - only groups. while participants were requested not to discuss the exercise experience, vicarious contamination of the control group by the active intervention fifthly, the leaders in this study were health professionals rather than peer leaders as is typical for other cdsmps. nevertheless, a recently published systematic review concluded that there were few differences between peer - led or health professional - led self - management programs,27 suggesting this was unlikely to be a source of bias. although this did not vary a great deal, it may have yielded information as to a differential effect of the intervention and would be a consideration for future research. finally, the cdsmp does not include a structured home exercise program, since under the license agreement, we were precluded from doing so. in conclusion, participants with copd attending a cdsmp can expect a small increase in their physical capacity, but there seems little point in adding a single supervised exercise session. either there needs to be a more intensive conventional exercise program as advocated in guidelines, or new ways need to be investigated for successfully fostering adequate amounts of home or community - based exercise which meet current recommendations for optimizing health benefits. before completely abandoning the cdsmp plus limited supervised exercise approach, we are currently undertaking such a trial using an additional community - based mentoring component. | purposeboth exercise and self - management are advocated in pulmonary rehabilitation for people with chronic obstructive pulmonary disease (copd). the widely used 6-week, group - based chronic disease self - management program (cdsmp) increases self - reported exercise, despite supervised exercise not being a program component. this has been little explored in copd. whether adding supervised exercise to the cdsmp would add benefit is unknown. we investigated the cdsmp in copd, with and without a formal supervised exercise component, to address this question.patients and methodsadult outpatients with copd were randomized to the cdsmp with or without one hour of weekly supervised exercise over 6 weeks. the primary outcome measure was 6-minute walk test distance (6mwd). secondary outcomes included self - reported exercise, exercise stage of change, exercise self - efficacy, breathlessness, quality of life, and self - management behaviors. within- and between - group differences were analyzed on an intention - to - treat basis.resultsof 84 subjects recruited, 15 withdrew. 6mwd increased similarly in both groups : cdsmp - plus - exercise (intervention group) by 18.646.2 m ; cdsmp - alone (control group) by 20.046.2 m. there was no significant difference for any secondary outcome.conclusionthe cdsmp produced small statistically significant increase in 6mwd. the addition of a single supervised exercise session did not further increase exercise capacity. our findings confirm the efficacy of a behaviorally based intervention in copd, but this would seem to be less than expected from conventional exercise - based pulmonary rehabilitation, raising the question of how, if at all, the small gains observed in this study may be augmented. |
multiple sclerosis (ms) is the most common nontraumatic neurological disease in young adults. it is an inflammatory demyelinating disease that is primarily associated with axonal loss and formation of lesions in the central nervous system. magnetic resonance imaging (mri) is the leading diagnostic tool in the context of ms. the extensive use of this imaging technique has significantly improved our understanding of the disease. mri is currently being used for ms diagnosis, assessment of disease progression, and evaluation of the efficiency of drug therapy. the most common quantitative parameter is the lesion burden (or load) of the disease expressed in terms of the number and volume of the brain lesions. however, it should be stated that the exact relationship between mri measured lesions and pathological findings is not entirely clear [911 ]. last but not least, it is sensitive to temporal variations that are typical to this disease. a correct segmentation of healthy tissues is crucial for successful detection and segmentation of ms lesions. moreover, one of the characteristics of the disease is a decrease in the volume of healthy tissues, especially gray matter (gm). having said that, it is clear why, in spite of the fact that the present work focuses on ms lesion detection and segmentation, the topic of healthy tissue segmentation should be addressed as well. first, mri images present various noise artifacts such as intratissue noise, partial - volume effects and thermal noise. these artifacts may cause unrealistic results, where tissue regions as well as lesion regions may appear granular, fragmented, or violating anatomical constraints. second, the geometry of the brain tissues is complex and can not be modeled by simple geometric primitives. also, as each mri modality reflects different physical properties, the exact lesion boundaries may vary between different mri channels. to complicate matters, the number of voxels that are associated with ms lesions is significantly smaller than the number of voxels that are associated with healthy tissues. thus, simple clustering algorithms (e.g., k - means), fail to discern ms - lesion voxels from the rest of the image. with notable exceptions (e.g., [15, 16 ]), segmentation of mri brain images of ms patients several authors suggested to model the intensity of the brain tissues using a mixture of gaussian probability density functions [1722 ]. in this model, heron termed gaussian mixture model (gmm), the intensity of a voxel is assumed to be drawn from the specific normal distribution associated with its parenting tissue. the gmm belongs to the category of parametric classification methods, since it is fully defined by its parameter set. other researchers have suggested using nonparametric methods such as the k - nearest neighbor (knn) classification rule, or parzen windows [23, 24 ]. these methods avoid making any assumption on the probability density function that is underlying the data. instead, they try to locally estimate the probability density function, based on some labeled prototypes. these prototypes are given manually by a human expert, so such methods are not completely automated. a combination of parametric and nonparametric methods has been suggested recently. while several authors (e.g., [13, 23 ]) model the ms lesions as a distinguished class in addition to the healthy tissue classes (csf, gm, wm), another approach is to model the lesions as outliers. for example, van leemput. treat voxels that are not well explained (by either of the healthy tissue models) as candidates to be classified as lesions. among these candidates, the separation into lesion and nonlesion voxels is made according to contextual information and a set of rules. it is also possible to treat lesions as outliers only in an intermediate stage, and then to build an explicit model for them. a segmentation method that relies solely on the voxel intensity is unlikely to produce sufficient results. as such a similar approach is the template - driven segmentation [20, 23, 24 ] that employs a deformable digital anatomical atlas to extract white matter masks using nonlinear registration. the main purpose is to reduce the number of misclassifications, by assuming that lesions are usually placed within the wm tissue. atlas / template based approaches make a powerful tool for image segmentation ; however, they suffer from several disadvantages. registration is especially problematic when the patient presents significant anatomical variability with respect to the atlas / template, which is often the case in ms patients. another conventional method to improve segmentation smoothness and immunity to noise is to use a hidden markov random field (hmrf), thus modeling neighboring voxel interactions [13, 18, 19 ]. smoother structures are obtained in the presence of moderate noise as long as the hmrf parameters controlling the strength of the spatial interactions are properly selected. hmrf based algorithms are computationally intractable unless some approximation is used which still requires computationally intensive algorithms. in very recent are used in a multifeature and multiscale approach combining segmentation and classification (supervised) for ms lesion analysis. in a 4d space (t1 intensity and spatial features) a large number of gaussians is used per brain tissue to capture the complex spatial layout. the intensity of a tissue is considered a global feature and is incorporated into the model by parameter tying of all related gaussians. the model, termed constrained gmm (cgmm), was shown in to provide accurate segmentation of t1 simulated and real mri brain images into the three healthy tissues, in particular in noisy and low - contrast mri images, without the need for coregistration of the input image and an atlas. in the current work we propose an extension to the cgmm model that can handle multisequence mri data with a focus on ms lesion detection and segmentation. another focus of this work is utilizing the cgmm as a good initialization for a level set method that provides a refined lesion boundary. we show that direct implementation of curve evolution methods to the complicated mri image provides poor brain segmentation and no detection of ms lesions. the lesion detection obtained using the cgmm makes it possible to apply active contour techniques for lesion detection. a preliminary version of this paper appeared in. lesion detection based on the cgmm and lesion boundary refinement via a level - set method, hereon termed cgmm - ce, are described in sections 3 and 4, respectively. the complex spatial layout of an mri brain image poses a challenge for conventional intensity based gmm modeling schemes. to accommodate the spatial complexity, we model an image as if its voxels were drawn independently from a mixture of many gaussians : (1)f(x, i(x))=i=1nifi(x, i(x) i,i) such that x is the 3d position information included in the feature vector, i(x) is the intensity vector associated with the voxel located at position x, n is the number of components in the mixture model, i and i are the mean and the covariance of the ith gaussian component fi, and i is the ith mixture coefficient. considering the location of the voxel as an additional feature enables us to incorporate spatial information into the probabilistic model. each gaussian in the gmm provides a probabilistic model for a specific small area in the mri image. however, since we use a mixture of many components, each gaussian component models a small local convex region. the intravariability of the intensity features within a tissue is significantly less than the intervariability among different tissues. it is therefore sufficient to model the intensity variability within a tissue by a single gaussian (in the intensity features). to incorporate this insight into the model, each gaussian is linked to a single tissue and all the gaussians related to the same tissue share the same intensity parameters. the above assumptions impose the following structure on the mean and covariance of the gaussian components : (2)i=(ix(i)i), i=(ix00(i)i), where x is the spatial feature vector, i is the intensity feature vector, (i) is the tissue that is linked to the ith gaussian component, i and i are the spatial mean and covariance parameters of the ith gaussian component, and j and j are the intensity mean and covariance parameters shared by all the gaussian components that are linked to the jth tissue. the gaussian component fi has, therefore, the following form : (3)fi(x, i(x) i,i)=(x;ix,ix)(i(x);(i)i,(i)i). the main advantage of the cgmm framework is the ability to combine, in a tractable way, a local description of the spatial layout of a tissue with a global description of the tissue 's intensity. the multigaussian spatial model makes our approach much more robust to noise than intensity - based methods. an illustration of the cgmm model applied to the three tissues (csf, gm, and wm) is shown in figure 1(a). in case of ms lesion segmentation we consider the lesion matter in the cgmm modeling as a fourth tissue in addition to the three healthy tissues (figure 1(b)). given an mri image with t voxels : { (xt, i(xt)) | t = 1,, t }, the likelihood of a given cgmm parameter set is (4)t=1 t i=1nifi(xt, i(xt) i,i). initially, the model parameters are, of course, unknown. to find the maximum - likelihood parameters we utilize the classical expectation - maximization (em) algorithm. the em algorithm handles the parameter estimation task by iterating the e and m steps. in the e - step, it treats the parameter set as given, and estimates the probabilities of each sample to be drawn from each gaussian. informally, this step can be seen as soft segmentation (but here, the number of segments is n, the number of gaussians, and not k, the number of the tissues). e - step : (5)wit = p(i xt, i(xt))=ifi(xt, i(xt) i,i)j=1njfj(xt, i(xt) j,j) i=1,,n, t=1,,t wit can be viewed as the posterior probability that voxel t was created using gaussian i. (5)wit = p(i xt, i(xt))=ifi(xt, i(xt) i,i)j=1njfj(xt, i(xt) j,j) i=1,,n, t=1,,t wit can be viewed as the posterior probability that voxel t was created using gaussian i. m - steplet (6)ni=t=1twit i=1,,n, kj=i(i)=jni j=1,,k with ni and kj interpreted as the expected number of voxels associated with the ith gaussian and kth tissue, respectively, and t is the number of voxels in the mri image. let (6)ni=t=1twit i=1,,n, kj=i(i)=jni j=1,,k with ni and kj interpreted as the expected number of voxels associated with the ith gaussian and kth tissue, respectively, and t is the number of voxels in the mri image. intensity parameters : (8)ji=1kji(i)=j t=1twiti(xt),ji=1kji(i)=j t=1twit(i(xt)ji)(i(xt)ji). note that while the spatial parameters are estimated separately for each gaussian, the estimated intensity parameters are the same for all the gaussians that belong to the same tissue. the grouping function that links between the gaussian components and the tissues is learned in the initialization step and is not altered by the em iterations. however, since the learning is performed simultaneously on all the tissues, voxels can move between tissues during the iterations. once the optimal set of parameters is obtained using the em algorithm, we can compute soft tissue segmentation maps. moving from soft segmentation to hard segmentation is straight forward, using the maximum - a - posteriori (map) criterion. tissue segmentation is achieved by the affiliation of each voxel to the tissue that maximizes the a posteriori probability : (9)tissue - label(voxelt)=arg max j p(tissue = j xt, i(xt))=arg max j i (i)=jifi(xt, i(xt) i,i).figure 2 illustrates the segmentation induced from the cgmm model and shows how the em iterations improve the segmentation quality. since a lot of the algorithmic effort is spent on finding a good initialization (see section 2.2), the em needs only few iterations to converge. in section 2.1, we saw how the em algorithm is applied to the mri data to find the maximum - likelihood parameter - set for the cgmm. hence, appropriate initialization is required. in the first step the k - means clustering is done based only on the intensity features (t1, t2, and pd), which gives a good initial segmentation into the three major tissue classes. we utilize t1 to give tissue labels to the three groups. given the intensity k - means clustering of the voxels into the three tissues, we want to model each tissue with many small locally convex gaussians. it is important that small gaussians will be allowed, as lesions are often very small, and we would like the gaussians layout to capture them. we have found empirically that randomly selecting 1/20 of the voxels as initial gaussian centers yields a resolution that is high enough for detecting the ms lesions. to ensure that small isolated areas are explicitly represented by local gaussians, we first apply a three - dimensional connected component process to each tissue. if a connected component contained less than three voxels, it was ignored and disregarded as noise. for each connected component (of voxels all from the same tissue) a subset of voxels is randomly chosen and a new gaussian is formed at each of the chosen voxels. to compute initial covariance values for each gaussian the gaussian covariance (both intensity and spatial) is computed based on all the voxels that are assigned to the gaussian center. as a result of the initialization process each gaussian is linked to one of the tissues and each voxel is affiliated with a selected gaussian. the strength of the proposed model relies on the highly detailed spatial representation obtained by using many small gaussians such that each gaussian represents a small coherent and convex region. of course, the number of gaussians that are required to cover the entire image increases as the size of the gaussians decreases. the main implementation problem is the large number of gaussians, that leads to high computational complexity of the em algorithm. the computational complexity involved with the e - step is o(nt) where t is the number of voxels and n is the number of gaussian components. a typical number of voxels in 3d mri brain images is on the order of 10. the number of gaussians in the cgmm framework may be as high as 10 or even 10 when segmentation of ms lesions is also required. it is clear that voxels that are very far away from the spatial mean of a particular gaussian i, have a small chance to originate from this gaussian. due to numerical limitations, the probability density of each gaussian vanishes whereas the actual distance from the spatial mean is still finite. thus, for practical purposes, there is no need to compute in the e - step the weights wit for every gaussian i and every voxel t. instead, for every voxel we maintain a small list of its nearest gaussians. our experiments showed that even a small number of gaussian neighbors (say 10) gives rise to very accurate approximations of the model and does not cause any degradation in the segmentation performance. the computational complexity is o(10 t) and therefore using this procedure, the number of computations in each e - step is reduced by a few orders of magnitude. we note in passing that this approach is superior to maintaining a list of voxels for every gaussian. the reason is that in the latter, we may find that there are voxels that are explained by no gaussian at all. admittedly, maintaining a list of nearest gaussians for every voxel may result in gaussians that have, if any, very little voxels. however, this should not compromise the results, since such gaussians will simply shrink and die off during the em process. the main problem is efficiently finding the list of nearest gaussians for each voxel. in this section the method is based on the k - distance transform (kdt), (and can be applicable to other gmm applications defined on a grid in which the number of gaussians is large). kdt, introduced by warfield and later improved by cuisenaire, is an extension to the distance transform (dt). the first map is a first nearest - neighbor (nn) map. in effect, each element in this map contains the identifier of the prototype that is the nearest to this element. the brute force approach for kdt computation is very simple and extremely inefficient : for every space element, compute its distances from all the given prototypes, sort them by the distances, and select only the nearest neighbors. clearly, this approach is not applicable to large datasets due to its high complexity. warfield proposed an efficient way for fast kdt computation, that is based on local propagations, rather than direct computations. the main point is that local propagations, implemented using several raster scans, require a number of computations that is only linear in the number of elements in the discrete space. in our work, the input prototypes can be taken as the spatial means of the gaussians and applying the kdt algorithm, we obtain for every voxel a list of its nearest gaussians. we can use the mahalanobis distance as the metric of choice. note that the mahalanobis distance depends not only on the locations of the prototype (in effect, the spatial mean of the gaussian) and the voxel, but also on the covariance matrix of the gaussian. experiments indicate that while em iterations modify the spatial parameters to some extent, they are unlikely to cause a gaussian to wander far away from its original vicinity. thus, it is enough to apply the kdt for computing the list of nearest gaussians only once. there is in fact a chance that the list of the nearest gaussians will change slightly, but the gaussians that will be replaced are likely to be far from the voxel so that their contribution is not important. here too, our experiments indicate that any discrepancy between the approximated model and the true model is intangible. note that in the present work, the importance of the kdt is primarily in approximating the exact computation of the probabilistic model. not only is it possible to know which are the k nearest gaussians for every voxel, but also their tissue affiliation. hence, the kdt immediately gives additional useful information : for every voxel it is possible to know, which tissue its nearest gaussians relate to. such knowledge can be beneficial to determine if a voxel is located within a wm area and is used in the following section to obtain a fast implementation of rules for detecting ms lesions. a major focus of the current work is to extend the cgmm framework to ms lesion segmentation. section 2.2 described that one of the very first initialization steps was intensity - based k - means clustering. as mentioned earlier, simple clustering methods for ms lesion detection are usually inapplicable, due to the small number of lesion voxels. we present a novel approach to ms lesion detection, that exploits the gaussians layout of the cgmm. first, the model is initialized with three tissue classes, and its parameters are learned. due to the fact that ms lesion voxels are significantly outnumbered by the healthy tissue voxels, the clusters still succeed in representing the three healthy tissues (csf, gm, and wm). the lesion voxels are of course misclassified at this point as one or more of the healthy tissues. moreover, at this stage there are misclassified gaussians, that is, gaussians labeled as healthy tissues that are supposed to be labeled as ms. the purpose of the current stage is to identify these gaussians, and change their labels accordingly. a major distinction of the present work from previous works is that ms lesion detection is performed on the gaussian level rather than on the voxel level. for each gaussian a decision is made, based on its features, whether it should in fact be labeled as ms, or remain labeled as one of the healthy tissues. both supervised and unsupervised approaches can be used to deal with this task. for example, a rule based system can be used to distinguish the ms lesion gaussians from the normal tissue gaussians. an example of such a rule set for gaussians labeled as gm is the following : t2 mean - intensity of the gaussian > t2 mean - intensity of the gm tissue + gm, t2, t2 mean - intensity of the gaussian > pd mean - intensity of the gm tissue + gm, pd, a large mahalanobis distance between the mean - intensity of the gaussian and the mean - intensity of the csf / gm / wm tissue, the majority of gaussian 's k nearest gaussians are labeled as wm, where gm, t2 and gm, pd are thresholds that can be tuned and optimized. the rules that rely on mahalanobis distance imply that only gaussians that are not well explained by the healthy tissue models are suspected as lesion gaussians. the last rule incorporates contextual information by reflecting our expectation to find lesions within the wm tissue. however, note that at this point decisions are made at the gaussian level rather than the voxel level. clearly, a rule - based system is only one option for ms gaussian detection. for example, a more supervised approach can be used : given examples marked by a human expert, standard classifiers can be applied to the gaussian features. while we do not claim that the rule - based system suggested here is optimal (although it is intuitive and easy to implement), we do suggest that the detection on the gaussian level provides greater strength in comparison to voxel - based detection. following the ms lesion gaussian - detection stage, all the relabeled gaussians now form a fourth class named ms lesion (msl), with its own global intensity parameters. the em is now applied to cgmm with four tissue types and segmentation of the tissues and the lesions is obtained as explained in section 2. an illustration of the relabeling of the gaussians can be seen in figure 1(b). this is most likely due to the fact that the area in the proximity of a lesion is somewhat different from the other tissues. in addition, the segmentation step of the cgmm model is done voxel - wise and does not take into account the smoothness of the lesion boundaries. we use a modified version of the variational framework for segmentation of vector valued images, suggested by rousson and deriche based on the chan - vese model. in a curve evolution equation for object - background image segmentation the intensity in each region is modeled using a gaussian distribution. in our case, the optimal lesion segmentation is defined by minimizing the energy : (10)f(1,1,2,2,)=i=12 iei(i(x))+length(), where 1 are the lesion region voxels, 2 are the nonlesion voxels, is the curve separating the two regions, i, i are the gaussians parameters of i and ei(i) = logf(i | i, i). in level - set formulation, the functional from (10) takes a new form : (11)e(,)=i=12 [ei(i(x))i((x))]dx+length(), where is the level - set function, is the parameter set { 1, 1, 2, 2 }, and 1, 2 are the characteristic functions of regions 1 and 2, respectively : 1() = h(), 2() = 1 h() such that h() is a regularized heaviside function. note that the level - set formulation enables performing integration on the entire image domain,. the energy (local) minimum can be obtained utilizing the level - set theory via a gradient descent (gd) method. from the gd equation, t = f/, we obtain the following curve evolution equation : (12)t(x)=(vdiv (||)+e2(i(x))e1(i(x)))((x)). since brain images are complex, and since lesions are relatively small, it is impractical to use the standard initial contour (small circles) [31, 32 ] to find the three tissues and the lesions, simultaneously. the method is highly sensitive to the initial conditions for complicated four - phase images (such as brain images) as a gradient - descent optimization finds only local minima of the functional. figure 3(a) shows a segmentation obtained from a four - phase curve - evolution using a standard circle - grid initialization. it can be seen that (even with extensive manual tuning of the curve - evolution parameters) due to the complexity of the image and the added noise, we obtain poor brain segmentation results and no detection of msl. using segmentation results of the three tissues and lesions obtained by cgmm just for initialization of a four - phase ce is not sufficient. even with the improved cgmm initialization, the four - phase ce fails, as the image is too complex and the lesions are relatively small. figure 3(c) shows an example of msl segmentation results based on a cgmm segmentation followed by five iterations (that were needed until convergence) of four - phase curve evolution (the initial cgmm segmentation is shown in figure 3(b)). as expected, and in agreement with rousson and deriche, the lesions gradually disappeared and the segmentation failed due to the complexity of the image. in our approach we use a two - phase ce : lesions (object) versus nonlesion (background). we use the cgmm not just a starting point but also for background and object modeling. we model the lesion in the curve - evolution process using an intensity based gaussian that is extracted from the lesion component of the cgmm model and is kept fixed during curve evolution iterations. we model the background using an intensity based gmm that is extracted from the cgmm model. since there are three tissues that belong to 2, we define e2(i(x)) to be min (ecsf, egm, ewm). this definition implies that the last two terms in the curve evolution equation are the log - likelihood ratio between msl and the most likely normal - brain tissue. in the energy is minimized with respect to both the gaussian parameters and the boundary position. in our case this enables us to continue enforcing the global constraints of the cgmm in the active contour step. for example, if one of the slices has an initial contour that encircles only false positives, then the constraint will usually cause it to die off, whereas parameter estimation during the curve evolution is likely to cause the system to learn the parameters of the false lesion. to summarize, three modifications are suggested in the current work, in applying the rousson - deriche framework to the msl delineation task. the background is modeled by a gmm (csf, gm, wm) instead of a single gaussian. parameter estimation during the curve evolution process is avoided ; cgmm intensity parameters are used instead. the lesion detection obtained using cgmm, therefore, makes it possible to apply active contour techniques for lesion delineation. we dub the proposed method, which is based on cgmm followed by a curve evolution refinement, cgmm - ce. the steps of the cgmm - ce algorithm that were presented in sections 2, 3, and 4 are summarized below. apply intensity based k - means to obtain an initial clustering of the voxels into three tissues. form the initial spatial gaussians based on clustering of the connected components of each tissue. set the gaussian labels according to the tissue they belonged to. first, with notable exceptions there are hardly any data sets that are publicly available for the research community. second, there is no clear convention for the best measures to quantify the results. it is a challenge to decide where the lesion ends and the healthy tissue starts, a fact that introduces variability in human - expert markings. this makes it difficult to produce ground truth segmentation for large data sets. in the current work, we demonstrate results on a the brainweb online database which is considered to be a standard benchmark upon which mri brain image segmentation algorithms are tested and compared. the cgmm - ce algorithm was tested on bias - free simulated mri data taken from the brainweb online database. voxel size was 1 mm. experiments were done on 61 slices (axial slices 60120) that contain 93% of the lesion burden. different levels of noise were added : 3%, 5%, 7% and 9%. according to brainweb 's web - site, the noise in the simulated images has rayleigh statistics in the background and rician statistics in the signal regions. the percent noise number (e.g., 5% noise) represents the percent ratio of the standard deviation of the additive white gaussian noise versus the signal for a reference tissue. in the brain images under observation the noise reference tissues were wm for t1, and csf for t2 and pd. visual inspection suggests that images with 9% noise present a challenge even for manual segmentation. we are aware that the noise level in real data is almost always less than 9%. we provide the 9% segmentation result to demonstrate the robustness of the cgmm - ce method. as preprocessing steps, the brain region was extracted and the intensity distribution of each channel was normalized to have zero mean and unit variance. then, a k - means intensity - based clustering was performed with k = 3, to achieve an initial crude segmentation (see figure 5). the global intensity parameters of each tissue were initialized as the sample mean and sample covariance of the extracted tissue segment. following the cgmm parameter initialization and kdt computation (with 10 neighbors), a set of decision rules was applied. if all the conditions described in this set held, and in addition the majority of gaussian 's 10 nearest gaussians were labeled as wm, then the gaussian was relabeled as msl. the velocity, v, in (12), was chosen as a constant : v(x, y) = const. the constant value was set to be 5, 3, 1, 1 for noise levels 3, 5, 7, 9, respectively (higher velocity for low noise levels). performance is evaluated via a comparison with van leemput 's state - of - the - art algorithm (hereon termed kvl) implemented by the ems software packagehttp (http://www.medicalimagecomputing.com/ems). in the kvl implementation, the statistical brain atlas of the spm99 (http://www.fil.ion.ucl.ac.uk/spm/) was normalized to the target brain volume images. note that kvl is the only one who actually made his code / toolbox available, so the comparison is fair. we have not found reported results on 9% (or in fact, even 7%) other than kvl. figure 6 shows segmentation results on a single slice. results are shown for both kvl, cgmm, and cgmm - ce methods. figures 6(a)6(c) shows a low noise case of 3%, figures 6(d)6(f) shows a high noise case of 9%, and figures 6(g) and 6(h) show the ground truth ms lesion segmentation. visual inspection indicates that for a low level of noise (3%), both algorithms present results that are close to the ground truth. for strong noise scenarios (9%), also note that in terms of detection of whole ms lesions (e.g., number of false positives), the cgmm presents more successful results. following the curve evolution step, the cgmm - ce framework presents further improved results. the need for boundary refinement following the cgmm segmentation, and the result of applying the curve evolution process are shown as a zoom - in in figure 7. two slice examples are shown including for each is the ground - truth (left), the cgmm segmentation result (center), and the refined cgmm - ce segmentation (right). in the bottom left of figure 7(e) we can see that two distinct lesions were joined together in the cgmm segmentation. figure 7(f) shows that the refined contour succeeded in separating these two lesions. comparing these two images further, we can also see that the overestimation of several of the lesions has significantly decreased. note that in figure 7(c) the refined lesion split to two very close adjacent lesions, this is not a general feature of the ce, as the fact that the cgmm (see figure 7(b)) produced a lesions whose two parts are almost disconnected. the reason for this is that only three main healthy tissues are modeled, but in fact the data presents about 9 or 10 healthy tissues, most of them are typically ignored. this particular lesion is on top such a tissue the glilal matter and therefore few pixels are missed, which resulted in a final insignificant splitting. a quantitative comparison between the algorithms was made using the dice overlap metric which measures the overlap between the automatic segmentation and the ground truth for each tissue j. this performance index is often used in assessment of medical image segmentation algorithms, and is given by (13)dice index=2vaejvaj+vej, where vas is the number of voxels that are assigned to tissue j by both the ground truth and the automated algorithm, va and ve denote the number of voxels assigned to tissue j by the algorithm and the ground truth, respectively. dice values vary between zero (no agreement with the ground truth segmentation) and one (perfect agreement with ground truth segmentation). table 1 shows ms lesion dice results for kvl and the cgmm - ce methods. in the 3% case, kvl is slightly better. for higher values of noise, it should be noted that for lesion segmentation, dice values above 0.70 are considered to be clinically stratifying. moreover, a comparison between two different segmentations given by two different human experts, is likely to yield dice index lower than 0.70. the computation time (using pentium (r) 4 cpu 3.40 ghz, 1.49 gb of ram) for each significant part of the algorithm is initialization : 3 minutes, kdt : 2 minutes, ms gaussian detection : 1 minute, and em iteration : 1 minute. this is partially due to the fact that the cgmm provided a starting point that is very close to the local minima of the functional and thus the number of curve evolution iterations was usually small. also, since we used the cgmm intensity parameters, there was no need to reestimate these parameters for every iteration. thus, the entire cgmm - ce process takes less than a quarter of an hour for brainweb data, slices 60120. in what is still a work in progress, these experiments were performed in a joint effort with the ms center at sheba medical center, israel. here, however, we have manual ms lesion segmentation, provided by a human expert. in these experiments, the fast flair (ff) modality, was added to t1, t2, and pd which were previously used. the ff provides a good contrast between csf and the other healthy tissues (gm and wm). to some extent, it also provides a good contrast between ms lesions and the three healthy tissues. the main purpose of ff in ms is to suppress false positive from csf signals. preprocessing steps include conventional procedures such as extraction of the brain region (also know as skull removal) ; coregistration of the images from the different channels ; and bias filed correction. once the preprocessing is finalized, we extract from each voxel both intensity and spatial features. unlike the isotropic voxel used in the brainweb data experiment, here the voxel size was 1 mm 1 mm 3 mm. the set of decision rules for gaussian detection was similar to the one used in the brainweb dataset, with the addition of rules that take the ff into account (e.g., lesions are brighter than all the other tissues). results on real data are shown in figures 8 and 9. in both examples it is possible to see a smooth and visually plausible segmentation of the healthy tissues. with regard to the lesion detection and delineation, the main lesions are in fact detected (except for one in figure 9). in, the cgmm was shown to be a successful framework for healthy tissue segmentation of t1 mri brain images. in the current work, the cgmm - ce framework was presented, as a methodology to detect and delineate ms lesions within multichannel input data. a novel model approximation scheme has been suggested to accelerate computing time by several orders of magnitude. finally, a curve evolution technique was applied to refine the segmentation of the ms lesions. cgmm was shown here to provide a successful parametric framework for healthy tissue and ms lesion segmentation in abnormal brains, without requiring prior registration to a brain atlas. unlike most ms lesion segmentation algorithms, here detection of ms lesions is done on the gaussian - level rather than the voxel - level. besides the fact that it ensures smoother segmentation and an inherent robustness to noise, gaussian - level reasoning incorporates spatial information in a simple and intuitive way (unlike mrf that is often used in the literature). one of the advantages of the suggested rule - based system for ms gaussian detection, in addition to its simplicity, is that it enables the user (the radiologist) to tune the parameters in a clear and understandable way. also, users can easily add or remove rules to match the rules that they himself apply for manual lesion segmentation. for example, they can decide to exclude all gaussians that are close to the centerline of the brain. while similar rules were used in the literature, here the rules are applied to gaussians (that correspond to small regions) rather than to voxels. of course, the rule - based system for ms gaussian detection is only one option for classifying gaussians. for example, it is possible to use the markings given by a human expert to train classifiers such as a support vector machine. the gaussian kdt enables a fast implementation of the cgmm framework and provides additional contextual information. the kdt can be useful for other gmm applications in which the number of gaussians is large. the curve evolution technique suggested by rousson and deriche was utilized to refine the cgmm lesion segmentation. since brain images are complex, this technique can not be directly applied to the mri data. the cgmm segmentation provides an excellent initial contour, so the gradient - descent limitations are reduced. the use of the optimal msl intensity parameters from the cgmm, eliminates the need to reestimate them in each curve evolution, thus speeding the process. in experiments with simulated data, both visual and quantitative comparisons with the kvl algorithm typical results of msl segmentation reported in the literature refer to 3% noise only (e.g., the sate - of - the - art algorithm suggested by akselrod - ballin.). we are currently extending the experiments to additional real brain datasets, in a joint effort with the ms center at sheba hospital. some additional work needs to be done to address the very small size lesions, and to achieve more accurate delineation. multiple - expert markings need to be compared as well, in order to estimate the observer variability on this data set, which can then be compared to the automated algorithm performance. | this paper focuses on the detection and segmentation of multiple sclerosis (ms) lesions in magnetic resonance (mri) brain images. to capture the complex tissue spatial layout, a probabilistic model termed constrained gaussian mixture model (cgmm) is proposed based on a mixture of multiple spatially oriented gaussians per tissue. the intensity of a tissue is considered a global parameter and is constrained, by a parameter - tying scheme, to be the same value for the entire set of gaussians that are related to the same tissue. ms lesions are identified as outlier gaussian components and are grouped to form a new class in addition to the healthy tissue classes. a probability - based curve evolution technique is used to refine the delineation of lesion boundaries. the proposed cgmm - ce algorithm is used to segment 3d mri brain images with an arbitrary number of channels. the cgmm - ce algorithm is automated and does not require an atlas for initialization or parameter learning. experimental results on both standard brain mri simulation data and real data indicate that the proposed method outperforms previously suggested approaches, especially for highly noisy data. |
the presence of hb barts in blood at birth is a finding in -thalassemia trait, intermedia (hbh disease) and major (hydrops fetalis). diagnosis of -thalassemia major is not a problem and it is incompatible with life as most of the hemoglobin will be barts. neonates with -thalassemia intermedia have hbf, hba and hb barts, usually more than 15%. this condition is easy to diagnose later in life, even if not investigated at birth, due to the presence of hbh. most of these cases have normal hba2 levels if checked after the age of six months, though red cell indices are thalassemic. this group, which makes up the bulk of gene carriers, will be difficult to confirm by hb electrophoresis (hbep) or by hplc in childhood and adult life. if one can check hbep or hplc at birth then presence of hb barts would put the diagnosis on solid grounds. it is too expensive to perform hplc or hbep for all neonates, even in areas where frequency of the -thalassemia gene is high. so, if we can predict, with cheap tests, the presence of -thalassemia then hb separation can be performed for the highly probable cases only. this issue was addressed by work carried out in thailand, and in jeddah, saudi arabia a few years ago. mcv below 90 and mch below 30 were found to be of very high predictive value in the jeddah study. there was a need to repeat the study in another setting, where thalassemics are of variable ethnic groups, like in dubai. -thalassemia is present in the uae nationals and residents of iranian, arab, pakistani and filipino origins in dubai and it was expected that neonates in such a population would make a good sample for repetition of the study and confirmation of the validity of using the mcv and mch as predictive parameters in individuals of various ethnic groups. counts were performed using a coulter machine model lh7d1 (beckman coulter, brea, ca, usa). thirty - four consecutive neonates, with mcv below 90 fl were checked by hplc, using waters 2695 machine (milford, ma, usa), and short -thalassemia program, to look for hb barts band. blood films of all babies included in the study were checked by the hematopathologist for red cell morphology. they were random and judged to be necessary by the attending neonatologists for various reasons. as a control group, 26 neonates, who had mcv between 90 and 95 fl were also checked by hplc. again, these were not selected and all consecutive cases with such an mcv level were included into the control group. it took eight months to collect the entire test and control cases because only a minority of neonates in dubai have blood counts checked. all individuals in the test group had mcv below 90 fl (mean 85.35, range 7788.9). it ranged from a trace (less than 1%) to 10.6% (mean 4.65%). the 10.6% figure was obtained in the baby with the lowest mcv (77) and mch (25.1) (table 1), who had hb level of 12.5 and later proved to have hb h disease. mcv in the control group ranged from 90.1 to 94.8 fl (mean 92.27) and mch from 28.7 to 31.7 (mean 30.33). the 11 barts - positive cases further showed that 7 of them had mch below 30 and 6 were preterm (3 of them had mch below 30). only one baby (3.8%) out of the 26 controls and positive barts had mch above 30 and was full term (table 2). blood films of all babies included in the test group showed numerous target cells and microcytosis, but no signs of active hemolysis. table 1results on cord blood of test group.hb bartsmcvmch<9090<3030total casespositive34033134%1000973negative0na000000 table 2results on cord blood of test group.hb bartsmcvmch<9090<3030total casespositivena117 (3 preterm)4 (3 preterm)11%42.326.915.342.3negativena1501515%57.6057.657.6 after the work carried out in jeddah, saudi arabia, where the population is basically local with a low expatriate mix, as well as some variation in the origin of the local population itself, it was thought useful to the medical literature to repeat the study at another site to confirm the remarkable results of the original study. however, there is no clear demarcation of the level of mcv and mch where one can expect, with high confidence, to find -thalassemia. the work carried out in thailand took the mcv cut - off value of 95. in our experience, in both works performed in jeddah and in dubai a mcv cut - off value of 95 will give a lot of negative cases and make the need for hplc / hbep much higher, thus increasing the cost while one of the objectives of this scheme is to lower costs. the advantage of using mcv and mch levels is that these values are easily available from a simple automated blood count (fbc). if one can prove that there are limit levels for these two parameters which carry a high probability of -thalassemia trait and hbh disease, then there will be a small proportion of patients for whom one needs to perform hb separation procedure, by alkaline electrophoresis or hplc, to demonstrate hb barts, and thus prove -thalassemia presence. it is confirmed from this study that if mcv is below 90 and/or mch is below 30 there is a high probability of over 95% that the case is -thalassemia trait. in such cases one can extend the mcv level to 95, whether or not mch is below 30 pg. it is noteworthy that hb barts is not only a diagnostic finding for -thalassemia, but it tends to disappear a few weeks after birth. trying to confirm -thalassemia trait at a later age, when suspicion is raised by low red cell indices leading to erythrocytosis, hematologists first resort to hbep or hplc, looking for low hba2. iron deficiency anemia may also lower hba2 to below normal level. failing to confirm the condition by this routine test, as in the majority of cases, one can try molecular studies on the -polypeptide gene to look for deletions or mutations known to lead to -thalassemia trait. these deletions and mutations are so numerous that, in practice, laboratories only try the common local deletions or mutations, using the proper probes for them, if these are known from previous studies. in a mixed population the cost of these molecular tests is certainly higher than hb separation studies. in parts of the world where -thalassemia gene is of high frequency it should be remembered that most countries harboring a high frequency for the gene are also poor in resources. the simple strategy we are recommending, proven by the two studies in two different locations, is valid and is recommended by the authors for who and local public health schemes to establish the diagnosis and gene frequency. further studies are being considered to try to split populations with one, two and three gene deletions / mutations from levels of mcv and mch caused by them and the hplc performed as a result of obtaining the low mcv and mch figures. the simple investigational scheme that we suggest to be followed for the -thalassemia screening of neonates is shown in figure 1. | thirty - four blood samples of neonates in dubai, uae, with an mcv below 90 fl were checked by high performance liquid chromatography (hplc) for hemoglobin variants to confirm a previous study carried out in western province of saudi arabia which showed a very high predictive index of such mcv for alpha (-) thalassemia minor (atm). mch below 30 pg was an additional factor which supported such a prediction. the dubai study confirmed the original finding with 100% of such neonates showing hb barts band. a control group of 26 neonates with an mcv between 90 and 95 fl showed hb barts in only 11 cases (42.3%). of these, 6 (23.1%) were preterm babies, expected to have higher mcv. five cases (19.2%) had an mch below 30 pg, though mcv was 90 or higher. three of the preterm babies also had mch below 30. the study confirmed the saudi results in neonates. it seems very highly probable that a term neonate with mcv below 90 and mch below 30 has atm. |
women are known to respond to infection, vaccination, and trauma with increased antibody production and more t helper (th)2 predominant immune response, whereas a th1 response and inflammation are usually more severe in men. systemic lupus erythematosus (sle) is a connective tissue disease of unknown etiology, generally considered a common occurrence in women of childbearing age and rare among men. it is estimated that out of every 10 people who have lupus only one is a man. symptoms are equivalent in men and women, particularly skin rash, extreme fatigue and joint pain, but findings suggest that the disease in men has a more complex clinical course and some studies have shown that renal impairment, central nervous system and vascular diseases are more common in men when compared to women. lupus erythematosus evolves with periods of activity and varying periods in which patients are asymptomatic or mildly symptomatic. constitutional complaints such as adynamia, malaise, fatigue, weight loss and fever are frequently observed in the active phase of disease. fever may be low and continuous, or high, peaked, and must be distinguished from intercurrent infection. weight loss is usually mild, however, in some cases it can be quite severe, leading to lupus cachexia. butterfly rash or vespertilio is characterized by the acute onset of an erythematous rash located on the cheeks and dorsum. other acute injuries are erythematous - macular, papular or maculopapular and bullous lesions, also with a preferential location to sun - exposed areas. arthritis in small joints of the hands, wrists and knees is usually symmetrical and intermittent. most cases of anemia in sle are normochromic and normocytic, and it has been attributed to the chronic aspect and the inflammatory activity of the disease. leukopenia and lymphopenia are listed in more than 50% of cases throughout the disease course and severe thrombocytopenia (less than 50,000 platelets / mm) occurs in less than 10% of cases. renal involvement is characterized by proteinuria > 0.5 g/24 h, casts and abnormal or rising serum creatinine levels, without other causes. pleuritis and/or pericarditis are found in about 50% of the patients during the disease progression. in recent decades, a 25-year - old male farmer presented with a 6-month history of swelling in the elbows, shoulders, hands, knees, metacarpophalangeal and proximal interphalangeal joints associated with lumbar pain, morning stiffness and an approximately 10 kg weight loss. twenty days later, the patient developed a dry ; sometimes yellow sputum - producing cough, evening fever and night sweats. hospitalized, he was emaciated, had a generalized muscle wasting, as well as bilaterally reduced breath sounds, especially on the right side and crackles on the left base ; spared joints. tst was non - reactive for afb and sputum analysis was negative for fungus, normal bone marrow examination, electrocardiogram revealed a sinus tachycardia ; endoscopy showed mild erosive antral gastritis without evidence of h. pylori. electromyography : pattern of axonal polyneuropathy that was moderate in the sensory component and mild in the motor component. computed tomography (ct) thorax : early lung inflammation, located in the anterior and lateral basal segments of the left lower lobe, as well as discrete fibroatelectasis - like laminar opacities in fibroatelectasis appearance, located in the posterior basal segment of the left lower lobe. pericardiocentesis was performed and the fluid analysis was negative for neoplastic cells and for tuberculosis, rare mesothelial cells, lymphocytes and cellular debris in a serohemorrhagic background. new analyses were conducted, which reavealed : hb 8.3g%, leukocytes 4000/mm (segmented 75, lymphocytes 24, monocytes 1), platelets 700,000, esr 118 mm/ h, glucose 95 mg / dl, creatinine 0.8 mg / dl, c - reactive protein 114 iu, total protein 8.0 g% (albumin 2.6 globulin 5.4) ; unremarkable urinalysis ; thorax radiography : left - sided pleural effusion ; echocardiography : pericardial effusion with mild thickening located above the right atrium ; rheumatoid factor reagent 1/2 ; anf 1/640 fine dense cytoplasmatic pattern, positive anti ena, positive anti rnp, positive anti sm, positive anti - ro (ssa). after the diagnosis of systemic lupus erythematosus, corticosteroids were prescribed and the patient evolved with clinical improvement and was discharged with an outpatient prescription of prednisone 20 mg / day and hydroxychloroquine 400 mg / day. it has an unknown, multifactorial etiology, in which the interaction of genetic predisposition and various hormonal, environmental and infectious factors appear to lead to a loss of immunological tolerance. the importance of sex hormones in the pathogenesis of sle is well shown in animal models. in humans, exacerbations of sle during pregnancy, post partum and menstrual periods due to rapid hormonal changes are well documented, as there are numerous reports associating the use of estrogen - containing oral contraceptives and disease exacerbations. in contrast, it has been shown that men with lupus do not produce abnormal estrogen levels, which suggests that estrogens can not be considered an isolated determinant of lupus. stahl and decker studied 12 men with sle and observed that there was no evidence of hypogonadism or androgen deficiency. men with lupus are fertile, sexually active and have normal reproductive history. because of the predominance of women in most studies, little is known about the disease in men. recent studies suggest that the impairment occurs in older men have higher mortality in one year than women with sle, suggesting that even men with lupus have a more complex clinical course than women. it has become increasingly clear that men with sle have more seizures, immune - mediated anemia (low hemoglobin level) and lupus anticoagulant level that may lead to thrombogenesis. on the other hand, men appear to be less likely to have sjgren 's syndrome and even though men appear to have more severe manifestations, these can occur in both sexes. miller. studied 50 male patients with lupus erythematosus and reported a lower prevalence of neurological involvement, thrombocytopenia and alopecia when compared to female patients. hochberg. observed no significant differences in the major organ involvement in 12 male patients and 138 female patients affected with sle, except for peripheral neuropathy, which appeared to be more common in men. in our case, the patient had electromyographic changes. in a multicenter study by cervera., no difference in vital organ involvement was observed among men and women affected with sle, although they observed that men had less serosistis early in the disease and more arthritis during the disease course. there is no consensus on the presence of differences in the clinical manifestations between men and women with systemic lupus erythematosus. further studies are warranted and more awareness should be given once sex differences affect the drug activity and it has been suggested that specific therapies should be developed based on gender. in addition, the diagnosis of sle should always be considered in patients with systemic involvement, which would certainly avoid delay in management and prevent irreversible damage to target organs. | lupus erythematosus is a chronic autoimmune inflammatory disease of unknown cause that involves multiple systems. autoimmune diseases that are prevalent in men usually manifest themselves clinically before the age of 50 years old and are characterized by an acute inflammation, whereas autoimmune diseases with predominance among women appear clinically in later stages of life, when chronic diseases, fibrosis and increased number of autoantibodies are present. lupus erythematosus is more prevalent among women during the reproductive period, but the cause of this predilection is not fully established and little is known about the disease among men. we report a case of systemic lupus erythematosus (sle) in a male patient whose diagnosis was delayed due to the systemic manifestations and because sle is considered an uncommon disease in men. |
human dirofilariasis is a zoonotic filarial nematode infection caused by members of the genus dirofilaria (order spiurida). the most frequently implicated dirofilariae, of more than 24 species, are d. immitis and d. repens. a wide variety of natural definitive hosts includes canines and felines, both wild and domestic.. parasites those that inhabit the heart and blood vessels of the natural host (e.g. d. immitis, agent of heartworm disease of dogs), and those that live in the subcutaneous tissues (e.g. d. repens, causing subcutaneous infection in dogs and cats). mosquitoes implicated in transmission are mainly aedes and culex species, but also include members of other genera (armigeres, anopheles, and mansonia). infective larvae gain access to human tissues when the vector feeds, and mature into the adult stage over several months, typically presenting as ocular, subcutaneous or pulmonary reactive nodules containing the worms. most worms found in humans are sexually immature and solitary. in patent infections in the natural animal host, while d. immitis affects animal and human hosts in tropical and temperate regions in many parts of the world (excluding southern africa), d. repens is limited to the old world, including africa. in south africa, a few cases have been reported in animals but the detection of the first local human dirofilariasis cases, as described here, demonstrates the need for increased awareness of the clinical presentation of this infection in apparently non - endemic areas of the world. to our knowledge a 63-year - old female, residing in kwazulu - natal province, south africa, presented to her general practitioner with temporal headaches, a cystic conjunctival swelling and itchiness of the left eye, which had started 2 days prior to presentation. she had travelled to europe, during the month before presentation, visiting the czech republic for 2 days, poland for 14 days and england for 11 days. clinical examination revealed a normal right eye, with an obvious conjunctival mass on the inferomedial aspect of the left eye. the clear sac - like swelling contained a large, white coiled mass (figure 1) that was mobile, but remained localised during the examination. a slender, white worm was removed completely and submitted to the laboratory for identification. the sac from which the worm was removed appeared to be well - defined and encapsulated. the macroscopic appearance was that of a slender nematode, measuring 69 mm by 0.56 mm (figure 2a). the identification was confirmed microscopically as dirofilaria species on the morphological criteria of a simple head structure without obvious appendages, and prominent multiple longitudinal cuticular ridges on a transversely - cut fragment of the body (figure 2b). briefly, dna was extracted using a macherey - nagel nucleospin tissue dna kit (duren, germany). pan - filarial primers were used to amplify a sequence from the 5.8s-28s region in the first - round confirmation of a filarial worm, producing the 542-bp fragment typical of d. repens. the sample was then subjected to species - specific amplification of a 5srrna gene sequence of d. repens, which resulted in 2 pcr bands, as previously described, although the larger product was somewhat smaller (175 bp) than expected (247 bp) ; the smaller product was the anticipated 153 bp in size (figure 3). both of these pcr products were sanger sequenced in forward and reverse directions and blasted against the genbank database. the only matches were to d. repens, the best being 100% match of the 175bp product to a 5srrna sequence from italian strains (genbank accession no. aj242966), and 95% match of the 153 bp amplicon to a presumed indian strain 5srrna sequence (genbank accession no. a 57-year - old woman, resident in a town east of johannesburg, gauteng province, south africa, complained of a swelling in the right groin. prior to her presentation, she had travelled to the coastal city of richards bay, on the kwazulu - natal province s north coast. sections showed fibrofatty tissue and foreign body granulomas, with the presence of giant cells, lymphocytes, plasma cells and a few neutrophils. sections of a female parasitic nematode were present, identified as a dirofilaria species on the basis of prominent longitudinal cuticular ridges, large lateral chords, internal lateral ridges, and typical coelomyarian muscle cell arrangement and gut and reproductive organs (figure 2b, c). the average spacing between the cuticular ridges was 11.6 m, consistent with published descriptions of d. repens (namely, about 12 m). human dirofilariasis presents typically as subcutaneous or ocular masses (d. repens), or as pulmonary nodular lesions (d. immitis). recently, however, more atypical sites have been reported where d. immitis has been found in cranial, hepatic and intra - ocular tissue, while d. repens has been identified from the lungs, scrotum, penis and female mammary glands. transient reactive swelling to migrating parasites may occur before they localise to produce inflammatory masses. typically, a cystic lesion is noted either subconjunctivally, as in this case, or in other structures of the eye. the definitive host for o. lupi are dogs, and human infection has been reported in europe. in south africa ocular loiasis, caused by the filarial nematode loa loa, has been diagnosed in immigrants from west and central africa, but it has a different clinical presentation. the adult worm moves across the eye under the conjunctiva over the course of a few hours, and the transient inflammation and swelling subsides as the worm leaves the area and continues its subcutaneous migration. the diameter of the nematode and prominent longitudinal ridges in our cases make the most likely identification to be d. repens, as d. immitis has a smooth cuticle. other species with obvious cuticular ridges (d. ursi, d. tenuis, d. subdermata) are only found in north america. although the recent travel history for the first patient included europe, where d. repens infections are well described, the relatively slow rate of development of the dirofilarial nematodes indicates that the infection was acquired before her trip to europe. unequivocal molecular identification of d.repens was demonstrated in this case ; a possible intraspecies variant was suggested by a smaller than anticipated 5srrna amplicon, but this needs to be further investigated and confirmed. although suitable hosts and vectors for d. immitis are present in south africa, the only known cases in dogs in this country have been imported. there is limited evidence that dog heartworm occurs in mozambique, and it is known to be present in tanzania and kenya. a report of subcutaneous dirofilariasis from zimbabwe, was subsequently shown on morphological grounds to be infection by an onchocerca species (j. frean, personal communication). cases of d. repens in a south african dog and cat suggest that occasional local human cases can be expected the worm is not always submitted as a whole specimen, or may have been damaged at surgery. some morphological features are shared between zoonotic dirofilaria species, confounding definitive identification in histological sections. geographical distribution may change over time, with climatic changes potentially affecting vector distribution, and global travel with pets occurring more frequently. adjunctive diagnostic laboratory methods include serological tests, such as enzyme - linked immunosorbent assays (elisas), and molecular tests. dna amplification and sequencing methods have the highest specificity, with the advantage of not requiring a complete or mature worm for species identification. anti - helminthic therapy is not recommended for human dirofilariasis, as surgery is adequate. some cases of ocular dirofilariasis have resolved without any intervention. correct diagnosis is imperative in these patients, as misdiagnosis may lead to unnecessary chemotherapy and extensive surgery. human dirofilariasis is uncommon, and therefore lack of awareness of this entity amongst clinicians frequently results in misdiagnosis and underestimation of the actual disease burden. seroprevalence studies, for determination of disease burden in animals in south africa, where prevalence is unknown, but host, vector and appropriate environmental conditions are present, may be useful. increased awareness of disease burden in the natural reservoir in the country and of the clinical entity of human dirofilariasis can prevent inappropriate chemotherapeutic and surgical interventions for patients. | humans are occasionally inadvertently infected with dirofilariae, the zoonotic nematodes. we report two cases of human dirofilariasis in south africa, an area apparently non - endemic for this infection. dirofilariasis is frequently misdiagnosed, so increased awareness of this entity in areas that are non - endemic is essential for prevention of inappropriate investigations and invasive therapy. |
the world health organization estimates that worldwide approximately 42 million abortions take place each year. nearly 90% of abortions are performed in the first trimester of pregnancy, before 14 weeks of gestation. there are two approaches to surgical abortion : vacuum aspiration and dilatation and evacuation (d&e). d&e is a commonly performed daycare procedure in obstetrics. due to requirement for an early discharge however, like any other surgical procedure, d&e involve pain, and therefore pain control measures have to be undertaken. many patients still find this surgical procedure extremely uncomfortable ; 78 - 97% report at least moderate procedural pain and, therefore, optimizing pain control should be a goal in every procedure. short acting narcotic analgesics are commonly used for intraoperative and postoperative pain relief in day care procedures. fentanyl is a synthetic opioid, which is highly lipid - soluble and rapidly acting drug. its onset of action is 2 min and duration of action is 30 - 60 min. its adverse effects are respiratory depression, pruritus, skeletal and thoracic muscles rigidity, etc., may delay discharge especially in day care surgeries. it rapidly passes through blood - brain barrier, reaches a high concentration in the cerebrospinal fluid and has an antinociceptive effect mediated by the central nervous system. injectable paracetamol was introduced for intravenous (iv) use in 2002, which provides the onset of pain relief within 5 - 10 min following administration. the peak analgesic effect is achieved in 1-h and the duration of this effect lasts 4 - 6 h. it is devoid of any major adverse effects like respiratory and circulatory depression and has no sedative effect making it ideal for day care procedure with mild to moderate pain. the hypothesis is that iv paracetamol is comparable with fentanyl for pain relief in d&e procedures. the objective of this study was to compare iv paracetamol with fentanyl for intra- and post - operative pain relief in dilation and curettage procedures. after approval from departmental research and hospital ethical review committee (2159-ane - erc-12) written informed consent was taken from all patients participated in the study. sixty adult american society of anesthesiologists (asa) i - ii female patients aged > 18 years who underwent elective d&e procedure requiring general anesthesia were enrolled. gestational age more than 12 weeks, hypersensitivity to any of the study drugs, anticipated difficult airway, morbid obesity, inadequate fasting, or hepatic disorders were excluded. each patient fulfilling the inclusion criteria was explained about study purpose and numerical rating scale by primary investigator in the wards / surgical day care unit. all patients were randomly allocated in two groups (30 each) by sealed envelope technique. the patients were explained in the ward about study purpose and assessment of pain by visual analog scale (vas) on 0 - 10 scales. the interpretation of pain scores was assessed as follows : 0 - no pain, 1 - 4 mild, 5 - 7 moderate and 8 - 10 - severe. all patients had received oral midazolam 7.5 mg as a premedication 30 min before procedure in the ward. group p had received iv paracetamol 15 mg / kg in the preoperative area 15 min before the start of surgical procedure. patients in group f were given iv fentanyl 2 g / kg at induction of anesthesia. the conduct and technique of general anesthesia was same for both groups. after application of standard monitoring (noninvasive blood pressure [nibp ], electrocardiogram, and pulse oximetry) (spo2), patients were preoxygenated for 3 min. nibp which includes systolic, mean and diastolic pressures, heart rate, pulse oximetry, respiratory rate, and end - tidal co2 were recorded. these readings were observed by one of the study author every 3 min from start of surgical procedure until the end. inadequate pain control during the surgical procedure was assumed if heart rate, blood pressure and respiratory rate are increased 20% above the baseline, which was regarded as preinduction time period. the rescue analgesia consisting of fentanyl was administered in 25 g increments in the intraoperative period for both groups. at completion of surgery, patients were allowed to regain consciousness and lma was removed when patients responded to verbal commands and thereafter transferred to the recovery room. same study author who recorded intraoperative observations visited the patients in the recovery room and observe pain scores on a numerical rating scale at 5, 15, and 30 min intervals after surgery. if pain score was greater than 3 according to numerical rating scale, rescue analgesia with fentanyl 25 g in increments was administered. all statistical analysis was performed using statistical packages for social science version 19 (spss inc., quantitative data were presented as mean and standard deviation and analyzed by student 's t - test, while qualitative data were presented as frequency and percentage and analyzed by chi - square test. we took percentage change (pc) in mean hemodynamics (heart rate, systolic blood pressure, and diastolic blood pressure) ranges from baseline to 20% and percentage change in variation (cv) of hemodynamics ranges from baseline to 15%. we calculated the sample size using n = (z/2 + z) 2 (cv) [1 + (1pc)/(pc) formula at 5% level of significance and 80% power, 60 patients with 30 in each group were selected for this study. a total of 60 patients who underwent elective d&e due to missed abortions requiring general anesthesia were studied. it was observed that there was no significant difference between the groups with respect to age, weight, height, and asa status as shown in table 1. the measured vital signs did not indicate any significant differences in mean heart rate, blood pressure (systolic, diastolic, and mean arterial pressure) and respiration between the two groups during the course of study as presented in figures 1 - 4, respectively. demographic and anesthetic measurements of the study patients comparison of mean systolic blood pressure between groups. error bars shows 95% confidence interval, which also indicate insignificant difference between groups at each point time comparison of mean diastolic blood pressure between groups. error bars shows 95% confidence interval, which also indicate insignificant difference between groups at each point time comparison of mean arterial pressure between groups. error bars shows 95% confidence interval, which also indicate insignificant difference between groups at each point time comparison of mean heart rate between groups. error bars shows 95% confidence interval, which also indicate insignificant difference between groups at each point time the pain score based on visual analog score (vas) were assessed at 5, 15, and 30 min in the recovery room. at 5 min mild pain was observed in both groups, mean pain scores were 1.57 1.1 and 0.97 1.3 in paracetamol and fentanyl groups, respectively except in 2 patients of paracetamol group who faced moderate pain and received rescue analgesia as shown in table 2, while at 15 and 30 min, pain was observed mild in all patients for both groups. poor pain control during the perioperative period leads to complications in both long- and short - term periods. with a good analgesic treatment plan, the anxiety, morbidity, cost and length of hospital stay in the postoperative period are reduced. d&e is a brief procedure, but it is associated with mild to moderate pain. short - acting narcotics are commonly used for perioperative pain relief in brief surgical procedures as these drugs provides good analgesia. however, resource variability is a major problem in developing countries, and working conditions may vary from excellent to poor. one of the working challenges in these places is the nonavailability or sudden shortages of opioids / narcotic drugs forcing anesthetist to look for safe alternatives. fentanyl unlike other opioids has fewer side - effects, but still can cause dose - dependent respiratory depression, which may contribute in delayed awakening, bradycardia, and hypotension, etc. the iv form of this agent has the theoretical advantage of greater predictability and acceptability compared with oral or rectal routes of delivery. it passes easily through the blood - brain barrier and shows its central analgesic effects within 15 - 20 min, which starts to decline after 4-h of administration. it is preferred in most surgical patients because it is does not affect mental status, bleeding, respiratory drive, gastric mucosa integrity, or renal function. we undertook this prospective randomized study to examine iv paracetamol as a suitable alternative to fentanyl for perioperative pain relief in dilation and curettage procedures. they found that iv paracetamol administered over a 24-h period in patients with moderate to severe pain after orthopedic surgery provided rapid and effective analgesia and was well - tolerated. a study in patients undergoing lower segment cesarean section, in which iv paracetamol was compared with oral ibuprofen, as the analgesic supplementation agent to morphine, indicated that patients in iv paracetamol had better pain control compared to ibuprofen group. tsang. did a study to see the opioids sparing effects of paracetamol in preoperative hip fracture patients, and they found that iv paracetamol had a significant opioid - sparing effect and satisfactory pain relief in preoperative hip fracture patients. a randomized, double - blind study had compared iv paracetamol and iv morphine for acute limb trauma in an urban united kingdom emergency department. approximately, half in each group had a fracture, and the other half had soft tissue trauma. the outcome measures were : pain score measured on a visual analog scale ; requirement for rescue analgesia ; and frequency of adverse reactions. there was no significant difference in the rescue medication, but there were significantly more adverse reactions in the morphine group. there were no significant differences between the groups in mean pain score and patient satisfaction. there was another randomized study of 84 patients undergoing out - patient knee arthroscopy comparing pain score and adverse reactions between 1000 mg iv paracetamol and iv morphine (0.1 mg / kg) given prior to awakening from general anesthesia. there was no difference in pain scores between those patients given the 2 medications, but there were more adverse reactions, dizziness, nausea, and vomiting, in the patients who received morphine. our study did not indicate any statistically significant difference between the iv paracetamol and iv fentanyl groups in pain scores. neither did we note any significant difference in physiological parameters and side - effect profiles clinically between the two groups. limitations of our study were small sample size, lack of the placebo arm, single - blinded nature and short follow - up duration. all of these could have improved the results of our study, but due to technical reasons and limited availability it was not possible. the study demonstrates the usefulness of iv paracetamol as it provides equivalent analgesia to fentanyl in dilation and curettage procedures. | background and aims : dilatation and evacuation procedure involves pain, for which pain control measures need to be undertaken. the purpose of this study was to compare paracetamol with fentanyl for pain relief in dilatation and curettage procedures.materials and methods : sixty female patients were randomly included during the period from march 1, 2012 to february 28, 2013. all patients had received oral midazolam 7.5 mg as a premedication 30 min before procedure in the ward. group p had received intravenous (iv) paracetamol 15 mg / kg in the waiting area of the operating room 15 min before starting the procedure. group f had received iv fentanyl 2 ug / kg / min at induction of anesthesia. pain scores on a numerical rating scale at 5, 15, and 30 min intervals after surgery were recorded.results:mild pain was commonly observed in both groups, an insignificant difference between groups.conclusion:the study demonstrates the usefulness of iv paracetamol which may be as effective as fentanyl in dilation and curettage procedures. |
a 14-year - old female patient presented to us with complaints of palpable purpura and ulceration over lower extremities for last 5 days. the patient was receiving anticonvulsive therapy (phenytoin) for epilepsy since she was 3 years old. a fortnight ago, her neurologist added levetiracetam 500 mg daily to phenytoin 50 mg thrice daily, owing to worsening of epilepsy symptoms. eight days after initiation of levetiracetam, the patient developed palpable purpura over lower extremities, associated with itching. she did not suffer from any preceding infection or illnesses and demonstrated the absence of systemic manifestations such as arthralgias, hematuria, melena, or abdominal pain. cutaneous examination revealed the presence of palpable purpura and large ulcers covered with necrotic eschars over the lower extremities [figure 1 ]. palpable purpura and necrotic ulcers over lower extremities routine laboratory investigations were detected to be normal. additional investigations demonstrated a negative antinuclear antibody, cytoplasmic antineutrophil cytoplasmic antibodies, perinuclear antineutrophil cytoplasmic antibodies and normal serum protein electrophoresis, hepatitis screen, urinalysis, antidouble - stranded dna antibody, and thyroid assay. a skin biopsy from the lesion revealed fibrinoid necrosis of walls of small vessels consistent with lcv [figure 2 ]. immunofluorescence of the biopsy specimen could not be done due to nonavailability in our setup. histopathological examination showing red blood cell extravasation, infiltration of neutrophils and eosinophils and fibrinoid necrosis (h and e, 40) in the dermis on the basis of temporal association and clinicohistopathological findings, a diagnosis of levetiracetam - induced lcv was established and levetiracetam was discontinued. adverse reaction severity was determined as level 4(b) according to modified hartwig and siegel scale as it was the reason for admission. the patient was treated with systemic corticosteroids (30 mg / day of prednisolone) along with topical corticosteroids (mometasone furoate 0.1% cream twice daily), emollients, and oral levocetirizine 5 mg twice daily. after 1 week of stoppage of levetiracetam and initiation of treatment, lesions had started subsiding. oral steroids were gradually tapered off over next 3 weeks. at 4 weeks ' follow - up, lesions had cleared completely with residual hyperpigmentation and scarring. causality assessment was carried out using naranjo 's scale and world health organizationuppsala monitoring centre criteria, which suggested that levetiracetam was the probable drugs can cause vasculitis of various morphological types. most commonly, these cause a superficial small vessel cutaneous lcv ; however, other patterns including systemic vasculitis also occur. more than 100 drugs are implicated as causes of drug - induced vasculitis although actual frequency of drug - induced vasculitis is low. commonly, implicated drugs include penicillins, sulfonamides, quinolones, analgesics, anticonvulsants, phenothiazines, allopurinol, and colony - stimulating factors. less commonly used drugs with a significant risk of vasculitis include thiouracil, hydralazine, and biologicals such as adalimumab, etanercept and infliximab, ephedrine, amphetamines, vaccines (especially influenza and hepatitis b vaccines), herbal remedies, and food additives. exact pathogenesis of drug - associated cutaneous vasculitis is unclear, but studies suggest that the offending drug may act as a hapten, which stimulates antibody production and immune complex formation. these immune complexes are subsequently deposited in postcapillary venules leading to complement activation and vascular damage. cutaneous manifestations of drug - induced vasculitis include palpable purpura, papules, and bullae. palpable purpura can occur hours to weeks following exposure to offending drug and most commonly develops on gravity - dependent areas, such as lower extremities and buttocks. this was also observed in the present case where lesions started appearing after 8 days of initiation of offending drug and were localized mostly to lower limbs. distinguishing drug - related vasculitis from other etiologies of lcv can be difficult. however, pattern of development and resolution of lesions with continuation or discontinuation of medication can help in diagnosis. withdrawal of offending drug and minimization of stasis by compression, elevation, and use of nonsteroidal anti - inflammatory drugs are employed. antihistamines and systemic corticosteroids may be required when the cutaneous lesions are progressive as in present case. levetiracetam is a pyrrolidone derivative that is thought to exert its antiepileptic effects through adherence to synaptic vesicle protein sv2a and modulation of neurotransmitter release. levetiracetam is an effective adjunctive therapy in patients with refractory focal epilepsy, myoclonic epilepsy, and primary generalized tonic drug interactions between lev and other medications, such as warfarin, digoxin, oral contraceptives, probenecid, and other antiseizure medications, are not reported. levetiracetam causes several adverse neurological effects including headache, somnolence, asthenia, dizziness, irritability, and behavioral changes. in addition, it causes hematological side effects such as mild thrombocytopenia, leukopenia, and anemia. cutaneous side effects with levetiracetam are less frequent compared to other antiepileptic medication, but adverse effects such as bullous pemphigoid, maculopapular drug rash, and drug reaction with eosinophilia and systemic symptoms have been reported. even though lcv is a common adverse effect of antiepileptics, but to the best of our knowledge, this is the first reported case of lcv with levetiracetam. event relationship, morphology, distribution, histopathological findings, and rapid response on withdrawal of the drug, we conclude that patient developed lcv due to levetiracetam. even though risk of cutaneous adverse effects with levetiracetam is less, clinicians should be aware of possibility of adverse effects such as small - vessel vasculitis or drug eruption occurring as a rare adverse effect with this drug. | drug - induced leukocytoclastic vasculitis is a small - vessel vasculitis that most commonly manifests with palpable purpuric lesions on gravity - dependent areas. vasculitis occurs within weeks after initial administration of medication and demonstrates clearance upon withdrawal of medication. levetiracetam, a pyrrolidone derivative, is used as an adjunctive therapy in patients with refractory focal epilepsy, myoclonic epilepsy, and primary generalized tonic clonic seizures. we present a case of a 14-year - old female, who developed cutaneous small - vessel vasculitis within 8 days of initiation of levetiracetam. vasculitis was successfully managed by discontinuation of medication and systemic corticosteroids. this adverse reaction, to the best of our knowledge, has not been previously reported in literature. |
the tousled locus was originally identified in a. thaliana and antirrhinum majus during a study of mutations leading to defects in meristem expansion. mutations of tousled produce a complex phenotype characterized by specific defects in development of leaf and floral organs. this was proposed to be linked to a replicative defect during organogenesis, but it may also result from failure to protect the genomefrom dna damage [2, 3 ], resulting in developmental aberrations [4, 5 ]. highly related tousled - like genes can be found in many organisms in both kingdoms, several of which encode multiple transcripts resulting in different protein isoforms. it was originally proposed that tousled (tsl) may be a component in a signal transduction pathway controlling cell proliferation and dna synthesis during organogenesis, and this immediately prompted a search for its substrates. however, unlike most kinases that usually display a propensity to phosphorylate numerous substrates, after many years of study, only a few direct interacting substrates of tlks have been identified, namely, the histone chaperone asf1, histone h3-s10, aurora b, and more recently rad9. this suggested a function for tlks in chromatin assembly [9, 10 ], during transcription [2, 11 ], dna repair [3, 9, 12 ], and condensation of chromosomes at mitosis [4, 5 ]. the latter function, which was found critical for proper chromosome segregation, prompted a search for additional indirect substrates and functions and resulted in the identification of an activity on myosin ii in mammalian cells and on the chromosome passenger complex in trypanosomes. the search for tlks functions at mitosis and meiosis is currently a very active pursuit in several labs in more genetically tractable organisms like drosophila and c. elegans (jill schumacher, personal communication). in addition, whereas only nuclear functions were initially proposed for these proteins, some splice variants localize also to the cytoplasm, perhaps due to their reported interaction with 14 - 3 - 3 proteins with their shuttling function and hence could play additional roles in potential cytoplasmic substrates, one of which was identified as the dead - box p68 rna helicase. more emphasis is presented next for three of the most important substrates of tlks : asf1, rad9, and histone h3. asf1 is a histone h3-h4 chaperone that is essential in mammals and other organisms [20, 21 ] but not in s. cerevisiae, although such cells deleted for asf1 are sensitive to genotoxins. a recent review on asf1 and other histone chaperones can be found in and its critical importance for epigenome maintenance in. asf1, in conjunction with another chaperone called caf1, promotes the assembly of nucleosomes onto newly replicated dna, but it can also promote nucleosome eviction at activated promoters [2527 ]. thus, asf1 is generally involved in chromatin remodeling, which also entails dna repair [22, 28 ]. the crystal structure of asf1 in complex with h3-h4 was solved at high resolution, and asf1 was found to cover the dorsal side of the h3-h4 dimer, thereby sterically preventing formation of the core tetramer. this is thought to be important for disrupting nucleosomes during transcription or remodeling of chromatin in damaged dna [3, 9 ], and the role of tlk1b in radioprotection was initially attributed to its effect on asf1, presumed to be via its phosphorylation. more recently, however, i showed that tlk1b can stimulate chromatin assembly in vitro in conjunction with asf1 regardless of its phosphorylation. this suggested that tlk1/1b act as chaperones in chromatin assembly, in addition to their kinase function. hence, an important role of tlks via asf1 is to promote nucleosomes eviction at dsbs and access of the repair machinery to unencumbered dna. rad9, rad1, and hus1 form a trimeric complex (termed 9 - 1 - 1) that is structurally similar [32, 33 ] to the pcna sliding - clamp, which encircles the dna conferring processivity to polymerases [3436 ]. 9 - 1 - 1 assembles in a complex at sites of damage, and it is the genotoxin - activated rfc - rad17 clamp loader that locks 9 - 1 - 1 onto dna. the 9 - 1 - 1 may then serve as a scaffold for assembly of dna repair proteins, flap endonuclease [38, 39 ], dna polymerase, dna ligase 1, and dna glycosylase muty, in addition to aiding processing of the dna ends by its own exonucleolytic activity [4345 ]. we showed that tlk1b phosphorylates rad9 at s328 and that this appears to play a key role in resumption of the cell cycle arrested after ir. however, tlk1b also had a function as a chaperone for rad9 assembly at dsbs that was independent of its kinase function. a possibility is that the regulated binding of 9 - 1 - 1 and tlk1b to dsbs recruits repair enzymes and a chromatin disassembly apparatus to facilitate access to unencumbered dna and promote efficient dsb repair, and only subsequently in the dna damage response (ddr) disengagement and deactivation of the checkpoint. rad9 participates in additional functions of the ddr and in repair and also in restart of stalled replication forks, along with numerous other proteins, like rhino and topbp1 or wrn. although the rad9 c - terminal tail (119 aa) shares no homology with pcna and is thought to be nonessential for the formation of the 9 - 1 - 1 complex, this region is multiply phosphorylated, constitutively and inducibly in response to genotoxic stress [49, 50 ]. rad9 is normally phosphorylated independently of the cell cycle at s277, s328, s336, t355, and s387. cell - cycle - dependent phosphorylation of rad9 at thr 292 occurs during mitosis in a cdc2-dependent manner. although damage - dependent phosphorylation of rad9 was initially believed to modulate the stability of the 9 - 1 - 1 complex, it is now believed that neither constitutive nor damage - induced phosphorylation influences the interaction of rad9 with its partners rad1 and hus1 [37, 49 ]. most studies point to the role of damage - activated rad9 phosphorylation in downstream cell signaling via activation of chk1, and it was reported that phosphorylation of rad9 influences cell viability after uv and hydroxyurea cell - cycle stalling. substitutions at all carboxy - terminal 8 phosphorylation sites compromised rad9 interaction with topbp1 and impaired the cellular response to dna damage. collectively, these results suggest that rad9 phosphorylation regulates protein interactions and downstream cell signaling from dna damage. our studies with reconstituted rad9/ cells indicated that s328 phosphorylation is not essential for rad9 interaction with hus1 and rad1, consistent with previous studies. however, phosphorylation at s328 appeared to be important for exiting cell - cycle arrest after production of dsbs and resulting in a smaller fraction of apoptotic cells and better clonogenic survival. some studies have implicated stress - activated rad9 phosphorylation to activation of chk1 [50, 53 ], which mediates the cell cycle checkpoint. indeed, current research in our lab has shown that inhibiting tlks with specific chemical inhibitors results in incapacity to exit the cell cycle checkpoint and high rates of apoptosis when combined with dsb - inducing agents (to be published elsewhere). we observed similar results by overexpression of a tlk kinase - dead (kd) and observed a delay in the release of the rad17-clamp - loader and rad9 from a single genomic dsb introduced with the ho nuclease transiently expressed from adenovirus. the most logical conclusion arising from these results, and from the specific pattern of activity of tlks (see below), is that the s328 phosphorylation of rad9 by tlks is critical for deactivation of the ddr checkpoint following dna repair. phosphorylation of histone h3 at ser10 was recognized as the first substrate of tlk1b, as demonstrated both biochemically and by direct mass - spec measurements ; this was further confirmed by genetic complementation of a yeast strain defective in ipl1 (aurora kinase of s. cerevisiae) that is the main h3-s10 kinase in this organism. tousled (tsl) could phosphorylate histone h3 in vitro, just like mammalian tlk1b, but the recombinant c. elegans tlk was not effective at phosphorylating directly h3 but was highly stimulatory in conjunction with air-1 (aurora kinase), which was found to be an interacting target of the single tlk protein in c. elegans. phosphorylation of h3 by aurora kinase is a hallmark of mitosis, and that specific phosphorylation is proposed to be critical for chromosome condensation at mitosis. in that context, it is not clear if the role of tlk - mediated phosphorylation of h3 is central to mitosis, although the phosphorylation of h3-s10 is reduced in nonsynchronized cells expressing the kd, and the condensation of chromosomes and phosphorylation of h3 is reduced at mitosis. but this of course could be also an indirect effect on aurora kinase activity [5, 55 ]. more importantly, however, inhibition of tlk activity by genotoxic stress (see below) by either ir or uv results in reduced levels of h3-s10 in unsynchronized cells [3, 8 ]. since mitotic cells represent only a small minority of cycling cells, even the large ~5-fold increase in h3p(s10) seen during mitosis would account for only a very small amount of the h3p from the total population. hence, it would seem logical to assume that tlk - mediated h3p phosphorylation probably accounts for some other function in chromatin maintenance. if for example tlk1/1b is the main h3 kinase involved in a chromosomal response to dna damage, then atm - mediated inhibition of tlk1 (see below) is expected to result in a loss of h3 phosphorylation by endogenous phosphatases and in altered kinetics of chromatin assembly during replication and/or repair. it is possible that physiologically the increased tlk1b synthesis following ir can help offset the loss of tlk activity resulting from ir and restore appropriate levels of h3p later on during the recovery. the reduction of h3p following genotoxic stress (ir) was previously reported also by another group. in any case, there are other situations where h3-s10 phosphorylation is induced beside mitosis, and a clear case is that of the nucleosomal response at the early - response genes following mitogenic stimulation. gene disruption in murine embryonic stem cells, and genetic evidence from coffin - lowry syndrome, has implicated rsk-2 as the kinase directly responsible for phosphorylating h3 following mitogenic stimulation. on the other hand, another kinase (msk1) was reported to phosphorylate h3 more efficiently and be sensitive to the kinase inhibitor h89, which impairs the nucleosomal response, whereas rsk-2 was insensitive to this inhibitor. however, it now seems clear that several families of h3-s10 kinases exist (e.g., ipl1/aurora and nima) and may be involved in different or partially overlapping functions [60, 61 ]. we have clearly shown that recombinant tlk1b phosphorylates h3 at s10 and could complement a temperature - sensitive mutant of ipl1 in yeast and restore h3p in those cells at the nonpermissive temperature. furthermore, it could do so with high specificity in a mix of core histones, and in cells overexpressing tlk1b we found increased levels of h3 phosphorylation. these findings, as well as the genetic complementation data, strongly suggest the inclusion of the tousled family of kinases to the list of h3-s10 kinases, even though their precise role during the cell cycle or perturbations of it (inhibitory or stimulatory) has not been fully elucidated. the use of newly identified specific chemical inhibitors of tlks could perhaps shed light on the role of tlk - mediated phosphorylation of h3-s10 and its significance in chromatin assembly during normal division or after dna damage. the first human tlk cdna to be cloned, what we later referred to as the tlk1b splice variant (kiaa0137), was first identified during the random cloning of novel cdnas from the human myeloid cell line kg-1. the cdnas for tlk1 (chr 2) and tlk2 (chr 17) were later cloned during a pcr - based search for human kinases and independently from an expression library screened on the basis of autophosphorylation activity (; named pku and pku by these authors). instead, we have independently cloned the tlk1b splice variant with a completely different screen, based upon polysomal redistribution of weakly translated transcripts that become preferentially recruited upon overexpression of eif4e. we subsequently found that tlk1b is synthesized efficiently in several cell lines overexpressing the translation factor / oncogene eif4e, and we then presented several lines of evidence to confirm its translational regulation, particularly after genotoxic stress. the significance of this translational regulation is discussed later when i emphasize the role of tlks in dna repair and protection from genotoxic agents, including ir and uv. below, we propose that an important role for tlks is as guardian of the genome, and we implicate a function in cancer development and progression. this derivation seems obvious given their role both in basic aspects of chromatin assembly, transcription, replication, and repair and also for their distinct role in chromosome segregation into daughter cells. a high percentage of human tumors, including cancer of the prostate (cap) and breast (bca), show mutations in dna repair genes and checkpoint functions that make them overly dependent on alternative pathways for survival. unfortunately, this can result in carcinomas that are highly resistant to radiation therapy (xrt) or radiomimetic therapy (rmt) from failsafe repair mechanisms also designed to contain excessive genomic instability. we propose that the addition of inhibitors of tlks to enhance response to radiochemotherapy will greatly benefit cap and bca patients ' therapy management. in fact, ameliorating the effects of standard therapy, and possibly reducing its doses while maintaining specific killing, still seems to be the one of most promising course of action for the near future. certainly, the success of parp inhibitors for triple negative bca seem to point in that direction. to recapitulate some basic information before addressing tlks in humans, the tlks are involved in chromatin assembly, dna repair, transcription, and chromosome segregation (and references therein). two tlk genes (tlk1 and tlk2) with several splice variants have been identified in humans. tlk1/1b interacts specifically with the chromatin assembly factor asf1 and rad9 [9, 46 ], and we have presented evidence that tlk1b promotes repair by processing of the double - strand break (dsb) ends and disassembly of chromatin near the dsb to facilitate recruitment of repair proteins. since rad9 is a critical mediator of the response to dna damage, ddr checkpoint, and in repair (specifically of dsbs), it seemed that the tlk1-rad9 interaction would be very important in implementing the mechanism of tlk1b - mediated radioprotection. the past few years have witnessed significant advances in understanding the roles of tlks in the ddr and in direct repair of dsbs, as well as their clinical relevance. in bca, elevated expression of the tlk1b splice form is found in ~30% of the patients and often corresponds to poor response to xrt and doxorubicin, presumably due to efficient repair of dsbs in the tumor cells. we postulated that its expression could serve as a marker for prognosis as well as a target for therapeutic intervention. in addition, there are bca cases where tlk1/1b is not elevated, but it is tlk2, which lies in a region of chr 17q23 nearby the brca1 locus, that is amplified and/or overexpressed in a significant number of bca specimens [69, 70 ]. thus, for a large proportion of sporadic bca, specific tlk inhibitors should be extremely beneficial as radio - chemosensitizers. the fact that tlks are overexpressed likely renders tumor cells more dependent on these kinases than normal tissues and, hence, their preferential tlk - targeted killing. in contrast to bca, in the most common human cap cell lines, only one or the other tlk gene is expressed, although typically at high level we do not have the story yet for the analysis of patient samples. the significance of the tlk / rad9 axis is perhaps even greater for prostate cancer for which several studies have implicated the critical role of rad9 in disease progression and prognosis. in one study, rad9 was associated with tumor stage and was reported to regulate tumor growth in mice. in another, the investigators found that rad9 contains androgen - responsive elements and that its expression is also androgen regulated. in a third study, rad9 acted as a corepressor of ar transactivation all of which suggest that rad9 expression may be a significant part of the androgen switch that leads to cancer cell survival and that rad9 has functions beyond dna repair that make it clinically relevant as a biomarker or in tumor growth control. additional implications for the role of rad9 in cap and other common cancers are reviewed in [75, 76 ]. correspondingly, elevated expression of tlks (along with rad9) may be a significant marker of radioresistance in cap cell lines and likely in cancerous samples and hence represents a hallmark of poor prognosis. in contrast to rad9, there is no report for the direct involvement of either of the two human asf1 genes in cancer development, perhaps due to the critical importance of these histone h3/h4 chaperones for all mammalian cells (normal and cancer). nonetheless, a recent report correlated the expression of asf1b in prediction of bca relapse, perhaps due to its higher importance for cell proliferation and chromosomes duplication. it is noteworthy that translocations / amplifications involving 17q23 that include the tlk2 gene are not unique to bca but are also found neuroblastomas and glioblastomas multiforme. the fact that overexpression of tlks (or more specifically the tlk1b splice variant) conferred a high degree of protection against ir in mouse cells was one of the first effects reported for these proteins. it was soon found that the protection involved increased and more efficient repair of dsbs in living cells [9, 12 ], and then more precisely with in vitro plasmid repair assays with defined components and recombinant proteins. in such reactions, the assembly of nucleosomes on the plasmid was simultaneously monitored as a decrease in the linking number via formation of high - mobility topoisomers in conjunction with repair of a dsb or of excision of uv - induced pyrimidine dimers. hence, the specific contribution of asf1 to repair of dsbs or uv damage could be studied in those conditions. for repair of the dsb, depletion of asf1 had some effect on supercoiling, but it had only modest effect on religation of the ends. quantitative analysis showed that conversion of the linear form to circular / relaxed and then supercoiled was complete after 20 min in control extract, but not until 40 min in asf1-depleted. hence, asf1 albeit likely involved, was not essential for these repair reactions nor for supercoiling, at least for the case of cohesive - ends repair. similarly, repair of uv - damaged plasmids did not absolutely depend on asf1, although the kinetics of repair were strongly delayed, consistent with a previous report that looked at the contribution of asf1/caf1, and even tlks, in repair of uv - damaged plasmids. the identification of asf1 and later rad9 as two main targets of tlks immediately suggested some plausible mechanisms for their role in dna repair. we believe that the binding of 9 - 1 - 1 and tlk1b to dsbs recruits repair enzymes in conjunction with the chromatin remodeling machinery to create limited repair regions of dna that is not encumbered by chromatin, similar to what has been reported in yeast for the repair of the single dsb at mat during mating - type switching. we should, however, stress that in such capacity, the role of tlks as kinases has not been fully elucidated, since for some of these repair functions, expression of the tlk1b - kd was capable of producing effects similar to the catalytically active protein, and in specific reactions of nucleosome assembly, even in the absence of atp [9, 31, 82 ]. on the other hand, it seems now clear that the kinase activity of tlks is very significant in ddr signaling, and most likely during deactivation of the checkpoint. this is the last topic of this paper and is described below. while studying the gene expression regulatory activities of the translation factor eif4e, we had originally identified an eif4e - regulated transcript encoding a protein kinase (tlk1b) that when overexpressed increases radioresistance in mammalian cells. tlk1b is translationally upregulated in response to the presence of dsbs via a mechanism that involves activation of mtor following that of a pi3k member, likely atm, which ultimately results in eif4e stimulation. a rapid response to dna damage at the translation level is a novel mechanism for cellular survival that has opened new areas of investigation in dsb repair and in damage signaling. indeed, while transcriptional responses todna damage are well known, there is no question that the induction of a repair protein (tlk1b) at the translation level would be a far faster response to the injury, and this mechanism of translational activation is now being much more appreciated. while it seemed clear that an increase in tlk1b expression could play some advantageous role during dna repair, other investigators soon discovered that the kinase activity of tlks is actually rapidly inhibited during genotoxic stress [85, 86 ], which initially seemed at odds with our observations for the obvious need for increased levels of tlk1b. why making more protein kinase if at the same time it is going to be inactive for phosphorylation ? of course this would not include the chaperone function of these proteins [9, 31, 82 ]. hence, evidence exists for a strong link between tlks (both level and activity) and a dna damage relay. this is inferred by the observation that the kinase activity of tlk1 is inhibited by ir and genotoxins. the inhibition is mediated by atm via chk1 by direct phosphorylation of tlk1 at s695. these findings identified a functional cooperation between atm and chk1 in propagation of a checkpoint response mediated by transient inhibition of tlk1, which may regulate processes involved in chromatin remodeling after damage. we believe that the main reason for the cycle of inactivation and then hyperactivation of tlks, due to the obvious increase in tlk1b expression following genotoxic stress, is to fine - tune chromatin disassembly / reassembly (via asf1 and/or other histone chaperones) and to mediate the cell cycle checkpoint (particularly its deactivation) via 9 - 1 - 1. a model for this asf1 is known to interact with rfc (subunits 25) tethered to pcna, and it is recruited to the replication forks. we propose that after dna damage, asf1 is similarly recruited to the lesions to prepare for repair. here, asf1 may be instead recruited by the rad17-rfc clamp - loader, just as rad9 is, in association with tlk1/1b. after dissociation from rfc, the recruited asf1 is positioned to disrupt the h3/h4 tetramer resulting in nucleosome eviction. as repair progresses, newly synthesized tlk1b induced by dna damage could lead to dissociation of the asf1/h3/h4 heterotrimer and promote reformation of the h3-h4 tetramer. many details remain to be filled in for instance the role that atm plays in modulating the two separate activities of tlk1/1b (kinase or chaperone). a possible outcome for the role of atm - mediated inhibition of tlk1/1b is that the reduction of asf1 phosphorylation would lead to a more stable association of tlk1/1b - asf1, instead of a kinetic association involving the ratio of unphosphorylated to phosphorylated asf1. another question is how the rad9-mediated checkpoint activation of atm and atr may affect the entire pathway and its own association with tlk1/1b and rad17. once tlk1/1b activity is restored after repair, rad9 may then be rephosphorylated, which could be an important mark for release of the clamp complex and signaling completion of repair and resumption of the cell cycle. indeed, at least 3 lines of evidence indicate that the tlk kinase activity plays a role in checkpoint establishment and/or its deactivation : (1) the rad9/ cells complemented with tlk - kd show a defect in reentering the cell cycle after g2 arrest induced by ir. (2) mm3mg - ho cells expressing the tlk - kd can repair the dsb induced with ho but 1015% of cells die of apoptosis two days later, which may indicate a defect in deactivating the checkpoint, as supported by the fact that rad9(s238) phosphorylation is impaired. (3) mammalian cells treated with a tlk inhibitor + doxorubicin (or ir) arrest preferentially in s phase and die of apoptosis (unpublished results). hence, we strongly believe that the rad9-s328 phosphorylation by tlk1 is a key in checkpoint deactivation and suppression of the atm / atr - chk1 signal that is propagated via the clamp / clamp loader and topbp1see [53, 88 ] for two possible models. this would likely involve most types of dna damage and genotoxic stress and also restart of stalled replication forks hence the great importance of tlks for dna repair and genome integrity. it would seem obvious that finding inhibitors of tlks could greatly improve current radio- and chemotherapeutic approaches to cancer treatment. and in fact, silencing tlk1 was highly effective in sensitizing cholangiocarcinoma (a rather incurable disease) cell lines to cisplatin - induced apoptosis. on the other hand, one could envision that exploiting the functions of tlks in dna repair could actually produce beneficial effects for normal tissues and organs exposed to the same genotoxic regimens : xrt, radiomimetic chemotherapy, or even daily skin exposure to uv damage. indeed such cases are being contemplated in our labs, and both gene therapy approaches aimed at sparing salivary glands from the damaging effects of xrt to treat head and neck cancer, as well as direct tat - tlk1 protein delivery to salivary glands, have been recently explored with a human clinical trial in sight. perhaps additional modes of delivery of these proteins, such as a topical skin delivery in a liposomal complex (of either the protein itself or via viral or plasmid gene delivery vehicle), will become feasible in the near future. a model for the participation of tlk in chromatin - remodeling linked to dna repair | the tousled - like kinases (tlks) function in processes of chromatin assembly, including replication, transcription, repair, and chromosome segregation. tlks interact specifically (and phosphorylate) with the chromatin assembly factor asf1, a histone h3-h4 chaperone, histone h3 itself at ser10, and also rad9, a key protein involved in dna repair and cell cycle signaling following dna damage. these interactions are believed to be responsible for the action of tlks in double - stranded break repair and radioprotection and also in the propagation of the dna damage response. hence, i propose that tlks play key roles in maintenance of genome integrity in many organisms of both kingdoms. in this paper, i highlight key issues of the known roles of these proteins, particularly in the context of dna repair (ir and uv), their possible relevance to genome integrity and cancer development, and as possible targets for intervention in cancer management. |
untreated hypertension may lead to many serious health conditions, including stroke, aneurysms, hypertensive heart disease, coronary artery disease, kidney disease, or peripheral artery disease [24 ]. hypertension has a major economic impact ranging from medical costs to human capital loss and decrease in productivity [5, 6 ]. the global burden of disease 2010 (gbd 2010) study estimated that hypertension was the leading risk factor for death in the kingdom of saudi arabia (ksa). hypertension accounted for about 24% of total deaths from cardiovascular and circulatory diseases and 1.87% of total deaths from hypertensive urogenital, blood, and endocrine diseases. from 1990 to 2010 these levels ranged from 26.1% among individuals 3070 years old in 19952000 to 25.5% among individuals 1564 years old in 2005 [10, 11 ]. in order to assess the current status of hypertension in ksa we selected a national sample to be representative of each of the 20 ksa health regions and the kingdom. we used an adapted standard questionnaire and took physical measurements and blood samples to examine blood pressure and chronic diseases. the software used for offline data collection allowed interviewers to upload data to our servers on daily basis and hence a rigid monitoring of data quality. the saudi health information survey (shis) is a national multistage survey of individuals aged 15 years or older. households were randomly selected from a national sampling frame maintained and updated by the census bureau. all regions were included, and a probability proportional to size was used to randomly select subregions and blocks. a roster of household members was conducted and an adult aged 15 or older was randomly selected to be surveyed. weight, height, and blood pressure were measured at the household by a trained professional. omron hn286 (sn : 201207 - 03163f) and omron m6 comfort (hem-7223-e) were used to measure weight and blood pressure. the survey included questions on sociodemographic characteristics, tobacco consumption, diet, physical activity, health care utilization, different health - related behaviors, and self - reported chronic conditions. we used measured weight and height to calculate body mass index (bmi) as weight (kg)/height (m). participants were classified into four groups : (1) underweight : bmi < 18.5 ; (2) normal weight : bmi within 18.525.0 ; (3) overweight : bmi within 25.030.0 ; or (4) obese : bmi was greater than or equal to 30.0. respondents were considered to be current smokers if they reported ever smoking any tobacco products and still currently smoke tobacco and past smokers if they reported smoking in the past but not anymore. we computed the servings of fruits and vegetables and red meats and chicken consumed per day from the detailed dietary questionnaire as the sum of the average daily consumption of fruits, fruit juices, and vegetables and red meats and chicken. we used the international physical activity questionnaire to classify respondents into four groups of physical activity : (1) met vigorous physical activity, (2) met moderate physical activity, (3) insufficient physical activity to meet vigorous or moderate levels, and (4) no physical activity. to assess diagnosed hypertension, diabetes, and hypercholesterolemia status, respondents were asked three separate questions : have you ever been told by a doctor, nurse, or other health professional that you had : (1) high blood pressure, otherwise known as hypertension ; (2) diabetes mellitus, otherwise known as diabetes, sugar diabetes, high blood glucose, or high blood sugar ; (3) hypercholesterolemia, otherwise known as high or abnormal blood cholesterol ? women diagnosed with diabetes or hypertension during pregnancy were counted as not having these conditions. those who were diagnosed with either of these conditions were further asked if they are currently receiving any treatment for their condition. similarly, the same type of questions was used to determine previous diagnosis of stroke, myocardial infarction, atrial fibrillation, cardiac arrest, congestive heart failure, chronic obstructive pulmonary disease, asthma, renal failure, and cancer. we considered a person to be diagnosed with a chronic condition if they reported being diagnosed with any of the conditions cited earlier. a total of three blood measurements were taken with the participant resting and at five - minute intervals. we followed the national health and nutrition examination survey (nhanes) for determining blood pressure levels. basically, respondents were considered to have hypertension if they met any of the following criteria : (1) measured diastolic or systolic blood pressure exceeding 89 or 139 mmhg, respectively, or (2) measured diastolic or systolic blood pressure not exceeding the appropriate threshold, but the respondent reported taking medications for hypertension. hence, respondents who were on drugs for hypertension were considered hypertensive even if their measured diastolic or systolic blood pressure did not exceed 89 or 139 mmhg, respectively. respondents were considered to have borderline hypertension if (1) they did not report taking drugs for hypertension and (2) their measured diastolic blood pressure was between 80 and less than 90 mmhg or systolic blood pressure was between 120 and 139 mmhg. we used sas 9.2 (sas institute inc., cary, nc, usa) for analyses and to account for the complex sampling design. between april and june 2013, a total of 12,000 households were contacted and a total of 10,735 participants completed the survey (response rate of 89.4%). a total of 1,957,191 (15.2%) saudis aged 15 years or older had hypertension (measured or reported taking blood pressure medication). of these, 1,119,027 were undiagnosed. characteristics of respondents with undiagnosed hypertension, hypertension, and borderline hypertension are presented in table 2. among participants diagnosed with hypertension, among all those who are hypertensive, 57.8%, 20.2%, 16.6%, and 5.4% are undiagnosed, treated uncontrolled, treated controlled, and untreated, respectively (figure 1). age, sex, and diagnosis history of diabetes and hypercholesterolemia were associated with hypertension (table 3). the risk of being hypertensive was lower among females (aor = 0.61 ; 95% ci : 0.500.74) but increased with age (aor = 1.07 ; 95% ci : 1.061.08), among obese participants (aor = 2.24 ; 95% ci : 1.892.65) and those who have been previously diagnosed with diabetes (aor = 1.95 ; 95% ci : 1.572.43) and hypercholesterolemia (aor = 1.94 ; 95% ci : 1.512.47). on the other hand, marital status, education, smoking status, diet, time spent watching tv, levels of physical activity, diagnosis history of prediabetes, or other chronic conditions were not associated with the risk of hypertension (table 3). being male, older, and obese and having a diagnostic history of diabetes also increased the risk of borderline hypertension. daily consumption of two to three servings of red meats and chicken and being moderately active was associated with the risk of borderline hypertension (table 4). a large percentage of hypertension was undiagnosed, as 57.8% of those with hypertension did not know of their condition, a total of 1,119,027 saudis. the likelihood of being undiagnosed decreased among women (aor = 0.54 ; 95% ci : 0.440.67) and increased with age (aor = 1.05 ; 95% ci : 1.05 - 1.06) and diagnosis history of diabetes (aor = 1.46 ; 95% ci : 1.091.94) (table 5). our findings are striking in a country with free medical care and high resources. indeed, these findings call for action to control the burden of hypertension in the kingdom. a national plan to increase awareness, early detection, and control of hypertension the most recent estimates date back to 2005 and provided a prevalence of 11.5% of reportedly diagnosed hypertension, among individuals aged 1564 years, a much higher prevalence than 5.6% from our study. data from 19952000 for saudis aged 30 years or older showed a hypertension prevalence of 26.1%. in comparison however, the 2005 steps survey and our study should be comparable and possibly indicate a leveling of the hypertension prevalence in ksa. our results for increased risk of hypertension with age and among men are similar to previous studies from ksa and other countries. however, literature on marital status and hypertension is inconclusive and mostly focused on comparing currently married to never married persons [14, 15 ]. our study did not show any association between marital status and high blood pressure after adjusting for confounders. previous reports on association between education and hypertension are mixed as some studies have reported the lack of association or an inverse relationship [17, 18 ]. while the findings on hypertension might be pointing toward stabilization or decline in prevalence, the ksa health system still has many challenges. for instance, the majority (57.8%) of hypertensive saudis are undiagnosed. the other 55.0% of those on treatment were not controlled. shis included questions on health care utilization, and only 14.8% reported visiting a health clinic for a regular checkup within the last year. it is probable that saudis are not engaged in preventive health care and only seek medical care for illnesses. understanding the barriers to seeking first, our data are cross - sectional, and hence we can not assess causality. second, many of our behavioral data, such as diet and physical activity, are self - reported and subject to recall and social desirability biases. on the other hand, our study is based on a large sample size and used a standardized methodology for all its measures. despite these limitations, our study remains nationally representative and has the merit of providing accurate data due to our near - real - time data quality monitoring through the whole survey period. the physical and blood measurements allowed us to control recall bias regarding diagnosed diseases and to uncover respondents who were affected by these chronic diseases but undiagnosed. our findings from this study along with those we reported previously in gbd 2010 call for a national plan to prevent and control the burden of hypertension in ksa. indeed, the plan has to be comprehensive to include programs to improve health behaviors such as diet and physical activity. moreover, the plan should increase health care utilization for preventive services. as uncontrolled blood pressure leads to catastrophic events such as stroke, heart attack, and renal failure, physicians in ksa should be encouraged to monitor their patients to ensure that their blood pressure is controlled. saudis should be encouraged to monitor their own blood pressure and seek medical care to control their conditions. ksa has a young population with 81% of the population under the age of 40. gbd 2010 reported that life expectancy has increased from 72.5 and 76.3 years in 1990 to 75.0 and 79.9 years in 2010 for men and women, respectively. as life expectancy increases and the size of population is on the rise, the burden of hypertension and other chronic diseases, if uncontrolled, will pose major challenges to the health system. as the ksa moh has successfully tackled the burden of infectious diseases through prevention, the same effort should be applied to reduce the burden of chronic diseases. | current data on hypertension in the kingdom of saudi arabia are lacking. we conducted a national survey to inform decision - makers on the current magnitude of the epidemic. we measured systolic and diastolic blood pressure of 10,735 saudis aged 15 years or older and interviewed them through a national multistage survey. we used multivariate logistic regressions to describe sociodemographic characteristics and risk factors of hypertensive, borderline hypertensive, and undiagnosed hypertensive saudis. we found that 15.2% and 40.6% of saudis were hypertensive or borderline hypertensive, respectively. risk of hypertension increased among men, with age, obesity, diabetes, and hypercholesterolemia. 57.8% of hypertensive saudis were undiagnosed. these were more likely to be male, older, and diagnosed with diabetes. among participants diagnosed with hypertension, 78.9% reported taking medication for their condition. about 45% of participants on medication for hypertension had their blood pressure controlled. the prevalence of hypertension and borderline hypertension is very high in saudi arabia. moreover, control of hypertension is poor. with the majority of hypertensive saudis being unaware of their condition, a national plan is needed to increase utilization of freely available screening, preventive, and medical services. |
the association of spirochetes with oral diseases has been known for many years. amongst the oral spirochetes, four species have been cultured widely and reliably by many laboratories : treponema denticola, t. pectinovorum, t. socranskii, t. vincentii.1 cultivation of oral spirochetes is difficult because of the unique and complex nutritional requirements and strict anaerobic conditions needed for growth. despite recent developments of improved culture conditions and artificial media, many spirochetes have still resisted efforts for long - term in vitro cultivation.2 t. denticola is a gram - negative, anaerobic, helically coiled, highly motile bacterium. this bacterium is by far the best characterized of the currently cultivable oral spirochetes species. nevertheless, it is usually difficult to grow.3,4 more than a hundred years ago, miller5 reported the occurrence of spirochetes in endodontic infections suggested that these bacteria were involved in the pathogenesis of pulpitis and apical periodontitis. spirochetes have then been found in necrotic root canals using microbiological methods,6,7 dark field microscopy8,19 and transmission electron microscopy.10 together, these publications suggest that spirochetes were only occasionally found and when they occurred, made up a small proportion of the flora.11 the advent of molecular genetic technology for microbial detection and identification has sparked a better understanding of the roles that difficult - to - grow or not - yet - cultivated bacteria, including spirochetes, play in diverse polymicrobial infections, including periradicular diseases.12,13 the application of molecular diagnostic methods for identification of spirochetes has demonstrated that occurrence of these spiral bacteria in infection of endodontic origin has been overlooked by technical hurdles of cultivation techniques.14 about 90% of the samples from different types of endodontic infections have been demonstrated to harbor at least one species of spirochetes.1,15,16 the molecular methods most often used for microbial identification are the polymerase chain reaction (pcr) method and its variations.17 the pcr is a simple method for amplification of specific segments of dna or cdna reverse transcribed from rna.18 numerous variations in the standard pcr procedure have been developed. single pcr, nested pcr, reverse transcriptase pcr, multiplex pcr, and real - time pcr are examples of approaches commonly used to identify target species.17 most pcr assays are qualitative or can be semi - quantitative. one exception is the real - time pcr, which is characterized by the continuous measurement of products throughout the reaction.13 real - time pcr assays allow the quantification of individual target species and total bacteria in clinical samples.17 periradicular lesions are classified into five main groups : acute apical periodontitis, chronic apical periodontitis, condensing osteitis, acute apical abscess, and chronic apical abscess. teeth associated with significant symptoms such as pain or swelling are referred to as symptomatic (acute), whereas those with mild or no symptoms are identified as asymptomatic (chronic).19 symptomatic apical periodontitis is localized inflammation of the periodontal ligament in the apical region.20 a tooth with a symptomatic apical periodontitis will give a very painful response to biting pressure or percussion. this tooth may or may not respond to pulp vitality tests, and the radiograph of this tooth will generally exhibit widened periodontal ligament space but no periradicular radiolucency.21 symptomatic apical abscess is an inflammatory process in the periradicular tissues of teeth, accompanied by exudates formation within the lesion.20 a tooth with a symptomatic apical abscess will be very painful to biting pressure, percussion, and palpation. this tooth will not respond to any pulp vitality tests and will exhibit varying degrees of mobility, and the radiograph can exhibit anything from a widened periodontal ligament space to periradicular radiolucency. swelling will be present in the mucobuccal fold and facial tissues adjacent to the tooth.21 due to difficulties in isolating and identifying of oral treponemes, the present study was undertaken to determine the presence of t. denticola in microbiological samples taken from symptomatic endodontic cases using a sensitive molecular method, real - time pcr. specimens were selected from adult patients that had been referred for endodontic treatment to the department of endodontics, dental school of ataturk university (erzurum, turkey). medical histories revealed that all patients were in good general health, and had no important systemic diseases such as diabetes. patients that had received antibiotic therapy during the last 3 months were excluded from the study. the ages of the patients ranged from 18 to 45 years. approval to undertake the study was given by the ethical committee in research of the dental school of ataturk university and informed consent was obtained from all patients. the following clinical features of each tooth were recorded : the type of restoration, pain on occlusion, tenderness to percussion or palpation, swelling, and the presence of periapical radiolucency. cases with a periodontal pocket probing depth greater than 4 mm and teeth in which proper rubber dam isolation could not be achieved were excluded from the study. sixty single - rooted teeth, all of them having carious lesions and necrotic pulps were included in this study. of these, thirty teeth showed both spontaneous symptoms and tenderness to percussion and thickening of the periodontal ligament space on radiographic examination were diagnosed as symptomatic apical periodontitis.21 the other thirty teeth showed tenderness to percussion and palpation, swelling, mobility and radiographic evidence of periradicular bone loss were diagnosed as symptomatic apical abscesses.21 samples from cases of symptomatic apical periodontitis were obtained from the root canals using strict asepsis as described previously.3,22 briefly, teeth were cleansed with pumice, isolated with rubber dam, and the surrounding field 3% hydrogen peroxide and then decontaminated with 3% hydrogen peroxide and then decontaminated with a 2.5% sodium hypochlorite solution. the operative field, including the pulp chamber, was then swabbed with 2.5% sodium hypochlorite. samples were initially collected by means of a # 15 k - type file (maillefer, ballaigues, switzerland) with the handle cut off. the file was introduced to a level approximately 1 mm short of the tooth apex, based on diagnostic radiographs, and a discrete filing motion was applied. afterwards, two sequential paper points were placed to the same level and used to soak up the fluid in the canal. each paper point was retained in position 1 min the cut file and two paper points were then transferred to cryotubes containing 1ml of 5% dimethyl sulfoxide (dmso) in trypticase soy broth (tsb). samples from cases of symptomatic apical abscesses were obtained by aspiration of purulent exudates from the swollen mucosa over each abscess. after disinfection of the overlying mucosa with 2% chlorhexidine, a sterile disposable syringe was used to aspirate the pus, which was immediately injected into cryotubes containing 1 ml of 5% dimethyl sulfoxide (dmso) in trypticase soy broth (tsb).16 samples were then immediately frozen at 20c. the frozen samples in 2 ml collection tube with tsb - dmso were left at room temperature for 20 min. after the temperature of the samples had been adjusted to room temperature, they were vigorously vortexed, centrifuged at 8,000 x g for 5 min, then the supernatants were removed and the pellets were used23 for dna extraction by using a qiaamp dna mini - kit (qiagen, gmbh, germany). the protocol recommended by the kit manufacturer for dna extraction from the tissue samples was followed precisely. the set of the species - specific primers and the taqman probe of real - time pcr amplification for t. denticola were designed as described previously by yoshida.24 briefly, a fluorescent probe was used to monitor pcr product formation continuously. the oligonucleotide probe was labeled at the 5-end with a reporter dye (fam : 6-carboxyfluorescein) and at the 3-end with quencher dye (tamra : 6-carboxytetramethylrhodamin). the primer - probe set comprised a forward primer (5-agagcaagctctcccttaccgt-3) and a reverse primer (5-taagggcggcttgaaataatga-3) and a probe (5-fam - cagcgttcgttctgagccaggatcatamra-3). the ubiquitous primers that match almost all bacterial 16s rrna genes at the same position but not 18s rrna gene from eukaryotic cells were used as a positive control for the dna extraction and probable pcr inhibitors. a pair of ubiquitous primers was 53 gat tag ata ccc tgg tag tcc ac and ccc ggg aac gta ttc acc g (base positions were 796818 and 13811399, respectively). the amplicon size was 604 bp.4 the amplification and detection of dna with species - specific primers by real - time pcr were performed with the icycler iq multicolor real - time rcr detection system (bio - rad laboratories, inc. hercules, ca usa). for each real - time pcr, 20 l of a mixture that contained 1x quantitect probe pcr buffer compositing of tris - cl, kcl, (nh4)2so4, 8 mm mgcl2, ph 8.7, dntps (contains ultra pure quality datp, dgtp, dctp and dttp / dutp) (qiagen gmbh, germany) 0.7 m for each forward and reverse primers and 0.24 m for the dual labeled probe, 5 l of target dna from samples were used. totally, 25 l pcr amplification volume for each reaction was placed in each well of a 96-well microamp optical reaction plate and covered with optical - quality sealing tape (bio - rad laboratories, inc. the dna amplification condition was used as following ; 1 cycle of 50c for 2 min, 1 cycle of 95c for 15 min, followed by 50 cycles of 95c for 30 s and 58c for 90 s, and infinite hold for 11c. specimens were selected from adult patients that had been referred for endodontic treatment to the department of endodontics, dental school of ataturk university (erzurum, turkey). medical histories revealed that all patients were in good general health, and had no important systemic diseases such as diabetes. patients that had received antibiotic therapy during the last 3 months were excluded from the study. the ages of the patients ranged from 18 to 45 years. approval to undertake the study was given by the ethical committee in research of the dental school of ataturk university and informed consent was obtained from all patients. the following clinical features of each tooth were recorded : the type of restoration, pain on occlusion, tenderness to percussion or palpation, swelling, and the presence of periapical radiolucency. cases with a periodontal pocket probing depth greater than 4 mm and teeth in which proper rubber dam isolation could not be achieved were excluded from the study. sixty single - rooted teeth, all of them having carious lesions and necrotic pulps were included in this study. of these, thirty teeth showed both spontaneous symptoms and tenderness to percussion and thickening of the periodontal ligament space on radiographic examination were diagnosed as symptomatic apical periodontitis.21 the other thirty teeth showed tenderness to percussion and palpation, swelling, mobility and radiographic evidence of periradicular bone loss were diagnosed as symptomatic apical abscesses.21 samples from cases of symptomatic apical periodontitis were obtained from the root canals using strict asepsis as described previously.3,22 briefly, teeth were cleansed with pumice, isolated with rubber dam, and the surrounding field 3% hydrogen peroxide and then decontaminated with 3% hydrogen peroxide and then decontaminated with a 2.5% sodium hypochlorite solution. the operative field, including the pulp chamber, was then swabbed with 2.5% sodium hypochlorite. samples were initially collected by means of a # 15 k - type file (maillefer, ballaigues, switzerland) with the handle cut off. the file was introduced to a level approximately 1 mm short of the tooth apex, based on diagnostic radiographs, and a discrete filing motion was applied. afterwards, two sequential paper points were placed to the same level and used to soak up the fluid in the canal. each paper point was retained in position 1 min the cut file and two paper points were then transferred to cryotubes containing 1ml of 5% dimethyl sulfoxide (dmso) in trypticase soy broth (tsb). samples from cases of symptomatic apical abscesses were obtained by aspiration of purulent exudates from the swollen mucosa over each abscess. after disinfection of the overlying mucosa with 2% chlorhexidine, a sterile disposable syringe was used to aspirate the pus, which was immediately injected into cryotubes containing 1 ml of 5% dimethyl sulfoxide (dmso) in trypticase soy broth (tsb).16 samples were then immediately frozen at 20c. the frozen samples in 2 ml collection tube with tsb - dmso were left at room temperature for 20 min. after the temperature of the samples had been adjusted to room temperature, they were vigorously vortexed, centrifuged at 8,000 x g for 5 min, then the supernatants were removed and the pellets were used23 for dna extraction by using a qiaamp dna mini - kit (qiagen, gmbh, germany). the protocol recommended by the kit manufacturer for dna extraction from the tissue samples was followed precisely. the set of the species - specific primers and the taqman probe of real - time pcr amplification for t. denticola were designed as described previously by yoshida.24 briefly, a fluorescent probe was used to monitor pcr product formation continuously. the oligonucleotide probe was labeled at the 5-end with a reporter dye (fam : 6-carboxyfluorescein) and at the 3-end with quencher dye (tamra : 6-carboxytetramethylrhodamin). the primer - probe set comprised a forward primer (5-agagcaagctctcccttaccgt-3) and a reverse primer (5-taagggcggcttgaaataatga-3) and a probe (5-fam - cagcgttcgttctgagccaggatcatamra-3). the ubiquitous primers that match almost all bacterial 16s rrna genes at the same position but not 18s rrna gene from eukaryotic cells were used as a positive control for the dna extraction and probable pcr inhibitors. a pair of ubiquitous primers was 53 gat tag ata ccc tgg tag tcc ac and ccc ggg aac gta ttc acc g (base positions were 796818 and 13811399, respectively). the amplification and detection of dna with species - specific primers by real - time pcr were performed with the icycler iq multicolor real - time rcr detection system (bio - rad laboratories, inc. 20 l of a mixture that contained 1x quantitect probe pcr buffer compositing of tris - cl, kcl, (nh4)2so4, 8 mm mgcl2, ph 8.7, dntps (contains ultra pure quality datp, dgtp, dctp and dttp / dutp) (qiagen gmbh, germany) 0.7 m for each forward and reverse primers and 0.24 m for the dual labeled probe, 5 l of target dna from samples were used. totally, 25 l pcr amplification volume for each reaction was placed in each well of a 96-well microamp optical reaction plate and covered with optical - quality sealing tape (bio - rad laboratories, inc. hercules, ca usa). the dna amplification condition was used as following ; 1 cycle of 50c for 2 min, 1 cycle of 95c for 15 min, followed by 50 cycles of 95c for 30 s and 58c for 90 s, and infinite hold for 11c. all amplifications were obtained using ubiquitous primers, suggesting that there was not any pcr inhibitor in the dna samples used (figure 1). pcr amplifications with species - specific primers gave the similar results when they were repeated. 68c of melting temperature for the pcr product obtaining with species specific primers was used to establish positive results. also 58c of melting temperature was proved by amplification of dna from t. denticola used as positive control dna. in general, real - time pcr method enabled the detection of t. denticola in 43 of 60 symptomatic endodontic cases (71.6%). t. denticola was detected in 24 of 30 cases diagnosed as symptomatic apical abscesses (80%), and 19 of 30 cases diagnosed as symptomatic apical periodontitis (63.3%). the development of effective strategies for root canal therapy is dependent upon understanding the composition of the pathogenic flora of the root canal system. identification of the root canal isolates from previous studies has traditionally been performed using standard microbiological and biochemical techniques.25 data on microbial morphology provides few clues for the identification of most microorganisms, and physiological traits are often ambiguous.26,27 in addition, several microorganisms are difficult or even impossible to grow under laboratory conditions.26 these factors are especially true in the case of spirochetes.1,12 recent studies using sensitive molecular diagnostic methods have allowed detection of microorganisms that are difficult or even impossible to culture in infections elsewhere in the human body, including within the root canal system.28 pcr techniques have been increasingly used in investigations of the periodontal and root canal flora and are able to detect the presence of genomic dna of bacteria present in the root canal space with a high degree of sensitivity and specificity.29,30 the real - time pcr method used in this study was a powerful technique combining sample amplification and analysis in a single reaction tube.31 the advantages of real - time pcr are the rapidity of the assay, the ability to quantify and identify pcr products directly without the use of agarose gels, and the fact that contamination of the nucleic acids is limited because of avoidance of post - amplification manipulation.32 the polymicrobial nature of the endodontic microbiota suggests that bacteria are interacting with one another and such interaction can play an important role for both survival and virulence.33 in a mixed bacterial community, it is likely that t. denticola has its virulence enhanced or it can enhance the virulence of other species in the consortium.34 oral treponemes can cause abscesses when inoculated in experimental animals.35 these microorganisms are reported to possess an array of putative virulence traits that may be involved in the pathogenesis of endodontic abscesses by wreaking havoc on host tissues and/or by allowing the microorganism to evade host defence mechanisms.16 its virulence factors include the major surface protein, the chymotrypsin - like protease complex, other extracellular or membrane - associated proteolytic and hydrolytic enzymes, lipoligosaccharide, lipoproteins, phospholipases, and metabolic end products (acetate, lactate, ammonia, and hydrogen sulphisde).35 in order to identify microorganisms and study their characteristics and pathogenic potential, it is essential that accurate methods be used for sampling.11 contamination of the samples is a significant problem to be considered in clinical studies.4 in our study, we used sampling methods described previously3,16,22 to reduce the risk of contamination in cases with symptomatic apical periodontitis and symptomatic apical abscesses. in the present study, t. denticola was detected in 80% of cases diagnosed as symptomatic apical abscesses, and 63.3% of cases diagnosed as symptomatic apical periodontitis. the differences between prevalence values of t. denticola may probably have been because of different environmental conditions (e.g. oxygen tension, nutrient availability, bacterial interactions).11 siqueira found t. denticola in 52% of symptomatic cases by means of single pcr assay. siqueira,36 in another study, found this bacterium in 4% of samples from acute periradicular abscesses by means of checkerboard dna - dna hybridization. jung have not found this bacterium in periradicular lesions using pcr amplification with bacterial universal primers and subsequent dot - blot hybridization. the disagreements in findings between our study and the three studies above mentioned can be explained by the different detection limits of the methods used. the real - time pcr used in the present study is more sensitive both single pcr assay and dna - dna hybridization methods. our finding disagrees with the findings reported by ras and baumgartner.2 ras reported the presence of t. denticola in 80% of samples from acute apical periodontitis using nested pcr. baumgartner reported the finding of t. denticola in 34.5% of samples from abscesses / cellulitis using nested pcr. such discrepant findings may be caused by different molecular techniques employed or geological difference.38 some researchers,16,39,40 using molecular methods, found t. denticola in 79, 78, and 75% of cases diagnosed as symptomatic periradicular abscesses, respectively, supporting our results. our findings suggest that t. denticola can participate in the pathogenesis of symptomatic apical abscesses. further investigations using animal model are required to further evaluate the mechanism of t. denticola pathogenicity. pcr based molecular techniques, even though expensive are very convenient for detection of t. denticola in dental and dental related samples. | objectives : the aim of this study was to investigate the presence of treponema denticola in symptomatic apical periodontitis and in symptomatic apical abscesses by real - time polymerase chain reaction (pcr) method.methods:microbial samples were collected from 60 single - rooted teeth having carious lesions and necrotic pulps. for each tooth, clinical data including patient symptoms were recorded. teeth were categorized by diagnosis as having symptomatic apical periodontitis or symptomatic apical abscess. aseptic microbial samples were collected using paper points from 30 infected root canals and from aspirates of 30 abscesses. dna was extracted from the samples by using a qiaamp dna mini - kit and analyzed with real - time pcr.results:t. denticola was detected in 24 of 30 cases diagnosed as symptomatic apical abscesses (80%), and 19 of 30 cases diagnosed as symptomatic apical periodontitis (63.3%). in general t. denticola was found in 43 of 60 cases (71.6%).conclusions : our findings suggest that t. denticola can participate in the pathogenesis of symptomatic apical abscesses. |
during the last twenty years the european community has issued a series of directives and horizontal and vertical regulations, which seek to ensure the welfare of livestock animal species in all steps of production, from breeding to slaughter, including transportation. 1099/2009 (european commission, 2009) on the protection of animals at the time of killing, in force as of january 1 2013, is the current reference standard for those who work in slaughterhouses. the main novelty of the regulation, which from 8 december 2019 will completely repeal the council directive no. 93/119/eec (european commission, 1993), is represented by the transfer of full responsibility for the protection of animal welfare to business operators (bos), which must apply specific haccp plans, similar to those implemented to ensure food safety. in particular, operators of slaughterhouses will have to develop and implement risk - based standard operating procedures (sops), able to ensure that, during the killing and related operations, such as handling, lairaging, restraining, stunning and bleeding, the animals are spared any pain, distress and unnecessary suffering. these procedures should include clear objectives, responsibilities, modus operandi, measurable criteria, as well as monitoring and recording procedures. in particular, as regards the stunning, the bo has the obligation to establish a representative sample of animals to check that they do not show signs of consciousness and sensibility in the period between the end of the stunning process and death. to enable companies to comply with the provisions of the new legislation, the eu will identify indicators of animal welfare as regards killing and related operations that are easily monitored within the slaughterhouses, and critical limits which meet the eu standards. the assessment of animal welfare is generally carried out applying two types of parameters : structural measurements (engineering measures) and assessments made directly on animals (animal - based measures). the former evaluate the adequacy of facilities and equipment, the latter the response and/or the effect of the environment and/or management practices on animals. it is precisely toward the animal - based measures that the efsa, appointed by the european commission to draft guidelines to harmonise the procedures for checking and monitoring the welfare of the different species of livestock animals, is turning its attention to (european food safety authority, 2012). a system based on measurements carried out directly on animals has been successfully used for a long time on a voluntary basis by major u.s. restaurant chains for audit activities on animal welfare at the slaughterhouse, aiming at the selection of suppliers (grandin, 2012a). this system, developed by temple grandin for the american meat institute (ami), takes into account the following five numerically scored key - criteria with specific limits of acceptability and specific classes of judgment : i) percentage of fallen animals during the handling ; ii) percentage of animals moved with an electric prod ; iii) percentage of animals that vocalise during handling and stunning ; iv) percentage of animals stunned effectively at the first attempt ; v) percentage of animals that remain insensitive during bleeding (grandin, 2010, 2012b, 2013). the purpose of this investigation was to test the applicability of the above - mentioned system of assessment of animal welfare during handling and only - head electrical stunning at a slaughterhouse for heavy pigs intended for processing. this survey was conducted by 3 observers in the a slaughterhouse area of a pork meat - curing factory in the province of ancona (italy), where 120 - 130 pigs per week with average live weight of 170 kg are slaughtered. it has the following facilities : an unloading platform ; a lairage stall of 137 m with 9 pens ; a single - file chute with a pneumatic guillotine gateway for transfer to the slaughter room, consisting of a straight ramp with a spray - sprinkling device for humidifying the skin of pigs and a floating raised curved tunnel (cp-0110 ; mancini spa, amandola (ap), italy) ; a stun box with a pneumatic guillotine gateway and a pneumatic rotating side wall (cp-0110 ; mancini spa) ; a conveyor belt for the sticking (rire-0055 ; mancini spa) and bleeding (tapp, ndt-050 ; mancini spa). the different batches of pigs are usually conferred on saturday morning and slaughtered the following monday, after a 42 - 45 hour rest. plastic bags are generally used for their handling and electrical prods (robset color ; plast micron, modena, italy), when necessary. stunning is carried out by means of tongs (tl002 ; gozlin, modena, italy) connected to an electronic stunner (te002 ; gozlin) which provides 50 hz sinusoidal alternating current. two operators are involved in the pre - slaughter stages : one is responsible for animal handling and the other for restraining, stunning, sticking and bleeding. due to the small number of observers and to the structural characteristics of the slaughterhouse which made it impossible to follow the animals during handling at an adequate distance, it was necessary to carry out the monitoring in 3 days from different fixed observation points. the first and second observations, carried out on 121 pigs, involved the animal handling from the unloading platform to the lairage entrance, and the handling of the same pigs to the slaughter room through the single - file chute and the immobilisation step, respectively ; finally, the third, performed on 126 subjects, involved the stunning, sticking and bleeding stages. the limited space of the lairage area and the lack of passing lanes reserved for the operators did not allow the pen filling and emptying steps to be observed, nor the monitoring of recovered animals. the assessment of animal welfare during handling was carried out by recording the number of fallen / slipped and prodded animals in two different check lists. limited to the handling along the single - file chute, the number of vocalising pigs and the time the pigs waited in the chute were recorded on a specific check list. the number of vocalising pigs at the entrance and the number of fallen / slipped animals inside the stun box were recorded in two different check lists to assess the animal welfare during immobilisation. the waiting time inside the stun box before the application of tongs was also recorded. as reported by grandin (grandin, 2012b), the sudden loss of the upright position in which a part of the body other than the limbs touches the ground, was considered as a fall, an extended sound of both high amplitude and high frequency produced with an open mouth (squeal) exclusively determined by operators or equipment was recorded as a vocalisation, and the touching of the body with an electric prod (whether energised or not) was counted as an electrostimulation. the narrow space of the stunning - sticking - bleeding area and the short stun - to - stick interval did not allow all clinical signs of effective stunning immediately after the release from the stun box to be observed. therefore, an observer positioned on the stun - sticking platform recorded the basic parameters of only - head electrical stunning, such as correct electrode placement, voltage and amperage, exposure time and stun - to - stick interval. other parameters detectable at a distance, such as vocalisations due to hot wanding (use of prematurely energised tongs), onset of clonic seizures (pdalage), and further stunning attempts before sticking. only the electrodes applied symmetrically on the temporal region or on either the hollow behind the ears, or as well as asymmetrically at the top and the bottom of the head were considered correctly placed (grandin, 2012b). another observer positioned on the bleeding platform recorded the number of sticked pigs during tonic seizure and the number of pigs that immediately after sticking and during the first 2/3 of bleeding showed the following signs considered as indicators of effective stunning and consciousness / sensibility recovery by efsa expert (efsa, 2004) : pdalage, fixed gaze, mydriasis, eyeball rotation, pupil, cornea and eyelid reflexes, nystagmus, pain reflex determined by picking the snout (nose pick), rhythmic breathing, vocalisation and attempts to raise. finally, a third observer positioned at the end of the bleeding platform, recorded the time interval between sticking and immersion into the scalding tank, and some signs of certain death, such as complete muscle relaxation and absence of breathing. the noise produced by the equipment in the slaughter room and the vibrations of the conveyor belt did not allow the absence of heartbeat to be verified, while horizontal bleeding made the lingual ptosis difficult to be detected. the most frequent event observed during handling in the unloading area was the use of electric prod, observed in 9.09% of pigs examined, followed by the slip (6.61%) and the fall (2.48%). the monitoring of the handling in the single - file chute, during which there was no fall / slip, showed a high percentage of prodded (90.91%) and vocalising (52.89%) pigs and an average waiting time of 107 (60). during immobilisation, whose average time was 26 (19), noticeable events were not recorded, except for a pig which vocalised upon entering the stun box (0.83%). as regards the monitoring of stunning a correct positioning of the tongs particularly, in most animals the electrodes were placed symmetrically behind the mandible s corner (47.67%), on either the hollow behind the ears (25.39%) or on the temporal region (20.63%). in a limited number of subjects the tongs were placed symmetrically on the dorsal part of the neck (3.97%), on the cheeks close to the snout (1.59%) and on the ears (0.79%). in two pigs where electrodes were placed on the cheeks the basic current parameters showed values between 192 and 220v (average 210.185.13), and between 0.67 and 1.73a (average 1.170.20). most animals (94.44%) were exposed to a current exceeding 200v, 22.31% received a current equal to or greater than 1.30a, the minimum level required by council regulation (ec) no. 1099/2009 (european commission, 2009), 56.20% between 1.00 and 1.29a, and the remaining 21.49% less than 1.00a. the exposure time was between 4 and 8. vocalisations due to hot wanding were detected in 14.28% of pigs examined. the stun - to - stick interval was between 6 and 26. most pigs (72.22%) were sticked within 15 from stunning, 26.20% within 18 and only two animals (1.58%) after more than 20. from the notes reported by the observer in the relevant check list it was possible to relate the prolongation of stun - to - stick interval over 15 to the need of manual release of the conveyor belt by the stunning operator. pdalage was observed in 44.44% and 98.41% of the animals examined before and after sticking, respectively. immediately after sticking, in most animals (88.09%) the eyes were closed. fifteen pigs showed open eye with rotated eyeball (10.31%) or fixed gaze and mydriasis (1.59%). the analysis of the crosscheck lists of the first and second observer showed that these two animals were sticked after more than 20 from the end of the tong application. soon, all pigs with closed eyelids opened their eyes, allowing the eyeball to be observed, which appeared rotated in 86.51%. as regards the clinical signs of consciousness and sensibility, 84.13% of pigs showed both corneal and blink reflex, associated in four subjects (3.17%) to pupil reflex and nystagmus, 80.16% showed rhythmic breathing, 30.16% reaction to nose pick, 10.32% vocalisation and 4.76% attempts to raise. in all subjects such signs appeared after variable times during the first 2/3 of bleeding, with the exception of two pigs in which the signs were observed immediately after sticking. despite the observation of a large number of animals with one or more signs of consciousness and sensibility, a second stunning was performed only on those pigs which attempted to raise immediately after sticking (1.59%) or during bleeding (3.17%). it is noteworthy that two of four pigs stunned for the second time during bleeding did not show pdalage and eyeball rotation, while pupil reflex and nystagmus were observed in all of them. finally, at the end of bleeding, whose average duration was 254 (32), all animals showed complete muscle relaxation and absence of breathing. in all pre - slaughter stages, frequencies of occurrence of one or more indicators of poor animal welfare above the acceptable limits defined for swine by grandin (grandin, 2012b) were recorded, with the exception of the immobilisation step. particularly, as regards the movement in the unloading area, the fall was the only parameter showing a slightly higher frequency than the critical limit proposed by the same author. almost all falls and slips, the latter considered as a secondary criterion for which there is no specific acceptability limit, occurred near the entrance of the lairage ; here some structural weaknesses, such as the limited width of the door, which requires pigs to form a single line, the smooth floor and the light / shadow contrast resulting in poor indoor lighting especially on sunny days, have been identified. therefore, it should be necessary to restructure the unloading area in order to allow the animals to walk side by side, to maintain floors in such a way to minimise the risk of slipping, falling and injuring, and to remove distractions resulting from the light / shadow contrast. 93/119/eec (european commission, 2009, 1993). during the handling in single - file chute, the percentage of prodded animals and those vocalising were the only two criteria that exceeded the limits of acceptability, grading both as serious problem (grandin, 2012b). the lack of fallen / slipped animals testifies the structural suitability of the single - file chute, designed and built for the exclusive handling of heavy pigs. whereas, the high variability of waiting times in the chute due to irregular animal flow to the slaughter room seems to be the main cause of the large number of prodded and vocalising pigs. in fact, the discontinuity of animal handling operations from the lairage boxes to the stun box led some groups of pigs to long waiting times in the single - file chute, improperly used as a sort of waiting pen. the animals repeatedly moved forward and backward, and overlapped, so most of them refused to enter at the opening of the stun box s door, forcing the operator to frequently use the electric prod. it must be emphasised that a steady supply of animals from the holding pens to the slaughter room is explicitly required by council regulation (ec) no. 1099/2009 (european commission, 2009). handling small groups of animals, slowing the speed of the slaughter line or, alternatively, increasing the personnel involved in the pre - slaughter stages are suggested as corrective measures to raise the level of animal welfare during this step. these measures would also reduce the waiting time in the stun box, too long for some subjects. indeed, as noted in the check list used for monitoring the immobilisation step, all the delays in the application of electrodes resulted from the need of manual release of the conveyor belt by the stunning operator due to the irregular pig flow. as regards stunning, animal - based measures indirectly used to assess the effectiveness of stunning, such as the percentage of vocalisations due to hot wanding and the percentage of correct positioning of the tongs, were considerably higher and lower, respectively, than the limits of acceptability, scoring both as serious problem (grandin, 2012b). the frequent wrong placement of the electrodes seems to be the main factor affecting the high percentage of vocalisations due to hot wanding and the percentage of pigs showing signs of consciousness and/or sensitivity recovery during bleeding ; according to this criterion, the presence of only one sensitive subject out of 100 animals checked is sufficient to score the slaughterhouse as not acceptable (grandin, 2012b). in fact, the positioning of the electrodes in different areas from those considered ideal implies a remarkable increase of the impedance ; this increase hinders the current flow and may delay or prevent the achievement of minimum amperage necessary to induce an effective and enduring stunning, leading to possible painful electric shocks and consciousness and/or sensitivity recovery before the death (efsa, 2004). even a malfunctioning of the stunning device may have contributed to the high percentage of conscious and/or sensitive pigs during bleeding. actually, the voltage was set to 245v but it never exceeded 220v, well below the recommended minimum value of 250v (anil and mckinstry, 1998 ; efsa, 2004 ; grandin, 2012b). therefore, effective corrective measures aimed at raising the level of animal welfare at this critical pre - slaughter stage may be represented by increasing the number of operators in the stun - to - stick area or, alternatively, by slowing down the speed of the slaughter line, in order to enable the stunner to pay more attention to the positioning of tongs, as well as by regularly calibrating the stunning device. finally, it must be emphasised that the efsa has recently urged a public panel discussion among all stakeholders to better define the indicators of effective stunning and of consciousness and/or sensitivity recovery. the observed frequencies suggest that in the electrical stunning of pigs by means of tongs followed by horizontal bleeding the pdalage, easily detectable even at a distance, is the best indicator of effective stunning, and that the corneal and eyelid reflexes as well as rhythmic breathing are the best indicators of sensitivity recovery after sticking. the results obtained at the slaughterhouse under investigation demonstrate the applicability and especially the usefulness of the animal welfare assessment system developed by grandin for ami. besides allowing the steps at greater risk to be identified, the animal - based measures allowed the causes of non - compliance with animal welfare to be determined and the corrective measures to be suggested. the main advantage of this system is represented by the small number of criteria considered, which makes the monitoring of pre - slaughter stages, particularly handling, easier. some concern arises about the acceptability limits defined by the same author on the basis of standards guaranteed by us industrial slaughterhouses which may be too restrictive for our production realities. in fact, especially light pigs are slaughtered in these establishments, where also the use of automated head - to - body electrical stunning is widespread. | the council regulation (ec) no. 1099/2009 requires slaughterhouse managers to implement specific standard operating procedures for all pre - slaughter stages considered at risk, aimed at achieving adequate levels of animal welfare. this survey was aimed at testing the applicability to an abattoir for heavy pigs of an assessment system of animal welfare through animal - based measures. in the monitoring of handling operations, the number of animals fallen / slipped and prodded, and that of vocalising pigs were recorded. in the monitoring of the immobilisation stage, carried out on the same pigs, vocalisations were recorded at the entrance to the box and falls / slips occurring inside it. animal welfare assessment during the stunning - sticking - bleeding steps, was carried out by recording the head - only electrical stunning basic parameters set by legislation, vocalisations resulting from hot wanding, and clinical signs of consciousness, sensibility and certain death. except for immobilisation, the percentage of occurrence of these events above acceptability limits was detected in all other pre - slaughter steps. the most critical stages were : handling in the unloading area and along the single - file chute, stunning and especially bleeding, where 84.13% of animals showed one or more signs of consciousness and/or sensibility recovery. wrong placement of electrodes observed in 53.98% of the animals, insufficient voltage and low amperage may explain why a high percentage of pigs recovered consciousness and/or sensibility before death. some simple restructuring of unloading area, slowdown of slaughter line speed, increase of personnel involved in pre - slaughter management and regular calibration of the electrical stunning device could be effectively corrective measures aimed at raising the animal welfare level at the slaughterhouse under study. |
endovascular coil embolization has emerged as the gold standard for treatment of intracranial aneurysms.1 2 the current standard of optimal endovascular treatment of intracranial aneurysms is based on achieving angiographic occlusion and high packing density (pd) in order to achieve a durable exclusion of the aneurysmal sac from the cerebral circulation.3 4 the goal of endovascular coil embolization is to reduce the flow of the aneurysm, which varies with coil properties such as permeability and therefore results in thrombus formation, exclusion from circulation, and a decreased risk of rupture.5 studies have shown that increasing pd resulted in lower intra - aneurysmal velocities and less fluid flow across the aneurysm.68 although an inverse relationship between pd and coil permeability has been observed, the pd of the coil mass may not be the only factor influencing its permeability. previous work on modeling the interactions between the coils within an aneurysm with the hemodynamic stress suggested that a coil mass at a given pd with low permeability may accelerate intra - aneurysmal thrombosis due to the drastic reduction in intra - aneurysmal velocities and increased residence times.5 a possible explanation for the variation in permeability at a given pd may be the differences in spatial distribution of the coil mass within the aneurysm.9 the goal of this study is to investigate the effects of both coil pd and spatial uniformity on aneurysm permeability. we have devised a repeatable in vitro assessment tool to objectively measure the coil uniformity in addition to the standard benchmark of pd. patient - specific a1 silicone aneurysms (5 mm dome, 3 mm neck, 2 mm parent vessel diameter), shown in figure 1, were prepared using a previously described technique.10 we incorporated a 1.5 mm channel emanating from the aneurysm dome for permeability assessment (blue arrow in figure 1). prior to aneurysm coiling, the aneurysm volume was calculated by measuring the volume of water needed to fill the aneurysm sac. silicone aneurysm models were packed by using guglielmi detachable coils (gdc, n=8) or target coils (n=8) (stryker neurovascular, fremont, california, usa). using an excelsior sl10 (stryker neurovascular), the gdc group was framed with a 59 mm gdc 360 detachable coil and finished using only gdc detachable coils. in the target group, aneurysms were coiled using the standard target 515 mm framing coils and were filled and finished with target detachable and target nano coils, respectively. the actual type, size, and number of coils used for both groups were determined by the operator 's preference. the coiling procedure was terminated after achieving the procedural end points namely, complete aneurysm obliteration, catheter dislodgement from the aneurysm, or herniation of coil loops into the parent artery.11 the number, size, and type of coils were recorded for each case and this information was used to calculate the pd of the aneurysm using angiocalc (http://www.angiocalc.com). the pd was determined by dividing the coil volume by the total volume of the aneurysm. coil permeability was obtained by evaluating the ability of fluorescent microspheres to pass through the coil mass. neutrally buoyant fluorescent microspheres of 1 m that approximate the size of platelets were used in this study. spheres of this size and specific gravity are often used for hemodynamic measurements in vitro.12 13 calibration curves were established to convert the recorded fluorescent intensities into microsphere concentration.14 the fluorescent microsphere solution was prepared by mixing 1 m (510 microspheres / ml) fluorescent microspheres (invitrogen flurospheres, eugene, oregon, usa) in a mixture of 0.15 m nacl, 0.05% tween 20, and 0.02% thimerosal. the coiled aneurysm was connected to the flow loop filled with the fluorescent microsphere solution. the channel emanating from the aneurysm dome was open to allow the microspheres to pass through for collection. the fluorescent intensity / concentration of the collected sample was recorded and normalized against the intensity / concentration of the stock solution. aneurysms were embedded in a low viscosity epoxy embedding medium, spurr 's resin (spi supplies / structure probe, west chester, pennsylvania, usa). coiled aneurysms were infiltrated with the resin and placed in low pressure to remove air trapped in the model. one neck and two dome sections were obtained from each aneurysm model using a low speed saw (buehler, lake bluff, illinois, usa) for a total of three sections. the two dome sections were obtained from two vertical cuts perpendicular to the neck of the aneurysm on either side of the center line. mounted aneurysm sections were imaged using a flat - bed scanner (canoscan 9950f ; lake success, new york, usa) and saved for image analysis. scanned images of aneurysm sections (figure 2a shows a dome section) were imported into matlab (mathworks, natick, massachusetts, usa), converted to a binary image (figure 2b), and custom fit with an elliptical mask that was truncated at the neck section (figure 2c) to provide a binary coil segmented image, c (i, j). the mask, m (i, j), is shown in figure 2d. the surface area fraction (saf) was calculated as shown in equation 1:1 image processing of aneurysm 6 (target) including (a) the scanned image, (b) the binary image, (c) the binary image with elliptical mask adjusted to account for the neck section, and (d) the mask. spatial uniformity of binary coil mass segmented images (eg, figure 2c) was determined using the fractal - based heterogeneity measure, lacunarity, represented as. values of approaching 0 imply spatially uniform distribution of coil mass with minimal gaps. lacunarity was determined using the fraclac plugin (v.2013april.b6, http://rsbweb.nih.gov/ij/plugins/fraclac/) for imagej (v.1.44p, national institutes of health). a sliding - box technique with a minimum region of interest of 55 pixels was translated by one pixel along i and j directions to determine lacunarity. while various metrics could be used for assessing spatial uniformity, lacunarity was chosen as it is a well - established image analysis metric1519 that has been applied in numerous studies including application in neuroimaging.20 further, the ready availability of this analysis tool would permit researchers to conduct similar investigations. the collected data were statistically analyzed using sas v.9.3 (sas institute, cary, north carolina, usa). statistical tests were conducted to assess whether coil mass permeability, packing, and uniformity measures differed with coil type (gdc vs target). it should be noted that the term coil type used in this paper refers to all the variations between the gdc and the target coiling systems, including but not limited to length of the framing coil, coil stiffness and configuration, and design of the coil / delivery wire junction. multivariate linear regression models were used to predict coil mass permeability as the outcome, with packing and uniformity measures as predictor variables. if the predictor variables exhibited statistically significant correlation, then partial least squares regression using principal components (pc - plsr) was used for statistical modeling with leave - one - out cross validation for extraction of factors. a linear regression model with coil mass permeability as the dependent variable and pd as the independent variable was used for comparative assessment. patient - specific a1 silicone aneurysms (5 mm dome, 3 mm neck, 2 mm parent vessel diameter), shown in figure 1, were prepared using a previously described technique.10 we incorporated a 1.5 mm channel emanating from the aneurysm dome for permeability assessment (blue arrow in figure 1). prior to aneurysm coiling, the aneurysm volume was calculated by measuring the volume of water needed to fill the aneurysm sac. silicone aneurysm models were packed by using guglielmi detachable coils (gdc, n=8) or target coils (n=8) (stryker neurovascular, fremont, california, usa). using an excelsior sl10 (stryker neurovascular), the gdc group was framed with a 59 mm gdc 360 detachable coil and finished using only gdc detachable coils. in the target group, aneurysms were coiled using the standard target 515 mm framing coils and were filled and finished with target detachable and target nano coils, respectively. the actual type, size, and number of coils used for both groups were determined by the operator 's preference. the coiling procedure was terminated after achieving the procedural end points namely, complete aneurysm obliteration, catheter dislodgement from the aneurysm, or herniation of coil loops into the parent artery.11 the number, size, and type of coils were recorded for each case and this information was used to calculate the pd of the aneurysm using angiocalc (http://www.angiocalc.com). the pd was determined by dividing the coil volume by the total volume of the aneurysm. coil permeability was obtained by evaluating the ability of fluorescent microspheres to pass through the coil mass. neutrally buoyant fluorescent microspheres of 1 m that approximate the size of platelets were used in this study. spheres of this size and specific gravity are often used for hemodynamic measurements in vitro.12 13 calibration curves were established to convert the recorded fluorescent intensities into microsphere concentration.14 the fluorescent microsphere solution was prepared by mixing 1 m (510 microspheres / ml) fluorescent microspheres (invitrogen flurospheres, eugene, oregon, usa) in a mixture of 0.15 m nacl, 0.05% tween 20, and 0.02% thimerosal. the coiled aneurysm was connected to the flow loop filled with the fluorescent microsphere solution. the channel emanating from the aneurysm dome was open to allow the microspheres to pass through for collection. the fluorescent intensity / concentration of the collected sample was recorded and normalized against the intensity / concentration of the stock solution. aneurysms were embedded in a low viscosity epoxy embedding medium, spurr 's resin (spi supplies / structure probe, west chester, pennsylvania, usa). coiled aneurysms were infiltrated with the resin and placed in low pressure to remove air trapped in the model. one neck and two dome sections were obtained from each aneurysm model using a low speed saw (buehler, lake bluff, illinois, usa) for a total of three sections. the two dome sections were obtained from two vertical cuts perpendicular to the neck of the aneurysm on either side of the center line. mounted aneurysm sections were imaged using a flat - bed scanner (canoscan 9950f ; lake success, new york, usa) and saved for image analysis. scanned images of aneurysm sections (figure 2a shows a dome section) were imported into matlab (mathworks, natick, massachusetts, usa), converted to a binary image (figure 2b), and custom fit with an elliptical mask that was truncated at the neck section (figure 2c) to provide a binary coil segmented image, c (i, j). the mask, m (i, j), is shown in figure 2d. the surface area fraction (saf) was calculated as shown in equation 1:1 image processing of aneurysm 6 (target) including (a) the scanned image, (b) the binary image, (c) the binary image with elliptical mask adjusted to account for the neck section, and (d) the mask. spatial uniformity of binary coil mass segmented images (eg, figure 2c) was determined using the fractal - based heterogeneity measure, lacunarity, represented as. values of approaching 0 imply spatially uniform distribution of coil mass with minimal gaps. lacunarity was determined using the fraclac plugin (v.2013april.b6, http://rsbweb.nih.gov/ij/plugins/fraclac/) for imagej (v.1.44p, national institutes of health). a sliding - box technique with a minimum region of interest of 55 pixels was translated by one pixel along i and j directions to determine lacunarity. while various metrics could be used for assessing spatial uniformity, lacunarity was chosen as it is a well - established image analysis metric1519 that has been applied in numerous studies including application in neuroimaging.20 further, the ready availability of this analysis tool would permit researchers to conduct similar investigations. the collected data were statistically analyzed using sas v.9.3 (sas institute, cary, north carolina, usa). statistical tests were conducted to assess whether coil mass permeability, packing, and uniformity measures differed with coil type (gdc vs target). it should be noted that the term coil type used in this paper refers to all the variations between the gdc and the target coiling systems, including but not limited to length of the framing coil, coil stiffness and configuration, and design of the coil / delivery wire junction. multivariate linear regression models were used to predict coil mass permeability as the outcome, with packing and uniformity measures as predictor variables. if the predictor variables exhibited statistically significant correlation, then partial least squares regression using principal components (pc - plsr) was used for statistical modeling with leave - one - out cross validation for extraction of factors. a linear regression model with coil mass permeability as the dependent variable and pd as the independent variable was used for comparative assessment. all aneurysms were coiled to a pd of at least 27%. during aneurysm sectioning, five of the 48 sections (3 neck and 2 dome) were damaged and hence the remaining 43 (89.5%) sections (13 neck and 30 dome) were included in the analysis. for the two dome sections from the same aneurysm, the saf and lacunarity measures were not statistically different (p>0.19, paired t test) and hence were averaged. where applicable, subscripts n and d are used to denote the neck and dome sections, respectively. summary statistics for all measured parameters are provided in table 1. despite relatively high pd, the paired t test indicated that safn and safd were statistically different (p0.18). summary statistics for measured parameters table 2 summarizes the pearson correlation coefficients between the packing and uniformity measures. pearson correlation coefficient correlation is significant (two - tailed test) at the 0.05 level. hence, statistical models using pc - plsr were used when two or more predictors were included in the model and linear regression was used when only one predictor was included in the model. five statistical regression models were considered with coil mass permeability (p) as the outcome variable (equation 2). model a assumes that the coil mass permeability is influenced by pd alone and was obtained by linear regression. since the permeability was statistically different between gdc and target coils, model b improves on model a by accounting for the differing coil types (ct). model c considers all variables studied as predictors in the regression model, model d considered the predictors from model b and the packing and uniformity measures from the dome section, and model e considered the predictors from model b and the packing and uniformity measures from the neck section. thus, models d and e address the question whether the permeability is better predicted when packing and uniformity measures from either the dome or the neck section are included with the ct and the pd. for all five statistical models the residuals were analyzed and found to satisfy the normality requirement (p>0.07, shapiro - wilks test). wold 's variable importance for projection criterion, in conjunction with the scaled and centered parameter estimates, was used to assess whether any of the predictor variables in each pc - plsr model needed to be excluded.2 linear regression (model a) indicated that the pd was a statistically significant predictor (p=0.005), confirming that the pd plays an important role in coil mass permeability. for each pls - pcr regression (models b e), the magnitudes of the model effects loadings or weights are summarized in table 3. for each model, the similar magnitudes of the predictors within that model indicate that all predictors within each model are equally important. the regression parameter estimates and the percentage variation in coil mass permeability accounted for by the models are summarized in table 4. for each pls - pcr regression (models b e), analysis of variance indicated that the slope, which represents the transformed predictor along the principal component, was statistically significant and different from zero (p0.07, shapiro - wilks test). wold 's variable importance for projection criterion, in conjunction with the scaled and centered parameter estimates, was used to assess whether any of the predictor variables in each pc - plsr model needed to be excluded.2 linear regression (model a) indicated that the pd was a statistically significant predictor (p=0.005), confirming that the pd plays an important role in coil mass permeability. for each pls - pcr regression (models b e), the magnitudes of the model effects loadings or weights are summarized in table 3. for each model, the similar magnitudes of the predictors within that model indicate that all predictors within each model are equally important. the regression parameter estimates and the percentage variation in coil mass permeability accounted for by the models are summarized in table 4. for each pls - pcr regression (models b e), analysis of variance indicated that the slope, which represents the transformed predictor along the principal component, was statistically significant and different from zero (p<0.0001). the negative parameter estimates for the packing measures (pd, safd and safn) indicate that denser packing of the aneurysm will reduce coil mass permeability, which is in agreement with current knowledge. the positive parameter estimates for lacunarity (n and d) indicates that reducing gaps or improving spatial uniformity will decrease coil mass permeability. the linear regression model with pd as predictor (model a) accounted for only 45% of the variation (r=0.449) in the measured permeability. addition of coil type (ct) in model b better accounted for the variation in coil mass permeability. symbol, indicating that the estimate is positive for the gdc coil and negative for the target coil. this implies that, when the aneurysms are coiled to the same pd, target coils reduce permeability compared with gdc coils. parameter estimates for the five statistical models for coil type (ct) parameter estimate denotes that ct is positive for gdc coil and is negative for the target coil. saf, surface area fraction. of the three statistical models that considered measurements from the dome and neck sections, model d incorporating measurements from the dome section (safd and d) along with pd and this implies that the dome of the aneurysm needs to be densely and uniformly packed for reducing permeability. interestingly, the percentage variation in permeability accounted for by model e, which considered measurements from the neck section (safn and n) along with pd and ct, was not better than model b which included pd and ct. plots of the measured versus the predicted permeability for four of the models (b e) are shown in figure 3 to aid in visual comparison. model a was not included in the plot for clarity and because the coil type used for embolization is always known. the solid lines represent the linear fits to the data and are color coded to match the symbols. among the four models, only model d exhibited a slope with 95% ci spanning one and an intercept with 95% ci spanning zero, indicating good agreement between measured and predicted permeability. slope and intercept values lcl, lower confidence limit ; ucl, upper confidence limit. the solid lines are linear fit to the data and are color coded to match the symbols. the goal of endovascular embolization is to prevent aneurysm growth and rupture by decreasing the intra - aneurysmal blood flow.5 11 using a patient - specific aneurysm model, the effect of pd, coil uniformity, and neck coverage on aneurysm permeability was observed. although previous literature indicated that higher pds result in lower intra - aneurysmal velocities and higher occurrences of stagnation,7 the study demonstrated that the permeability was influenced equally by uniformity and pd, as observed by similar loading factors or weights in table 3. this implies that pd alone may not necessarily be an accurate predictor of aneurysm exclusion from coiling. in previous studies, fluorescent microspheres were used to estimate regional perfusion and blood flow.21 22 the concept was extended to the investigation of aneurysm permeability in this study. the size of the microspheres (1 m) used in this study was smaller than the pore size of the coiled aneurysm23 and of similar size to platelets (3 m). therefore, the microspheres did not disrupt the coil mass or block the flow through the aneurysm. although the described technique was useful for studying coil mass permeability, intra - aneurysmal thrombosis in response to the coil mass was not modeled in this in vitro experiment. importantly, our study to measure coil mass permeability required an outlet, and therefore a pressure drop, across the aneurysm dome. the permeability measurements were therefore obtained in an idealized worst - case model system that is not representative of true intra - aneurysmal flow conditions. however, this design allows us to reproducibly study coil mass resistance to flow and to study the coil mass characteristics that are involved in reducing permeability. the coil mass within the aneurysm acts as a porous medium, with voids in the coil mass creating channels for fluid travel. with a higher coil pd, the tortuosity or weighted ratio of the length a fluid must travel to move across the media to the length of the media increases. because the porosity of a coiled aneurysm is imperfect, tortuosity can not be assumed to be consistent throughout the aneurysm, supporting the idea that pd alone is not adequate for predicting treatment efficiency. when local porosity and tortuosity within an aneurysm (ie, of individual volumetric nodes) are considered, it becomes clear that the uniformity of the coil mass distribution will have an effect on the range of local porosities observed. thus, in order to maximize tortuosity and maximize residence times as a result the porosity range should be minimized and pd maximized. the range of local porosities can be minimized by taking care to ensure uniform distribution of the coil within the aneurysm is achieved. clinical implications of this study may include achieving a higher pd, and coil mass uniformity may be inversely related to the rate of aneurysm recurrence or coil compaction. a higher pd and uniform coil construct reduce aneurysm permeability, resulting in rapid exclusion of the aneurysm from the circulation. in this study a single operator coiled all the aneurysms, and thus dependence of homogeneity of the coil mass on the operator or technique can not be assessed. however, two different coil types were used and we found that, compared with gdc coils, target coils reduced coil mass permeability, primarily by improving packing measures (pd and safd). the improved packing measures observed in the target group may be expected because of the use of the longer framing coil, which is a limitation of this study. since there is an upper bound to pd before coil herniation into the parent artery or catheter dislodgement occurs, our study indicates that designing coils to improve spatial distribution uniformity is a potential avenue for reducing aneurysm permeability. this study presents a bench - top method to rigorously study coil uniformity and its impact on permeability. the selected aneurysm dimensions were based on our clinical database, which shows a mean diameter for coiled aneurysms of approximately 5 mm. this relatively small diameter is probably the result of numerous small ruptured aneurysms that are coiled as well as a shift for larger aneurysms to be treated with flow diverters. future work should consider larger aneurysm models as well as other commercially available coils being specifically designed for large aneurysms.24 also under development are methods to reduce coil mass artifacts on 3d imaging.25 26 these techniques may ultimately lead to assessment of coil mass distribution using minimally invasive or non - invasive imaging modalities. however, until these developments are realized, bench - top experimentation can serve to provide feedback into the coil development process to produce coil technology that considers spatial distribution as a design input. the results of this study reinforce the prior knowledge that pd influences coil mass permeability, with higher pd resulting in lower permeability. importantly, the study showed that achieving spatially uniform distribution of coil mass with minimal gaps in the aneurysm dome is equally important in reducing coil mass permeability. a model incorporating pd, saf of the dome section, and a spatial heterogeneity measure (lacunarity) of the dome section predicted coil mass permeability better than a model with pd alone. | backgroundrates of durable aneurysm occlusion following coil embolization vary widely, and a better understanding of coil mass mechanics is desired. the goal of this study is to evaluate the impact of packing density and coil uniformity on aneurysm permeability.methodsaneurysm models were coiled using either guglielmi detachable coils or target coils. the permeability was assessed by taking the ratio of microspheres passing through the coil mass to those in the working fluid. aneurysms containing coil masses were sectioned for image analysis to determine surface area fraction and coil uniformity.resultsall aneurysms were coiled to a packing density of at least 27%. packing density, surface area fraction of the dome and neck, and uniformity of the dome were significantly correlated (p<0.05). hence, multivariate principal components - based partial least squares regression models were used to predict permeability. similar loading vectors were obtained for packing and uniformity measures. coil mass permeability was modeled better with the inclusion of packing and uniformity measures of the dome (r2=0.73) than with packing density alone (r2=0.45). the analysis indicates the importance of including a uniformity measure for coil distribution in the dome along with packing measures.conclusionsa densely packed aneurysm with a high degree of coil mass uniformity will reduce permeability. |
a 25-yr - old relatively healthy male (weight 62 kg, height 178 cm) visited our pain clinic complaining of posterior neck pain and mild limitation of motion for a couple of days. neck pain was initially of a waxing and waning nature and the numerical rating pain scale (nrs) was 5 - 6/10. physical examination revealed no lymphadenopathy but reduced range of motion and moderate tenderness over the posterior neck area. under the initial impression that the problem was myofascial neck pain syndrome, a trigger point injection with 0.5% mepivacaine 6 ml and dexamethasone 2 mg was performed around the splenius capitis and splenius cervicis muscle area. additional treatments, including a soft cervical collar, non - steroidal anti - inflammatory drugs (nsaids), and muscle relaxants were prescribed for pain control. several days later, the patient revisited our clinic with the complaints of worsening neck pain (nrs 7 - 8/10) and neck stiffness, combined with dysphagia and odynophagia symptoms. he described it as severe neck pain extending into the deep neck and radiating upward to the occiput and vertex of the head. he also complained of difficulty in opening his jaw and swallowing due to throat pain. on physical examination, there was a severely limited range of all neck motion in flexion, extension and rotation due to pain. the laboratory tests showed a mild elevation of white blood cell count at 10.1 10/l and an increase of the erythrocyte sedimentation rate (25 mm / h) and c - reactive protein (3.11 mg / dl) accompanied by low grade fever (37.3). an imaging study with a plain radiograph of the cervical spine showed a large area of prevertebral soft tissue swelling from c1 to c5 and focal calcifications anterior to the c1 (fig. 1). given the clinical presentation and plain radiographic finding, a cervical mri was performed with contrast medium. a sagittal t2 weighted mr image showed a high signal retropharyngeal effusion with acute inferior margin extending from the skull base to the inferior border of c5 (fig., the patient was referred to a radiologist and otolaryngologist to investigate a further definitive diagnosis. a cervical ct was performed with contrast medium and the bone sagittal view revealed retropharyngeal fluid collection and three calcific deposits anterior to the c1-c2 level (fig. because of dysphagia and odynophagia complaints, we consulted the otolaryngologist and he performed a flexible nasopharyngolaryngoscopy which showed posterior pharyngeal wall swelling in the arytenoid (fig. 4). after considering the clinical presentation and reviewing various radiologic studies in the inter - department conference, the patient was diagnosed with acute retropharyngeal calcific tendinitis of the longus colli muscle rather than myofascial pain syndrome. because of the retropharyngeal fluid collection and concern over the possibility of a retropharyngeal abscess, the patient was admitted to the hospital and started on regimens of antibiotic and nsaids such as intravenous amoxicillin, clindamycin, aconitum, acetylcysteine and intramuscular diclofenac. the posterior neck pain and limitation of neck motion were resolved progressively within approximately a couple of days after the initiation of therapy, and the dysphagia and odynophagia symptoms were also improved within a few days. the patient was discharged with a marked improvement of his disturbing symptoms several days after admission. during two months of follow - up visits, the symptoms were resolved without any significant sequelae, and the follow - up radiograph showed that the preverteral swelling was much improved (fig. 5). in pain clinic practice, it is generally common to meet patients who complain of posterior neck pain that refers to the occiput, shoulder or other areas. myofascial pain is a one of most common causes of posterior neck pain syndrome with referred pain. sometimes, differential diagnosis of posterior neck pain is very challenging based on symptoms and physical examination only. yoon. also reported a case of an intraspinal tumor during treatment of myofascial pain syndrome. an additional difficulty to this process of diagnosis is that clinical manifestations might be the summation of referred pain from multiple myofascial trigger points and even from other structures including bones, joints and visceral organs in patients with musculoskeletal pain. retropharyngeal calcific tendinitis, also known as calcific tendinitis of the longus colli muscle, is often unrecognized or misdiagnosed because of its relatively rare occurrence. determined that the disorder was caused by calcium hydroxyapatite deposition in the longus colli muscle. some suggest that repetitive trauma, recent injury, tissue necrosis, or ischemia may play a role in the pathogenesis of calcification. in this patient, there was no recent history of trauma or upper respiratory infection. it is believed that once the calcium crystals have deposited within the longus colli muscle, they provoke a painful inflammatory response and secondary retropharyngeal effusion. clinical manifestations of retropharyngeal calcific tendinitis are relatively nonspecific symptoms such as posterior neck pain and tenderness, motion limitation of the neck, dysphagia, and odynophagia, combined with mild fever, leukocytosis, and an elevated erythrocyte sedimentation rate or c - reactive protein. oropharyngeal symptoms of dysphagia and odynophagia are assumed to be caused by the close proximity of the retropharyngeal space to the adjacent pharyngeal constrictors. in this case, the patient also complaint of dysphagia and odynophagia associated with posterior neck pain on the second visit to the hospital. the pathognomonic radiographic findings of retropharyngeal calcific tendinitis hold true for plain film, ct, and mri : prevertebral soft - tissue swelling and amorphous calcification anterior to c1-c2 body. the soft - tissue swelling represents either discrete effusion or diffuse edema, which could be discriminated in ct or mri study. the lack of enhancement surrounding the effusion can be helpful in differentiating a reactive effusion from an infectious abscess. in this patient, we had a dilemma in diagnosis because there were findings of low signal components and heterogenous enhancement within the effusion on enhanced cervical spine mri. differential diagnosis between retropharyngeal calcific tendinitis and other pathological entites showing similar features, such as an abscess, meningitis, neoplasm, disc herniation, and myofascial neck pain, is considered important concern to avoid unnecessary managements or interventions. definitive diagnosis of retropharyngeal calcific tendinitis can be established by computed tomography, the gold standard examination for identifying the presence of prevertebral effusion and calcific deposits around c1 and c2. as also shown in this case, the definitive diagnosis was confirmed from the typical radiologic findings of ct and mri. in general, the natural progress of retropharyngeal calcific tendinitis is usually a benign and self - limiting course with conservative care. however, antibiotic treatment or surgical intervention is required in cases of retropharyngeal abscess with high morbidity. nsaids are the first - line treatment of retropharyngeal calcific tendinitis, but additional drugs, such as antibiotics, corticosteroids or opioids may be prescribed in severe cases. in this case, fearing retropharyngeal abscess, the otolaryngologist recommended admission and the prescriptions of antibiotics in addition to conservative treatments. immobilization with a soft cervical collar is another useful method to avoid aggravation of posterior neck pain. usually, the disturbing symptoms begin to be resolved within a couple of days from the initiation of treatment and the patients become symptom - free after 1 - 3 weeks of management. thorough understanding of this abstruse disease is clinically important, as it can prevent unnecessary evaluation and invasive interventions. in conclusion, retropharyngeal calcific tendinitis is a cause of acute or subacute neck pain that can be mistaken for myofascial pain syndrome or more ominous diagnoses such as retropharyngeal abscess, meningitis, neoplasm, or other diseases. we report a relatively rare but serious case of retropharyngeal calcific tendinitis which was initially diagnosed and evaluated as a myofascial neck pain syndrome. so, we recommend that a cervical spine ct or mri should be performed to exclude the possibility of retropharyngeal calcific tendinitis in cases when severe refractory neck pain is combined with oropharyngeal symptoms such as dysphagia and/or odynophagia. in pain medicine practice, the critical importance of recognizing this rare entity lies in preventing the misdiagnosis of posterior neck pain associated with retropharyngeal fluid collections, which might result in unnecessary surgical intervention rather than conservative managements. | differential diagnosis of posterior neck pain is very challenging based on symptoms and physical examination only. retropharyngeal calcific tendinitis is a rare and frequently misdiagnosed entity in various causes of neck pain. it results from calcium hydroxyapatite deposition in the longus colli muscle which is characterized by severe neck pain, painful restriction of neck movement, dysphagia, and odynophagia. we herein report a case of a patient with acute retropharyngeal calcific tendinitis, who complained of posterior neck pain, initially diagnosed and treated as a myofascial neck pain syndrome. |
after sixty years of development, the treatment of hyperthyroidism remains dependent upon three measures including antithyroid drugs (atds) and radioiodine and surgery. currently, methimazole (mmi) and propylthiouracil (ptu) are the most commonly prescribed atds. side effects of atd frequently appear in clinical practice and include rash, fever, hepatic injury [24 ], and leukopenia or agranulocytosis [58 ]. immunoallergic hepatitis is also a common side effect of atd therapy, affecting 1% of patients treated with atd. one study reported that the use of atd causes agranulocytosis in 0.10.5% of treated patients. clinical studies have found that agranulocytosis occurred in 0.37% of patients who received ptu and in 0.35% who received mmt. lithium carbonate has been used in the treatment of mania and in the prophylaxis against recurrent manic - depressive disorders, but it is also used as an adjunct drug for the treatment of hyperthyroidism in clinic. lithium carbonate inhibits the release of thyroid hormones and inhibits the synthesis of thyroid hormones [11, 12 ]. it has been reported frequently that patients with graves ' disease (gd) were treated with lithium carbonate in combination with radioiodine [13, 14 ]. however, the potential toxicity of lithium limits its application in hyperthyroidism, and its clinical effectiveness has not received much attention. this is the first report to study the effects of lithium carbonate on patients with gd as well as hepatic injury or leukopenia. in the study, the aims of this study were to investigate the clinical effectiveness of lithium carbonate on hyperthyroidism and to evaluate its safety. fifty - one gd patients with hepatic injury or leukopenia were recruited from january 2010 to january 2014 in department of endocrinology and metabolism. the study was approved by the ethics committee of the jiangsu province hospital on integration of chinese and western medicine, nanjing university of traditional chinese medicine. the patients included 8 males and 43 females and were 2058 years of age. according to liver function and leukocyte count, 51 patients with gd were enrolled in this study, including 33 gd patients with hepatic injury (5 males and 28 females, 64.7%) and 18 gd patients with leukopenia (3 males and 15 females, 35.3%) (table 1). we excluded the patients with thyroid crisis, who were pregnant woman, with psoriasis or a history of psoriasis, with renal insufficiency, with severe heart failure (nyha functional classification is more than class iii), with hepatic injury or leukopenia that have been induced by other reasons and patients who have contraindications of lithium carbonate. the signs and symptoms of hypermetabolism due to thyrotoxicosis, a diffuse goiter, increasing free t4 and decreasing tsh levels, an elevated radioiodine uptake, with or without orbitopathy and thyroid autoantibodies (trab, tpoab, and tgab). the concentration of alt (aminotransferase) and/or ast (aspartate aminotransferase) exceeded 2 times that of a normal lever and exclusion of viral hepatitis and other liver diseases. a decrease in the circulating wbc (white blood cell) count to less than 4.0 10/l, and agranulocytosis is defined by a reduction in the peripheral neutrophil count to less than 0.5 10/l and exclusion of hematological system disease. all patients were followed up for at least 1 year after stopping treatment ; thyroid function (ft3, ft4, and tsh), liver function (total bilirubin, alt, and ast), and white blood cell count were measured every 4 weeks. patients were considered to be in remission if the ft3 and ft4 level were within the normal range at the last visit. patients were considered to have relapsed if the ft3 and ft4 level exceeded the upper limit of the normal range and tsh levels were low during the follow - up. fifty - one patients with antithyroid drug - induced hepatic injury or leukopenia were treated with lithium carbonate for 36 weeks. on the other hand, gd patients with hepatic injury were treated with a hepatoprotective drug, and patients with leukopenia were treated with drugs to increase white blood cells, simultaneously (table 2). thyroid function, liver function, and white blood cell counts were measured every 4 weeks. pmol / l), ft4 (normal range : 11.5022.70 pmol / l), tsh (normal range : 0.554.78 miu / l), and trab, tgab, and tpoab (siemens healthcare diagnostics, new york, usa) were detected by chemiluminescence immunoassay. < 40 u / l), aspartate aminotransferase (ast, normal range : < 40 u / l), total bilirubin (tbil, normal range : 8.5021.0 mol / l), (roche diagnostics, mannheim, germany), and white blood cells (wbc, normal range : 4.010.0 10/l), and neutrophils (n, normal range : 2.57.5 10/l) were assessed. serum lithium concentration was measured by a colorimetric assay (roche diagnostics, mannheim, germany). side effects were observed during treatment, such as neuropsychiatric disorders, rash, nausea, vomiting, altered kidney function, and abnormality of blood glucose. the data analysis was evaluated using spss 16.0 ; p values < 0.05 were considered statistically significant. all values are expressed as the mean sd for the quantitative variables and as a percentage for the qualitative variables. the characteristics of the two groups were compared by t - test or nonparametric mann - whitney test for the quantitative variables and fisher 's exact test or test for the qualitative variables. factors associated with the outcome of hyperthyroidism were estimated using univariate analysis by logistic regression. the 51 patients were divided into two groups : gd with hepatic injury and gd with leukopenia. no significant differences in age, course, and thyroid parameters were observed between the two groups. we also recorded the using of atds before lithium carbonate treatment (table 1). the ft3 and ft4 values in gd patients with hepatic injury before the lithium carbonate treatment were 10.12 4.58 pmol / l and 27.46 8.94 pmol / l and 17.24 4.31 pmol / l (p < 0.01) after the treatment for 36 weeks. in gd patients with leukopenia similar changes in ft3 and ft4 were observed, before 10.86 5.35 pmol / l and 28.52 10.23 pmol / l and after 6.15 1.27 pmol / l and 16.31 4.19 pmol / l (p < 0.01), respectively (figure 1). the tsh value in gd patients with hepatic injury before the lithium carbonate treatment was 0.05 0.05 miu / l and significantly increased to 0.83 0.52 miu / l (p < 0.05) after the treatment for 36 weeks. in gd patients with leukopenia similar changes in tsh were observed, before 0.06 0.07 miu / l and after 0.92 0.65 miu / l (p < 0.05) (figure 1). thirty - three gd patients with hepatic injury were treated with an additional hepatoprotective drug (diammonium glycyrrhizinate, polyene phosphatidylcholine). the levels of alt, ast, and tbil decreased and remained at the normal level after treatment for 36 weeks. eighteen gd patients with leukopenia were treated with additional drugs to increase their white blood cells (leucogen, granulocyte colony - stimulating factor). the levels of wbc and n increased significantly and remained at the normal level after treatment for 36 weeks (data not shown). according to patient 's condition, it can not be denied that we used additional glucocorticoids including prednisone and methylprednisolone for 37% of the patients in the short term and propranolol was prescribed in 94% of the patients (table 2). blood pressure and blood glucose levels were not changed from the baseline and during the study period in the two groups (data not shown). one patient with mild heart failure (nyha functional classification is class ii) improved, 1 patient sustained atrial fibrillation, and they received radioiodine therapy later. overall, 12 patients (23.5%) obtained clinical remission at the 1-year follow - up, 6 patients (11.8%) relapsed after withdrawal and continue to receive lithium carbonate treatment, 25 patients (49.0%) received radioiodine therapy after lithium carbonate treatment, and 8 patients (15.7%) received surgical treatment after lithium carbonate treatment (figure 2). the liver function and white blood cells returned to normal levels in all patients. in comparing the remission with nonremission (radioiodine, surgery, and relapse) of the patients, we found that the failure to respond to lithium carbonate treatment may be correlated with several factors, including course of gd, thyroid hormone levels at baseline and 36 weeks, trab, and thyromegaly. no correlation was found between serum lithium concentration, lithium dose, age, treatment course, and glucocorticoid use (table 3). serum lithium concentration in gd patients with hepatic injury before the lithium carbonate treatment was 0.45 0.09 mmol / l and significantly increased to 0.56 0.08 mmol / l (p < 0.05) after the treatment for 36 weeks. in gd patients with leukopenia similar changes in serum lithium level were observed, before 0.41 0.06 mmol / l and after 0.54 0.06 mmol / l (p < 0.05). we did not find that there was a change in serum concentrations of lithium after patients are treated with corticosteroids. no abnormalities of renal function and blood glucose were found, and the side effects of psoriasis have not been observed. several patients appeared to have abdominal distention, vomiting, nausea, and so forth (table 4). lithium carbonate is usually used to treat manic - depressive and depressive disorders, but lithium carbonate can also decrease the levels of thyroid hormones and lead to hypothyroidism during the treatment [16, 17 ]. so it is also used as second - line drug for the treatment of hyperthyroidism. the relevance of the relationship between lithium treatment and thyroid function [18, 19 ] is well documented. hepatic injury and leukopenia may occur in untreated patients with thyrotoxicosis and patients treated with mmt or ptu. gd with atd - induced hepatic injury or leukopenia occurs frequently in clinical practice and treatment becomes more complex if these patients are unable to accept radioactive iodine therapy or surgery therapy ; thus, we were prompted to analyze the clinical effect of lithium carbonate on these patients. the study showed that the symptoms of hyperthyroidism were controlled, thyroid hormones decreased to a certain extent in all patients, and thyroid function was maintained at normal levels in 12 patients (23.5%) who discontinued drug therapy after 36 weeks. only 6 patients (11.8%) relapsed after lithium carbonate withdrawal for six months and continue to receive lithium carbonate treatment. overall, 33 patients (64.7%) could not reach a satisfactory target ; these patients were treated with radioactive iodine (25, 49.0%) or operation therapy (8, 15.7%). these findings indicate that lithium carbonate may be an effective therapeutic agent in the treatment of hyperthyroidism due to gd, especially in patients with hepatic injury or leukopenia. additionally, our results indicated that lithium may have an effect on treating mild or moderate hyperthyroidism but a poor role in treating severe thyrotoxicosis. in the present work, after 36 weeks of treatment with an additional hepatoprotective drug and a drug for increasing the white blood cells, liver function and white blood cell counts of patients improved. the levels of alt, ast, and tbil decreased in patients with hepatic injury, and the white blood cell counts increased in the patients with leukopenia. lithium carbonate does not injure the liver in the routine dose and is a perfect substitution for atd. study has found that an additive effect of increasing white blood cells was observed in patients treated with lithium carbonate because it may modulate granulocytopoiesis. moreover, lithium salts can not only increase the level of cd34 but also induce granulocyte colony - stimulating factor and increase the number of neutrophils by stimulating bone marrow ; thus, it is a good choice in patients with leucopenia or agranulocytosis [2123 ]. on the other hand, failure of lithium carbonate treatment may be influenced by several factors, including course of gd, thyroid hormone levels at baseline and 36 weeks, trab, and large goiter. in this study, 25 patients required radioactive iodine treatment after being treated with lithium carbonate, and no patient presented with thyroid crisis. data show that the thyroid iodine uptake rate (raiu) can be significantly increased in patients treated with lithium carbonate. study showed the addition of lithium is beneficial for gd patients treated with rai. compared with the patients treated with rai alone, the cure rate was higher in patients treated with rai plus lithium, and lithium therefore, lithium helps make the rai therapy more effective and the dose of i can be reduced. eight cases that required surgery after hyperthyroidism symptoms did not improve completely with lithium carbonate treatment, and these patients were stable during the intraoperative and postoperative periods. report that lithium carbonate is safe to treat patients with preoperative hyperthyroidism when atds are not effective and show adverse effects. it is concluded that the using of lithium alone or in combination with atds is an effective way for controlling hyperthyroidism before operation [26, 27 ]. in general, lithium carbonate requires specific care as it has a narrow therapeutic range, with the therapeutic levels for psychiatric disorders ranging from 0.6 to 1.2 mmol / l. the serum monitoring of lithium levels is important for safety of patients and clinical effectiveness. several patients had some side effects in this study, such as abdominal distention, vomiting, nausea, and asthenia (table 4) ; the symptoms disappeared when the lithium carbonate treatment was stopped. these patients were treated with radioiodine ; other patients did not appear to have side effects. inhibition of synthesis and secretion of thyroid hormones are the critical mechanism in the development of hypothyroidism [2931 ]. it reduces thyroidal iodine uptake and also inhibits coupling of iodotyrosine, and it can promote the thyroxine conversion into triiodothyronine in thyroid gland. in addition, the lithium salt can reduce thyroid adenylate cyclase activity, inhibit adenosine monophosphate, prevent the release of thyroid hormones, and decrease serum thyroid hormone levels. however, studies show that lithium affects the hypothalamic - pituitary - thyroid axis to decrease thyroid hormone levels [29, 33 ]. in conclusion, it is also effective for the preparation of radioactive iodine and surgical treatment in patients with thyrotoxicosis. our clinical study shows that lithium carbonate is safe and has no severe side effect on the treatment of hyperthyroidism. hence, we think that the antithyroid effect of lithium carbonate should be further studied, including pharmacologic mechanism, course of treatment, long - term efficacy, and safety. lithium carbonate has effects on the treatment of mild - to - moderate hyperthyroidism caused by gd, and it is particularly suitable for patients with atd - induced hepatic injury or leukopenia. it is also effective for the preparation of radioactive iodine and surgical treatment in patients with thyrotoxicosis. | objective. gd with atd - induced hepatic injury or leukopenia occurs frequently in clinical practice. the purpose of the present study was to observe the clinical effect of lithium carbonate on hyperthyroidism in patients with gd with hepatic injury or leukopenia. methods. fifty - one patients with gd with hepatic injury or leukopenia participated in the study. all patients were treated with lithium carbonate, in addition to hepatoprotective drugs or drugs that increase white blood cell count. thyroid function, liver function, and white blood cells were measured. clinical outcomes were observed after a 1-year follow - up. results. after treatment for 36 weeks, symptoms of hyperthyroidism and the level of thyroid hormones were improved and liver function, and white blood cells returned to a normal level. twelve patients (23.5%) obtained clinical remission, 6 patients (11.8%) relapsed after withdrawal, 25 patients (49.0%) received radioiodine therapy, and 8 patients (15.7%) underwent surgical procedures after lithium carbonate treatment. conclusion. lithium carbonate has effects on the treatment of mild - to - moderate hyperthyroidism caused by gd, and it is particularly suitable for patients with atd - induced hepatic injury or leukopenia. |
the diagnosis of mci due to ad (the symptomatic predementia phase of ad) has been recently proposed by the national institute on aging and the alzheimer 's association. diagnostic criteria include concern regarding a change in cognition, impairment in one or more cognitive domains, preservation of independence in functional abilities, and not demented. within the generally accepted framework clinical studies of prodromal stages to ad tend to focus on persons with amnestic mci (amci). both the alzheimer 's disease neuroimaging initiative (adni) and the mayo clinic cohorts typify this population. in the mayo clinical study of aging, persons in the age range of 7089 years are enrolled, with 2/3 of the mci group being amci. adni enrolls participants between ages 55 and 90, and mean age for the mci group is 74.7 years. researchers from the mayo clinic proposed quantitative criteria for identifying mci as a prodromal stage to alzheimer 's disease in 1999. they stressed the importance of memory impairment and proposed quantitative criteria specifying the level of memory deficit relative to global cognitive functioning. in adni, mci is defined as a clinical dementia rating of 0.5, and performance on the free recall measure of a neuropsychological memory test (logical memory ii) is below a given threshold. since the vast majority of the subjects characterized as amci will develop ad, amci may be defined as a prodromal condition of ad. the pathophysiological basis and prognosis of nonamnestic mci remains unclear, and the group is probably heterogenous [6, 7 ], including patients with frontotemporal dementia, parkinson 's disease, dementia with lewy bodies, vascular dementia, and neuropsychiatric conditions (depression). change in the frontostriatal network supporting executive functions may occur as a part of healthy aging [13, 14 ]. thus, an mci subgroup with isolated executive difficulties may be an extreme group of normal aging. the objective of this paper is to review executive / attentional impairment as an important aspect of mci or pre - mci in terms of symptom manifestation and importance for disease progression. we will focus on recent research on mri and genetic markers that may serve to further understanding of the pathophysiological processes underlying executive mci (emci) and its relation to ad. attention and executive impairment are frequent and disabling symptoms in mci when measured with neuropsychological tests ranging from simple processing speed tasks to tasks of complex problem solving. the distinction between clinical tests of attention and test of executive function is a fuzzy one and they are here treated as on the same continuum. there is no consensus on how executive function should be tested in clinical studies, and studies that take into consideration developments in cognitive psychology find high frequency of executive impairment in both amnestic and nonamnestic mci, with some subfunctions more affected than others. alzheimer 's disease (ad) is the most common cause of dementia and accounts for approximately 6070% of all dementia cases, and deficits of episodic memory are a cognitive hallmark of the disease. executive dysfunction is evident in the prodromal stage of ad [2022 ] and appears predominantly in tasks requiring cognitive flexibility, inhibition, and self - monitoring. there is evidence that the commonly reported impaired ability to perform two tasks simultaneously in ad reflects a specific deficit in dividing attention, rather than the result of a more general processing speed deficit. according to the model of cognitive decline leading to ad presented by perry and colleagues, executive problems appear after memory problems in time, but before typical parietal lobe symptoms (aphasia, visuospatial deficits). when executive dysfunction is present, it has a clear negative influence on ability to manage activities of daily living and may thus add to the risk of conversion from mci to ad. predictive accuracy for conversion from mci to ad for one set of cognitive variables (composed of episodic memory and processing speed measures) has been found to be as high as 0.86 (sensitivity, 0.76 ; specificity, 0.90).. found that a test of executive function and assessment of baseline functional capacity predicted conversion from mci to ad after 2 years better than biomarkers (mr and csf). both amci and attention / executive mci subtypes have been regarded as important predementia subtypes, at risk for ad. while amci is usually defined as a prodromal, at - risk condition of ad, isolated executive dysfunction can be a prodromal stage for several neurodegenerative diseases. in cases of predementia ad, it is not clear if amci and emci may be two different categories / subtypes of ad or represent different phases of ad development. if attention / executive mci is not a distinct ad subtype, but an earlier stage of ad than amci, then amci should be expected to have executive / attentional deficits in addition to memory impairment. it has been reported that attention and executive functions may be impaired in the incipient stages of ad and may contribute to the observed memory deficit. it has been argued that even patients defined as pure amci on screening tests may have executive impairment, when a comprehensive neuropsychological examination of executive cognition is performed. the existence of nonamnestic attention / executive mci has been recorded in several mci studies [29, 3237 ], where a comprehensive neuropsychological test battery has been utilized for classification purposes. the prevalence of attention / executive mci will vary widely in different samples based on recruitment criteria and assessment methods but has been reported as from 3 to 15%. in the sample studied by us attention / executive mci without amnestic deficit constitutes about 30% of the total mci group, which on the whole is 1015 years younger than the adni study group. in a study of johnson and colleagues, of the 12 executive mci patients who progressed clinically after two years, 2 converted to probable dementia with lewy bodies, 10 retained the clinical diagnosis of mci, and none reverted to normal. patients with single domain executive mci who progressed quickly over two years had more temporal lobe atrophy on mri and slightly lower scores for visual memory recall when compared to the stable executive mci patients, possibly suggesting that converters may be at their later stages of clinical progression. the executive mci patients who progressed reported fewer dysexecutive symptoms than nonprogressors, while there were no differences in informant - rated dysexecutive symptoms and baseline performance on all four executive tests. nine subjects with pure attention / executive mci were identified in the longitudinal study of whitwell and colleagues. in this study, almost 70% of mci patients within an attention / executive subgroup progressed to dementia in the period of four years, suggesting that the group is at high risk of developing dementia. three patients converted to dementia with lewy bodies and three patients converted to ad dementia. the prognosis for other patients with isolated attention / executive dysfunction in the study of whitwell and colleagues is not clear, but they may also convert to other dementias or remain stable over many years. by using similar criteria for classification of subjects as those used in the study of whitwell and colleagues, the attention and executive functions depend on distributed networks, encompassing both frontal and parietal associative cortices, as well as subcortical structures and white matter (wm) pathways. the executive functions control and monitor task performance and depend critically on the frontal lobes. three fronto - subcortical circuits (originating in the prefrontal cortex) have been identified as responsible for executive control functions, that is, the dorsolateral prefrontal cortex (working memory), the lateral orbital cortex (inhibition), and the anterior cingulate cortex (response conflict) [41, 42 ]. findings from functional imaging indicate that during prodromal ad, the brain network involving the dorsolateral prefrontal cortex and the anterior cingulate, is affected. while amci is characterized by medial temporal lobe affection, atrophy in the basal forebrain has been found to be characteristic for the mci groups with isolated attention / executive deficits [29, 33 ]. some studies have reported an association between prefrontal cortical changes and attention / executive impairment in mci [29, 34, 46 ]. significant cortical atrophy in frontal regions has been found in predementia ad. it has been argued that prefrontal damage, in combination with cingulate damage, has predictive value for the conversion from mci to ad. another recent study indicates that white matter (wm) pathology in ad is distributed in all lobes of the brain but it is most prominent in the frontal wm. in addition to frontal wm changes, mci patients may have wm changes in both anterior and posterior cingulate regions. the anterior cingulate region is regarded as belonging to a network responsible for executive control function while the posterior cingulate belongs to a memory network. thus, it has been hypothesized that the caudal portion of the anterior cingulate plays a major role in executive function abilities, primarily through its reciprocal connections with the prefrontal cortex [53, 54 ]. in our recent study on attention / executive mci, we have demonstrated consistent relationships between neuropsychological function and the microstructural properties of the wm brain pathways measured by diffusion tensor imaging (dti), as well as cortical - morphometric parameters. executive impairment in mci patients with unaffected memory performance has been associated with reduced wm tract integrity (increased radial diffusion (dr) and mean diffusivity (md)) in frontal and cingulate regions and cortical thinning in caudal middle frontal region. we have found that wm dr / md increases in frontal, cingulate, and entorhinal regions in patients with attention / executive mci, but cortical thickness was not different from controls in any of the studied regions. the findings may thus indicate that the relative importance of grey matter versus wm changes may differ at different stages of predementia cognitive impairment [55, 56 ]. frontal and temporal wm diffusivity changes have been previously described in amci patients. by using dti to characterize executive networks in mci, we found wm dr / md changes in both the anterior and posterior cingulate regions in emci suggesting that both regions may contribute to attention / executive impairment in mci. the cingulate cortex projects into the striatum, and both the anterior and posterior cingulate cortices receive mediodorsal thalamic afferents, which are part of fronto - subcortical circuits, involved in executive function. some attention / executive subfunctions correlated significantly with imaging findings in frontal and cingulate regions in the emci group, but no significant correlations were found in the controls. in attention / executive mci, response inhibition was associated with wm dr / md underlying the superior frontal cortex, and response inhibition / switching was associated with wm dr / md underlying the superior frontal, rostral middle frontal, lateral / medial orbitofrontal, and retrosplenial cortices. test scores for attention and divided attention were associated with the cortical thinning of the caudal middle frontal region. the study results thus support the results from previous mci studies, where associations between prefrontal changes and attention / executive impairment have been reported [29, 34, 46 ]. in addition, the results confirm that cingulate changes are associated with executive impairment in mci. in one recent study, mci patients with isolated executive dysfunction had cerebral hypoperfusion in bilateral middle frontal cortex, bilateral posterior cingulated, and the left precuneus relative to controls. relative to amci patients, emci patients had hypoperfusion in the left middle frontal cortex, left posterior cingulate, and the left precuneus, supporting the existence of pathophysiologically distinct mci subgroups. in the study of pa and colleagues, executive nonamnestic mci subgroup had significantly less grey matter in the left dorsolateral prefrontal cortex compared with control subjects. the emci subgroup had less volume in the caudate nucleus compared with amci group, but the differences for prefrontal cortex in emci versus amci were not significant, which could be due to some reduction of prefrontal cortex volume in amci as well. in contrast, the amci patients had less volume in the right inferior parietal cortex, typical for ad, than emci. these neuroimaging findings also suggest that some of the emci patients may represent a distinct subgroup of mci. we have found increased entorhinal wm dr and md in both patients with memory impairment and those with attention / executive dysfunction without objective memory impairment [37, 58 ], suggesting a common affection of regions known to show changes in early ad. attention / executive mci may be an earlier stage of ad than amci and the patients with nonamnestic attention / executive impairment may develop memory problems later. it is also possible that patients with attention / executive mci may progress to non - ad dementias or ad with disproportionate neuropathology in the frontal cortex. to study very early development of ad, neurobiological markers of high risk in asymptomatic individuals may be used. sperling. use the term ad - p to denote pathophysiological factors that are significant for the development of clinical ad (ad - c), but each factor in isolation does not cause ad - c. amyloid accumulation may be measured with positron emission tomography (pet) or with cerebrospinal fluid (csf) analyses, but both are invasive and costly procedures that are not suitable for screening. genetic risk can be assessed relatively simply, and followup studies of healthy at risk populations are not prohibitively expensive although they have ethical problems. apolipoprotein e (apoe) and e4 allele carrier status confer a significant increase in risk of developing ad. recent studies of relative risk based on large samples argue that the impact of apoe e4 on ad risk is similar to that of major genes in mendelian diseases and comparable to genetic risk of breast cancer. amyloid load in cognitively normal persons above age 60 correlates positively with apoe e4. in mci patients pib - positive pet scans are more frequent in apoe e4 carriers. in patients with ad, progression of cerebral amyloid load is associated with e4 gene dose. there is thus evidence that a major genetic risk factor for ad is associated with preclinical accumulation of beta amyloid, the most significant pathophysiological causal factor for developing ad. greenwood. have argued that in view of the complexity of apoe mechanisms affecting cognition, it would be misleading to view all cognitive effects of e4 as evidence of incipient ad, and they argue that in normal aging there is an accumulating effect of inefficient neural repair mechanisms associated with the e4 allele. these changes make the brain more vulnerable to pathological processes, including accumulation of amyloid beta 42 in ad, but do not cause this process to occur in all e4 carriers. severe cholinergic changes are found in advanced ad, with loss of cholinergic neurons and receptors [66, 67 ]. the authors propose that the loss of this apparent compensatory response may mark the conversion of mci to diagnosable ad. recent evidence indicates that complex interactions between apoe e4 and cholinergic genes (buche) affect the conversion rate of mci to ad and that the level of beta amyloid accumulation in the brain is related to both apoe and cholinergic activity [70, 71 ]. thus we see evidence of a negative interaction between apoe, beta amyloid accumulation, and cholinergic dysfunction. there have been numerous studies of cognitive symptoms associated with apoe e4 carrier status in nondemented persons. used a meta analysis of more than 2000 participants and found significant positive effect size for memory and global intellectual function. the effect sizes are moderate, and the studies are influenced by choice of methods, especially in nonmemory cognitive domains. parasuraman and collaborators have taken a cognitive neuroscience approach to study effects of genes involved in risk of ad (apoe) or mechanisms involved in cognitive deficit in ad (cholinergic genes they concluded that intact focusing and impaired disengagement of visuospatial attention may be linked to dysfunction in early ad of corticocortical networks linking the posterior parietal and frontal lobes. greenwood. found that healthy middle - aged adults without dementia who carry the apoe e4 allele show deficits in spatial attention and working memory that are qualitatively similar to those seen in clinically diagnosed ad patients. greenwood. used an experimental paradigm measuring working memory for dot locations in a spatial array and found that accuracy was reduced in healthy e4 homozygotes, of mean age 5760. reinvang. found that e4 carriers performed worse on letter - number span, another working memory task, and in addition on the stroop color - word interference task. wishart. studied cortical activation pattern in a working memory task and the e4 group showed greater activity during working memory in the medial frontal and parietal regions bilaterally and in the right dorsolateral prefrontal cortex. there were no regions in which the e3 group showed greater activation than the e4 group. by measuring event - related potentials (erps) while mci patients 5076 years of age worked on an experimental attention task (auditory three - stimulus oddball). reinvang. performed an event related potential (erp) study with mci patients and found attenuated n1 and n2 amplitudes in e4 carriers. in a follow up study with only normal controls covering the same age range working on the same auditory oddball task, furthermore, n2 latency was longer for e4 carriers, and this latency predicted memory decline 3.5 years later, suggesting that attention - related functions may presage memory decline in those with elevated risk for ad. the later component p3 has been shown to be associated with apoe in healthy controls. irimajiri. found reduced amplitude among healthy female e4 carriers in auditory task, and espeseth together, these findings indicate a potential clinical significance of individual differences in the attention - related erp components n1, n2, and p3. these findings of apoe - related changes in attention are associated with apoe - related differences in brain structure. found that healthy e4 carriers had thicker cortices than noncarriers in regions of the brain known to be involved in attentional function. however, an age by apoe interaction showed that this effect was specific for the middle - aged participants. the crosssectional data indicated that there might be an accelerated thinning of the cortex for e4 carriers, suggesting that the thicker cortex among the middle - aged might be associated with a dysfunctional process. espeseth. showed that cortical thickness in regions with significant carrier versus noncarrier diffrerences was negatively correlated with p3a amplitudes, suggesting that the increase in cortical thickness was indeed dysfunctional. [84, 85 ] who showed that while symptomatic psen1 mutation carriers had widespread cortical thinning compared to healthy controls, asymptomatic mutation carriers had thicker cortices, suggesting that high risk for ad may be associated with a temporary thickening of the cortex. cognitive functions are sensitive to interaction of apoe with other factors, including interaction with other genes. have argued that interaction (epistasis) of apoe and chrna4, a nicotinic receptor gene, influences function in the domains of attention and executive function. chrna4 has been shown to be related to attentional function in several studies [8693 ]. the search for cognitive markers of very early ad, possibly predating amnestic mci, should therefore take account of the cognitive neuroscience literature on the role of cholinergic systems in attention and executive function. furthermore, tasks from cognitive neuroscience research that have proven to be related to specific cortical - subcortical activation patterns or neurotransmitter systems may in general be more sensitive to subtle cognitive changes than tests derived from clinical studies of advanced pathology. problems of attention and executive function are common in mci, and in patients with amci they are the most important additional symptom domain in multidomain amci. it is generally believed that in this group, executive deficit appears after memory impairment in the sequence of cognitive decline leading to full blown dementia. executive mci may occur without memory impairment, and there is evidence that although etiology is heterogenous, a significant proportion of these patients develop ad. how large this group is in mci samples varies and is dependent on several factors. the strong focus on memory problems as key symptom in early ad, and the wide normal variation in attention and executive function in aging, may indicate a high threshold for these patients to seek medical service. our own data indicate that in a relatively young mci population investigated with comprehensive neuropsychological testing, emci is a common variant. attentional and executive dysfunctions may remain undetected even though a thorough neuropsychological examination is performed. difficulties in dividing attention and manipulating remembered information may be reflected in everyday tasks, such as packing a bag, keeping track of conversations, or walking whilst talking. patients with executive mci may show increased behavioral symptoms on questionnaires that specifically measure executive behaviors compared with amci and control subjects. knowing that decreased awareness of cognitive symptoms has been reported in some patients with mci and executive mci, it may be helpful to ask other informants to rate executive symptoms of the patient. current conceptions of early mr changes emphasize hippocampal and cortical atrophy as the significant pathological event, closely linked with emergence of memory impairment [59, 96 ]. mr analysis yields sensitive measures of a range of pathognomonic events, and recent publications from our group and others indicate that reduced quality of the connectivity of brain networks may compromise cognitive function. this is true, both for the memory network of the brain, including posterior cingulate, and additionally for frontal networks. our studies and those of others indicate that in identifying the brain changes underlying emci one should emphasize fiber integrity as measures with dti as well as frontal lobe cortical thinning. in their report on the status of preclinical markers, ad sperling and collaborators use the concept of alzheimer 's disease - pathophysiological process (ad - p) to denote different processes that may contribute to development of clinical alzheimer 's disease (ad - c). furthermore they point to studies combining biomarkers (of ad - p) with measures sensitive to very subtle cognitive decline as clearly needed. large - scale longitudinal studies of biomarker - positive populations raise enormous problems in terms of ethics, costs, and logistics. we suggest that healthy apoe e4 carriers are a realistic and highly relevant study group. subclinical genetic effects on mr - morphometry, dti, and default mode have been shown. experimental cognitive studies have identified specific attention and executive subfunctions as sensitive to apoe allele variation. the paradigms need to be further developed and standardized for use in clinical / epidemiological studies. this development could be modeled on the effort to standardize cognitive neuroscience paradigms for application in schizophrenia research. a great advantage is that they are suited for computerized administration and scoring, so that one may foresee study participants in a longitudinal study logging on to the internet and complete a set of standard tasks at regular intervals. | mild cognitive impairment (mci) may take several forms, and amnestic mci (amci) has been recognized as an early stage of alzheimer 's disease (ad). impairment in executive functions including attention (emci) may be indicative of several neurodegenerative conditions. executive impairment is frequently found in amci, it is significant for prognosis, and patients with emci may go on to develop ad. recent studies have found changes in white matter integrity in patients with emci to be more sensitive than measures of cortical atrophy. studies of genetic high - risk groups using sensitive cognitive neuroscience paradigms indicate that changes in executive function may be a cognitive marker useful for tracking development in an ad pathophysiological process. |
the mucociliary clearance system is a first line of defense against inhaled agents, and so its compromise can adversely affect health. the purpose of this paper is to provide a review of data on the effect of in vivo air pollutant exposures on the clearance of test particles from airways. data from both animals and humans are compared whenever possible, so that estimates of human health effects may be made. mechanisms of action are also discussed, presenting the view that for low level exposures, changes in secretions are probably responsible for most observed changes in clearance. the pollutants pertinent to this review are those that are common in the environment and most likely to have impacts on large numbers of people : sulfur oxides, sulfuric acid mist, o3, no2, particulates, diesel exhaust, and cigarette smoke.imagesfigure 1.figure 2. afigure 2. bfigure 4. |
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many authors found higher glucose salivary levels in diabetic patients than in nondiabetics [111 ]. such investigations aimed mainly at exploring whether diabetic control could be monitored by a noninvasive method of salivary glucose measurement [14 ]. several factors may account for the poor correlation between blood and saliva glucose concentrations prevailing in diabetic subjects. they include oral retention of alimentary carbohydrates [12, 13 ], glucose utilization by oral bacteria, release of carbohydrates from salivary glycoproteins [15, 16 ], and contamination of saliva by a large outflow of crevicular fluid in patients with a poor gingival status [17, 18 ]. in considering the relationship between salivary glucose concentration and salivary flow, the present study mainly aimed at re - evaluating salivary glucose concentration and excretion in unstimulated and mechanically stimulated saliva in both normal and diabetic subjects. the first set of experiments was conducted in 38 normal subjects, including 16 males and 22 females with respective mean ages (sem) of 24 2 and 23 2 years. the second set of experiments was conducted in 84 diabetic patients, including 36 males and 48 females with mean respective ages of 56 3 and 55 2 years. the third set of experiments was restricted to 9 normal subjects and 18 diabetic patients. at variance with the first two sets of experiments, it did not include measurements of salivary flow and, hence, glucose excretion rates. the fourth set of experiments consisted of an oral glucose tolerance test conducted in 4 normal subjects and 2 diabetic patients. the last set of experiments concerned 3 healthy subjects and 2 diabetic patients, examined at the occasion of successive samplings of stimulated saliva in the absence of any change in glycemia. the diabetic patients were treated and appropriate control subjects were recruited from the endocrinology department, istanbul university medical school, istanbul, turkey, and the stomatology department, erasmus hospital, universit libre de bruxelles, brussels, belgium. all experiments and sample collections, as well as saliva glucose measurements, were performed by the same investigator either in turkey or belgium. the present research was conducted in full accordance with ethical principles, including the world medical association declaration of helsinki. to collect saliva, both the cotton and two - compartment tube were obtained from the same manufacturer (salivettetm, starstedt, nmbrecht, germany). the upper part of the tube containing the cotton presented a hole, so that, after centrifugation, the saliva was recovered in the lower part and became available for analysis. saliva was collected in fasting subjects, immediately after rinsing the oral cavity two times with 150 ml of water and drinking this water, by means of cotton kept in the oral cavity for 1 to 3 minutes either in the unstimulated state or during mastication (stimulated saliva). salivary flow was determined by weighing the device with the cotton before and after saliva collection, assuming that 1 g of saliva corresponds to 1 ml. centrifugation of the device at 2000 g for 5 minutes allowed the saliva adsorbed to the cotton to pass through the orifice into the lower compartment of the device, the saliva being then immediately frozen at 20c. although salivary flux could be affected by the use of salivette, the latter was used to standardize the collection of saliva, for hygiene reasons, and to remove particles from the saliva. blood was taken from finger tip, and blood glucose concentration was measured by the glucose oxidase method. 100 l centrifuged saliva was mixed with 95 l of reagent medium containing 2.0 mm mgcl2, 0.5 mm atp, 0.5 mm nadp, and 0.06 units of yeast glucose 6-phosphate dehydrogenase in tris - hcl buffer (200 mm, ph 8.1). after a first reading of the absorbance at 340 nm, the reaction was started by the addition of 5 l yeast hexokinase in reagent medium (0.06 units). the assay was simultaneously conducted on glucose standards (final concentration comprised between 5 and 250 m). the results were calculated as nmol of glucose / ml saliva after the subtraction of reading in the absence of hexokinase and taking into account glucose standards and saliva volume. the coefficient of variation is, respectively, 3.3 0.4% (n = 25) and 5.4 0.6% (n = 51) for d - glucose standards and saliva samples. the standard curve of glucose between 5 to 250 m is linear with a correlation coefficient of 0.999. our method can measure as little as 0.5 nmol of glucose with a variation coefficient of 4.3%. all results are presented as mean values (sem) together with the number of individual determinations (n) or degree of freedom (d.f.). the statistical significance of differences between mean values was assessed by the use of student 's t - test. in a large series, the glucose concentration was 76.4 3.8 (111) and 32.4 2.4 m (126), respectively, in unstimulated and stimulated saliva from normal subjects. a first study was conducted in 38 normal subjects, including 16 males and 22 females. the glucose concentration averaged 79.4 5.8 m (n = 33) in unstimulated saliva, as distinct (p.3) in unstimulated saliva (66.7 6.1 nmol / min ; n = 33) and stimulated saliva (57.1 8.3 nmol / min ; n = 38), with a mean paired difference between unstimulated and stimulated saliva of 14.1 11.7 nmol / min (n = 33 ; p >.2). as a rule, these variables did not differ significantly in male versus female subjects (table 1). the stimulated salivary flow, however, was higher (p.4) in the unstimulated saliva (223.0 35.6 nmol / min ; n = 74) and stimulated saliva (255.7 28.1 nmol / min ; n = 83). none of these variables differed significantly in type-1 and type-2 diabetic patients of the same gender. likewise, the glucose concentration failed to differ significantly in male and female diabetic patients, whether in unstimulated or stimulated saliva. the basal salivary flow and glucose excretion rate were lower (p.1) from that in glycemia. the unstimulated saliva flow was also somewhat higher (p the glycemia averaged in diabetic patients 8.76 0.77 mm (n = 17) as compared (p.9) to that of blood glucose concentration, with an overall diabetic / control ratio of 178.8 24.2% (n = 53 ; p.3) recorded in normal subjects when comparing stimulated to unstimulated saliva. it also documents the increase in salivary flow (p.99) and glucose excretion rate (p >.4) observed under the same experimental conditions in diabetic patients. last, it illustrates the marked increase (p.2) of the corresponding mean values recorded under the same experimental conditions in normal subjects (100.0% 5.9% ; n = 76). despite the high number of individual determinations, no significant correlation was found in the diabetic patients between glycemia and either glucose concentration (r = 0.1614 ; d.f. = 91 ; p >.1) or glucose excretion rate (r = 0.0629 ; d.f. likewise, no significant correlation could be found in the diabetic patients between glycemia and either glucose concentration (r = 0.1545 ; d.f. = 98 ; p >.1) or glucose excretion rate (r = 0.1522 ; d.f. in the present study, the unstimulated salivary flow was higher compared to the unstimulated saliva flow of about 0.4 ml / min observed in many studies [2126 ]. this situation is probably linked to the use of a salivette for the collection of saliva. the present results confirm that the glucose concentration in saliva is higher in diabetic patients than in control subjects [111 ]. it also confirms that, in both normal subjects and diabetic patients, the salivary flow is higher in stimulated as compared to unstimulated saliva [7, 2128 ]. despite such an increase, the glucose excretion rate, taken as the product of saliva glucose concentration multiplied by salivary flow, failed to differ significantly under unstimulated and stimulated conditions, whether in normal subjects or diabetic patients. the latter finding argues in support of a dissociated regulation of salivary flow (increased by mechanical stimulation) and glucose release by salivary glands (unaffected by mechanical stimulation). the dependency of saliva glucose concentration on glycaemia was further documented by the time course of changes in the former variable during an oral glucose tolerance test, as documented in both normal subjects and diabetic patients. during the glucose tolerance test (ogtt), the salivary glucose level increased twofold within 60 minutes, as observed previously [29, 30 ]. the measurements of saliva glucose concentrations made during such an oral glucose tolerance test led us to observe, in a further set of experiments, that such a concentration decreases at the occasion of successive samplings of stimulated saliva, such a decrease occurred despite unchanged salivary flow. its pattern was reminiscent of the rapid clearance of exogenous glucose from the saliva of human subjects otherwise observed during the first 6 to 8 minutes, followed by a much slower clearance thereafter [3032 ]. no significant difference between type-1 and type-2 diabetic subjects was detected in the present study, and no significant correlation between glycemia and glucose saliva concentration or glucose excretion rate was found in the diabetic patients, whether in unstimulated or stimulated saliva. these findings confirm the poor link between glycaemia and glucose concentration or excretion in saliva, at least on an individual basis [58 ]. nevertheless, the present study may well set the scene for further investigations on the regulation of glucose output from salivary glands, as well as on the potentially unfavorable effect of a high glucose salivary concentration on selected variables of oral health status in diabetic patients. | the present report aims mainly at a reevaluation of salivary glucose concentration and excretion in unstimulated and mechanically stimulated saliva in both normal and diabetic subjects. in normal subjects, a decrease in saliva glucose concentration, an increase in salivary flow, but an unchanged glucose excretion rate were recorded when comparing stimulated saliva to unstimulated saliva. in diabetic patients, an increase in salivary flow with unchanged salivary glucose concentration and glucose excretion rate were observed under the same experimental conditions. salivary glucose concentration and excretion were much higher in diabetic patients than in control subjects, whether in unstimulated or stimulated saliva. no significant correlation between glycemia and either glucose concentration or glucose excretion rate was found in the diabetic patients, whether in unstimulated or stimulated saliva. in the latter patients, as compared to control subjects, the relative magnitude of the increase in saliva glucose concentration was comparable, however, to that of blood glucose concentration. the relationship between these two variables was also documented in normal subjects and diabetic patients undergoing an oral glucose tolerance test. |
bulk metallic glasses (bmgs), because of their long - range atomic disorder, deform uniquely [1 - 4 ] : the plastic deformation is highly localized into narrow shear bands at room temperature [5 - 8 ]. under deformation - constrained loading modes such as compression [9 - 11 ] and nanoindentation [12 - 19 ], serrated plastic flow phenomena have been widely observed and found to be rate - dependent : as strain rate decreases, the flow serrations become more distinct. moreover, rate - dependent shear - band patterns were observed on the surfaces or inside the post - deformed specimens under compression and nanoindentation [15 - 17 ]. therefore, currently, it is well accepted that the macroscopic serrated plastic flow behavior is associated with the shear - banding operations on a nanoscale within bmgs. schuh and nieh and zhang. suggested that the simultaneous operations of multiple shear bands at high strain rate result in the smooth plastic flow ; with increasing strain rate, the deformation mode transits from inhomogeneous to homogeneous. however, jiang and atzmon, greer., and jang. considered that the disappearance of flow serrations at high strain rate arises from the limitation of data acquisition and the instrumental response ; even at extremely high strain rate, the plastic deformation is still inhomogeneous. since these hypotheses were mainly gained from ex situ experimental observations on shear - band patterns, the physical origin of rate - dependent serrated plastic flow, even if well accepted as a temporal event or process behavior, is still not well grounded. very recently, infrared camera technique has been used for in situ observing dynamic shear banding operations in the study of the serrated plastic flow during compression by jiang.. based on the information from experimental observations, they conjectured a spatiotemporal picture of shear - banding during serrated flow, and further explained the inhomogeneous deformation during the indentation. however, as they pointed out the propagation of a shear band is very fast ; hence only mature shear bands can be captured, not to mention the fine shear - banding events on atomic scale. in addition, during indentation, since the shear bands develop underneath indenter, direct observation in situ on them is very difficult, even impossible. the molecular dynamics (md) simulation is generally believed to be an effective way in modeling various indentation processes [20 - 22 ], providing an in situ observation on atomic motions. in this aspect [20 - 23 ] by developing argon s shear transformation zone (stz) concept. they found that the load - drop events in a load - displacement curve, i.e., serrated plastic flow, correspond closely to the bursts in deformation activity (irreversible atomic rearrangement) associated with shear bands. spatially, the suppression of wing - like shear bands of post - deformed specimen at higher strain rate leads to milder serrations. however, how do the strain rates affect this inherent correlation between serrations and atomic rearrangements ? how does the temporal behavior of such rearrangements produce the final shear - banding patterns after loading ? these questions are still not well understood and deserve further investigation. in this letter, we rely on md computer simulations to roundly probe how spatiotemporal distribution of shear - banding events on an atomic scale is related in situ to macroscopic rate - dependent serrated plastic flows in bmgs undergoing nanoindentation. strain rate effect on this relationship and its underlying physics are discussed. a binary amorphous alloy system, cu46zr54, was used in our md simulations. in this system, atoms interact via a modified lennard - jones 48 potential of the form : (1) where rij is the distance between the atoms i and j, a, b, c, and d are constants whose values are available in ref., and rt is the truncation distance with the values of 5.08, 5.58, and 6.00 for cu the motion of each atom was evaluated by integrating the newtonian equations of motion using velocity - verlet method with a time step of 1 fs. to form an amorphous sample, an initial structure for the sample was built by placing all atoms into a face - centered cubic (fcc) crystal lattice in a random order, and the initial velocities of all atoms were set to be zero. the initial structure was gradually heated to 2400 k for sufficiently melting, and then cooled to 1 k with the cooling rate of 25 k / ps. in this process, the npt ensemble was used, and the pressure was kept at zero ; periodic boundary conditions were used in all three directions. then, for the subsequent indentation simulations, we set the top boundary free and fixed a layer of 6.0 in thickness at the bottom. another 100 ps was carried out to a new equilibration. finally, a three - dimensional sample (sample i) which contains 432,000 atoms with the size of 250 250 125 and a two - dimensional sample (sample ii) which includes 250,000 atoms with the size of 1950 1050 were formed in this way. the indenter was modeled by a purely repulsive potential with the form : (2) where r is the distance from the indenter center to a sample atom, r is the radius of the indenter which is chosen as 20 for sample i and 400 for sample ii, (r r) is the standard step function, and e is a constant which is equal to 3.0 and 3.9 nn / for a cu atom and a zr atom, respectively. the indenter was displaced toward the top surface of the sample at a constant strain rate by keeping an invariable displacement interval of 0.1 and adjusting the relaxation time for each displacement interval. in this process, the control to the temperature (1 k) was only allowed in a layer of 10.0 in thickness which is just above the fixed layer at the bottom. for sample i, the total indentation depth was 15, and three strain rates, 10, 10, and 10 s, were executed ; for sample ii, the total indentation depth was 50, and three strain rates, 4 10, 4 10, and 4 10 s, were performed. parallel computing was used in all the simulation processes. the load displacement (p h) curves for the indentation simulations are shown in fig., the rate - dependent serrated plastic flow phenomena can be observed : when strain rate decreases from 1a c, flow serrations become more prominent. the result is consistent with a series of experimental observations for real metallic glasses under indentation [11 - 18 ]. it has been recognized that the serrated plastic flow, relating to shear - banding operations, occurs as a result of a number of structural rearrangements at atomic scale. the parameter dmin(tt), therefore, is adopted to identify such irreversible rearrangement with the form : (3)(4)(5)(6) where the subscript n runs over the atoms within the interaction range of the reference atom (n = 0) and rn(t) is the position vector of the nth atom at time t. the parameter dmin(t tt) then denotes the local deviation between the true deformation denoted by rn(t) r0(t) ] and the affine deformation indicated by (x y) rn(t t)r0(t t) ] during the time interval t tt. we calculated dmin values of all atoms during each displacement interval (0.1) to get information of in situ deformation. we selected 1.5, which is about half of the average distance between a cu atom and a zr atom in the samples, as a cutoff of dmin to characterize the rearrangements that make up a plastic event at all strain rates. it is important to point out that the method of choosing the cutoff may be a little rough, considering its value may be affected by strain rates ; nevertheless, it is efficient to judge the plastic deformation, and should not significantly change the trend. in addition, we find that choosing different cutoffs can not change the trend of plastic flow under various strain rates. any atom whose dmin value is greater than the cutoff is considered to be rearranged, and the numbers of the rearranged atoms at all intervals are displayed in fig. 1 as histograms under p h curves. note that the numbers of the rearranged atoms get larger, but their distribution becomes more inhomogeneous when strain rate decreases. moreover, when comparing the histograms with the p h curves, a strong correlation between them was surprisingly discovered : the load - drop events (i.e., flow serrations) in the p h curves correspond to the peak values (i.e., large numbers of the rearranged atoms) in the histograms ; the more obvious the flow serration is, the larger the number of the rearranged atoms in that interval is. in fact, the number of the rearranged atoms can be regarded as an indication of the degree of plastic deformation in the displacement interval. thus, we can conclude that the serrated plastic flow strongly depends on the temporal characteristic of the atomic rearrangement underpinning plastic deformation : successive low degree of plastic deformation at high strain rate leads to less pronounced serrated flow, and intermittent high degree of plastic deformation at low strain rate results in more distinct serrated flow. the load displacement curves and the temporal distribution of the number of rearranged atoms at various strain rates for sample i. the strain rate decreases from (a c) to ferret out how shear bands operate during the indentation processes, we examined the spatial distributions of the rearranged atoms when flow serrations occur. 2, we found that, during a displacement interval, at high strain rate, few rearranged atoms form many small atomic clusters, and at low strain rate, many rearranged atoms form few large atomic clusters. the figure indicates that the degree of atomic rearrangement underlying flow in an individual loading step varies with strain rate or loading timescale. this kind of discrete flow event (i.e., rearranged atomic cluster) finally leads to distinguishing shear - band patterns at the maximum indenting depth, which are displayed in fig. 3 : more and thinner shear bands formed at high strain rate, while fewer and coarser shear bands nucleated at low strain rate. the patterns are drawn by coloring the atoms according to their dmin(0, t) values ; here, the darker the color the larger the dmin(0, t) value. they are quite similar to those observed from instrumented [15 - 19 ] and simulated indentations. the results in figs. 2 and 3 were taken from sample ii for the reason that larger planar size is available to display the shear - band patterns., the potential energy grows more slowly, but fluctuates more prominently ; the energy - drop events essentially correspond to the load - drop events. since our md processes proceed at very low temperature, the potential energy which is large compared to the thermal energy must dominate the flow. thus, the unique spatiotemporal characteristic of deformation can be understood in terms of potential energy landscape (pel) theory [30 - 33 ]. the rate - dependent spatial distributions of the rearranged atoms in the displacement intervals where flow serrations occur for sample ii. the strain rate decreases from (a c);astrain rate 4 10 s, bstrain rate 4 10 s, cstrain rate 4 10 s the rate - dependent shear - band patterns of the maximum indenting depth for sample ii. the strain rate decreases from (a c);astrain rate 4 10 s, bstrain rate 4 10 s, cstrain rate 4 10 s (a) the potential energy versus displacement curves at different strain rates for sample i ; (b) the schematic of the disappearance of a local energy minimum induced by loading ; (c) the schematic of the rate - dependent systematic energy states when the system transits to a new energy basin before loading, the system stays at a local minimum of the potential energy surface (pes), and the configuration of the system is metastable. loading will tilt the pes, and induce the disappearance of some local minima. as a result, the system will became unstable, and move to a new energy local basin. at the same time the disappearance of a potential energy basin induced by loading is schematically shown in fig. when transiting from one energy basin to another, the system at high strain rate will stay at a higher energy state in the new basin in a displacement interval than at low strain rate because of the shorter relaxation time it has. thus a whole transition of the system at high strain rate usually costs more displacement intervals, and the degree of plastic deformation at a particular displacement is relatively lower. on the contrary, at low strain rate, the system experiences less displacement intervals to reach the new basin, and it is this temporal characteristic of atomic rearrangement that dominates the macroscopic serrated plastic flow. on the other hand, since the potential energy of the system at the same indenting depth at high strain rate is higher, there will be more atoms at high energy state in the plastic zone. thus, the rearrangement occurs at multiple regions, but the number of the rearranged atoms is small (see fig. when next loading step is applied, atoms preferentially rearrange at the same positions owing to their relatively higher local energy. in other words so it is hard to conjecture from the shear - band patterns how many shear bands operate in a moment. on the other hand, the system has lower potential energy at low strain rate, so the number of the atoms with high energy state will be smaller nevertheless, the rearrangement in these few regions can develop more sufficiently, finally producing coarse shear bands (see fig. 3c). in addition, since local atoms rearrange sufficiently, leading to a lower local energy, the atomic rearrangement at the next step must occur at other regions having high energy level. in other words, the shear bands nucleate and develop sufficiently at different positions under low strain rate. if the plastic deformation zone or spatial distribution is not related to its time, such as the uniaxial compression case, more and finer shear bands can finally be produced at low strain rates. however, in nanoindentation, the unique spatial patterning, fewer and coarser shear bands (see fig. the temporally inhomogeneous characteristic of the plastic deformation was revealed as the main determining factor of serrated flow behavior. it is not proper to directly relate the numbers of shear bands with the flow serrations. for example, although there is no serration in some fe - based or ce - based bmgs during nanoindentation, a number of fine shear bands are observed under the indents. the unique rate - dependent spatiotemporal distributions of shear banding can be understood in terms of pel theory. we believe that these findings can shed light on the relationship between microstructure and inhomogeneous plastic flow in bmgs. the work was supported by the natural science foundation of china (grants nos. 10725211, 10721202, 10534030, 10674163), the ministry of science and technology of china (2006cb921300, 2007cb925000), and the knowledge innovation project & key project of chinese academy of sciences (nos. all computation of this work were carried out by supercomputer deepcomp 6800, and we thank dr. yangde feng of super computing center of chinese academy of science for his help in the computations. | nanoindentation simulations on a binary metallic glass were performed under various strain rates by using molecular dynamics. the rate - dependent serrated plastic flow was clearly observed, and the spatiotemporal behavior of its underlying irreversible atomic rearrangement was probed. our findings clearly validate that the serration is a temporally inhomogeneous characteristic of such rearrangements and not directly dependent on the resultant shear - banding spatiality. the unique spatiotemporal distribution of shear banding during nanoindentation is highlighted in terms of the potential energy landscape (pel) theory. |
encapsulating peritoneal sclerosis (eps), a condition in which a fibrous cocoon has surrounded the bowel loops, is an uncommon but devastating complication of chronic peritoneal dialysis (pd). long pd duration and chronic exposure to dialysis solutions are considered risk factors for its development [24 ]. clinically, patients can present with symptoms of abdominal pain, nausea, vomiting, repeated bowel obstruction, blood - stained effluent and loss of ultrafiltration capacity. the diagnosis of eps is based on clinical symptoms in combination with pathological findings and abdominal imaging. recently, stuart. case reports, case series and some larger studies have been published over the years. an increased awareness of computed tomography (ct) as imaging modality for diagnosing eps has developed. in a recent paper that reviewed the clinical significance and implications of eps, imaging modalities were described in short and ct scanning was suggested as the investigation of choice in patients with established eps. the present review focusses on all imaging modalities specifically used to diagnose eps, nowadays and in the past, and discusses their features, qualities and shortcomings. the medline database was searched for relevant reports and studies on imaging modalities to diagnose eps. plain abdominal radiography can show air fluid levels and signs of bowel dilation, indicating obstruction [911 ]. however, plain abdominal x - ray films can appear normal even though eps is present [12, 16 ]. although it is readily available and helpful in establishing bowel obstruction and peritoneal calcifications, it does not provide conclusive or sometimes not even additional information on the presence or absence of eps ; therefore, we conclude that when eps is suspected, an abdominal x - ray has no additional diagnostic value in diagnosing eps. ultrosonography (us) has been used in the past when eps was suspected. us characteristics of eps are best appreciated with peritoneal fluid in situ. in one study, us abnormal small bowel activity was present in 12 patients, tethering of bowel to the posterior abdominal wall in 10, intraperitoneal echogenic strands in 7 and membrane formation in 5. in another study by krestin disturbed motility during real time, us was observed in all 13 patients, signs of intestinal obstruction in 9 and bowel wall thickening in 5. reviewed us images of five patients that died from eps, four patients with eps suspicion and six patients considered to be at an increased risk for eps due to prolonged pd therapy. they found a characteristic appearance in several patients consisting of an echogenic membrane in the bowel wall. a major limitation is that the interpretation of the images is very dependent on the radiologist. abdominal ct scans of two patients with a clinical suspicion of eps revealed loculated ascites, adherent bowel loops, narrowing of bowel lumen and a thickened peritoneum. several other case reports described similar ct findings and other features such as bowel dilation [9, 18 ] and the presence of peritoneal calcification [1315, 18, 19 ]. signs of disturbed motility indicated by dilated bowel loops and air fluid levels were seen and in half of the cases, loculated fluid and contrast - enhanced thickening of the peritoneum were present. also reviewed ct scans of five eps patients, four patients with eps suspicion and six patients considered to be at an increased risk for eps and found peritoneal thickening and calcifications in some cases. three studies compared ct scans of eps patients to those of other pd patients. in the first study, ct findings of 10 eps patients were compared to those of 71 control pd patients. peritoneal calcifications, peritoneal thickening, fluid loculation and tethering of small bowel loops were considered diagnostic for eps. in the second study, abdominopelvic ct scans of 27 patients with eps were compared to ct scans of 15 hemodialysis and 20 pd patients by using a severity scoring system. scoring parameters included peritoneal calcification and thickening, bowel wall thickening, bowel tethering and dilation and fluid loculation. a highly significant difference was found between total ct scan scores of eps patients and scores of controls. the clinical outcome of eps patients varied and the total ct scan score did not show a correlation with this outcome, making this score unsuited for predicting the clinical course. the authors also showed that ct scans could not be used for screening purposes because eps patients had only mild abnormalities in 9 of 13 cases on ct scans that were performed > 4 months before the diagnosis. in the third study, performed by our own group we found that contrast - enhanced ct had a sensitivity of 100% and a specificity of 94% for diagnosing eps when experienced radiologists applied a combination of specific ct findings. a cut - off point for a positive test was set at positively scoring three of the six following items : peritoneal enhancement, thickening and calcifications ; adhesions of bowel loops ; signs of bowel obstruction and fluid loculation / septation. a representative example of a ct scan of an eps patient is shown in figure 1. diagnosis of eps is based on clinical features of intestinal obstruction accompanied by radiological imaging of bowel encapsulation. this means that only these patients are labelled as having eps in the described studies and that subsequently, less severe cases are not taken into account. the value of ct scanning in this last group of patients has not been evaluated in these studies and one could speculate that its value is much less. it shows ascites and bowel loops that are drawn into the centre of the abdominal cavity indicating adhesions and an enhanced thickened peritoneum with calcifications both visceral and parietal. ct peritoneography, a technique in which a ct scan is combined with peritoneal contrast medium inserted through the peritoneal catheter, can demonstrate scar tissue and pathological peritoneal recesses. however, calcifications can be overlooked because they can be obscured by high attenuation of contrast medium. it might be valuable to evaluate the presence of eps with this technique but to our knowledge, no studies have been published. major advantages of ct are that it is well tolerated by patients and readily available in most hospitals. shortcomings of ct are radiation burden and risk of loss of residual renal function due to contrast - induced nephropathy. despite these shortcomings, it is considered a safe technique. when used in the right clinical setting in symptomatic patients, danger of radiation exposure of ct in general to prevent contrast - induced nephropathy, patients should be well hydrated before, during and after the procedure. although the incidence is relatively low in well - hydrated patients, the risk is increased in patients with a severely decreased kidney function. in a recent study, 7 of 58 patients with a residual renal function of < 30 ml / min developed contrast - induced nephropathy. if a long - term pd patient has no residual renal function anymore, it is of no concern. in any other case, small bowel distension and circumscribed focal wall thickening were described in one patient and massive lobulated ascites in the omentum with wall enhancement of the lobulated ascites and compression of the bowel in another. magnetic resonance (mr) peritoneograhy has been used to detect complications of pd [30, 31 ] but to our knowledge, it has never been used for the purpose of diagnosing eps. gadolinium - containing mr contrast media are associated with nephrogenic systemic fibrosis and should therefore be avoided in patients with renal failure [3234 ]. also, mri is a time consuming and rather costly technique, which is not yet as available as ct, making widespread use less appealing. follow - through examinations of small and large bowels have been performed in eps patients. in one case, a small bowel follow - through revealed bowel wall thickening of a distal jejunal loop followed by a cauliflower - like formation of ileum loops. in another case, small bowel follow - through with barium showed bowel dilation and encapsulated loops. in the study by krestin., an upper gastrointestinal follow - through examination was performed with barium in three patients and water - soluble diatrizoate in five before surgical intervention took place. all cases demonstrated a delayed transit time but no clear evidence of compressing intraperitoneal bands was present. in the study by campbell. follow - through examinations can provide information on bowel function and may be helpful in locating the obstruction site. however, they are invasive, time consuming and require preparations that could interfere with fluid restrictions of dialysis patients. nowadays, the usefulness of fluordeoxyglucose positron emission tomography (pet) in diagnosing eps was studied in three eps patients and five asymptomatic long - term pd patients. for this technique, the authors showed that this technique detects the inflammatory phase, if present, of sclerosing peritonitis because of an increased tracer uptake in the peritoneum. however, a positive scan could also occur as a result of an acute peritonitis ; therefore, the clinical presentation should be taken into account in its interpretation. recently, a case report was described in which radiolabeled dialysate was inserted in the peritoneal cavity after which a peritoneal scintigraphy was performed because peritoneal adhesions were suspected. non - uniform distribution of the dialysate in combination with loculated tracer accumulation confirmed the presence of adhesions. it might be possible that that this technique could be effective in detecting eps but no studies have been published. biomarkers in peritoneal effluent have a potential role in early diagnosing eps but, until now, no imaging screening methods are available a variety of imaging techniques, invasive as well as and non - invasive, have been used and studied to diagnose eps. in this review, we have provided an overview of these modalities and discussed their specific findings, advantages and limitations. it is the only technique that has been investigated in case control designs [2022 ] and for which data on sensitivity and specificity are available. although we have discussed several shortcomings, ct has been shown to accurately diagnose eps. we advocate that ct with contrast enhancement should be the modality of first choice when eps is suspected. evaluation of the ct scans should preferably be performed by experienced radiologists with knowledge of pd and eps. in conclusion, ct is the definitive imaging modality for eps at the present time. however, it should be noted that data of other imaging techniques, such as mri, are lacking. due to the shortage of publications, drawing certain conclusions studies comparing different imaging modalities with one and other in patients with and without eps should be conducted to solve this issue. | encapsulating peritoneal sclerosis (eps) is a rare but very severe complication of long - term peritoneal dialysis (pd). since the first reports on this disease in the eighties, several imaging techniques have been used for its diagnosis. because of the rarity of this condition, uniformity in modality and protocols for abdominal imaging for diagnosis has been lacking overtime. nowadays, computed tomography (ct) is most often used. in this review, we provide an overview of all imaging modalities that have been used overtime to diagnose eps as a late complication of pd. imaging features characteristic for eps and advantages as well as shortcomings of all modalities are discussed. we believe that when eps is suspected, ct with contrast enhancement should be the modality of first choice in clinical practice. |
gene transfer that is mediated by retroviral vectors has been successfully and extensively undertaken. however, gene transfer technology carries the risk of insertional mutagenesis that results from proviral integration. several studies have found that premalignant clonal proliferation of t cells or t cell acute lymphoblastic leukemia occurs in some patients with severe combined immunodeficiency (scid) and wiskott - aldrich syndrome (was) treated with a -retroviral vector (mlv). this procedure is often associated with vector - mediated insertional activation of the lmo2 oncogene. thus, studies on the selection of integration sites for retroviral or gene therapeutic vectors in the host genome are particularly important. previous studies have shown that integration site selection of some retroviruses or retroviral vectors is not random. different retroviruses or retroviral vectors have different integration preferences in human and animal genomes : (i) those found within genes (i.e., transcription unit - like lentiviruses such as hiv, siv, eiav, and fiv) ; (ii) those found near transcription start sites and cpg islands (-retroviruses such as mlv, xmrv, perv, mscv, fv and herv) ; and (iii) those that display only weak preferences for transcription units, transcription start sites and cpg islands or that are randomly dispersed (-retroviruses such as aslv ; -retroviruses such as mmtv ; and -retroviruses such as htlv-1 and blv). of course, host cells may also affect integration site selection of the retrovirus or retroviral vectors. for example, mitchell. found that tissue - specific transcription resulted in tissue - specific integration that was targeted by the hiv-1-based vector in the human lymphoid supt1 cell - line, human peripheral blood mononuclear cells (pbmcs), and imr-90 lung fibroblasts. lentiviral vectors are a very potent and versatile class of retroviral vectors that are derived from hiv, siv, eiav, and fiv, among others, or a number of ex vivo or in vivo gene transfer applications into dividing and non - dividing cells, and are promising candidates for use in the gene therapy of human skin inherited diseases. however, the characteristics of lentiviral integration site selection in the genomes of human skin cells, particularly in the keratinocyte genome, have until now been poorly defined. it might be more advantageous to study vector integration site selection in host cells after prolonged growth. thus, it may be necessary to explore the prolonged genomic toxicity of retroviral vectors. it has been demonstrated that mouse bone marrow cells containing lentiviral vector genetic integration sites become progressively less common after prolonged growth. however, an unresolved question concerns integration sites and their relevance in human keratinocytes. to address this issue, we identified 874 hiv - based lentiviral vector integration sites in hacat human keratinocytes after prolonged growth, and evaluated the distribution of integrants in relation to normal healthy genes, cancer genes, transcription start sites, cpg islands, and repetitive elements. these data will strengthen our capacity to study the relative risk of the lentiviral vector system in the setting of skin cell gene therapy. in our laboratory, hacat human keratinocytes were cultured as a monolayer at 37c in a 5% co2/95% air atmosphere in 25-cm culture flasks with defined keratinocyte - sfm culture medium (gibco - brl, usa) supplemented with penicillin (100 iu ml) and streptomycin sulfate (100 g ml). the 293 ft cell line (invitrogen) was maintained as a monolayer at 37c in a 5% co2/95% air atmosphere in 25-cm culture flasks in a standard culture medium of dmem (gibco - brl, usa) supplemented in 7% fbs, 2 mml - glutamine, and antibiotics (50 u ml penicillin and 50 mg ml streptomycin sulfate). to produce lentiviral vectors, 293 ft cells were co - transfected with the following 3 plasmids by using the calcium phosphate method : i) a plasmid that was encoded by the hiv-1-based lentiviral sin vector segment (phser - ef1-gfp, which was described previously, a kind gift of dr. guangqian zhou, queen s university belfast, belfast, uk) ; ii) the packaging construct (pspaxi, preserved by our laboratory) ; and iii) the envelope protein - producing construct (pmgid, preserved by our laboratory). forty - eight hours after transfection, the viral supernatant was harvested, centrifuged to pellet cellular debris, and filtered through a 0.45-m filter unit. the vector titer was determined by transduction of 3.010 hacat cells with dose - dependent quantities of vector supernatant and polybrene (8 g ml). cells were collected 96 h post - transduction and analyzed by fluorescence - activated cell sorting for expression of green fluorescent protein (gfp). in these studies, 3.010 hacat cells at 6080% confluence were incubated with 1.8810 infection units per ml of the lentiviral sin vector supernatant. cells were incubated with the supernatant for 48 h in the presence of polybrene (8 g ml). transduced hacat cells were trypsinized and then seeded into a 96-well plate with defined keratinocyte - sfm culture medium (gibco, usa) containing g418 (500 g ml) by limiting dilution. after 2 weeks of continuous culture, the concentration of g418 in the medium was changed to 200 g ml. then, a typical single - cell clone appeared in 1 well after 58 weeks of continuous culture. this gfp - positive keratinocyte clone was selected and expanded in a 24-well plate with defined keratinocyte - sfm (gibco, usa), and then sub - cultured to a passage of 48 for further analysis when the cells had grown in static culture for about 6 months. proviral integration sites were cloned by ligation - mediated pcr (lm - pcr) as described previously. briefly, genomic dna was purified from 1 to 510 cells that were digested with msei and psti to prevent amplification of internal 3ltr fragments and ligated to an msei double - strand linker. lm - pcr was performed with nested primers (supplementary table 1) specific for the ltr and the linker. pcr products without purification were directly shotgun cloned by using the topo ta cloning kit for sequencing (invitrogen, usa) and transformed into top10-competent cells to form libraries of integration junctions, which were then sequenced to saturation by the gs - flx genome sequencer (roche/454 life sciences) pyrosequencing platform following the manufacturer s instructions. sequences were trimmed to remove the linker and viral dna sequences using the software program primer premier 6.0 and mapped onto the human genome (ens62, apr 2011, grch37.p3/hg19) using the online tool gtsg - quickmap, which is a system that can automatically identify genuine integration sites (target set) and calculate the frequencies of integration within or near various genomic features of interest (genes, transcription start sites, and cpg islands). in addition, gtsg - quickmap spontaneously generated a reference set of 1 million random integration sites as the matched control. spss software (version 18.0, spss, usa), and chi - square analysis was then used to compare the integration frequencies of the target versus control set in our laboratory, hacat human keratinocytes were cultured as a monolayer at 37c in a 5% co2/95% air atmosphere in 25-cm culture flasks with defined keratinocyte - sfm culture medium (gibco - brl, usa) supplemented with penicillin (100 iu ml) and streptomycin sulfate (100 g ml). the 293 ft cell line (invitrogen) was maintained as a monolayer at 37c in a 5% co2/95% air atmosphere in 25-cm culture flasks in a standard culture medium of dmem (gibco - brl, usa) supplemented in 7% fbs, 2 mml - glutamine, and antibiotics (50 u ml penicillin and 50 mg ml streptomycin sulfate). to produce lentiviral vectors, 293 ft cells were co - transfected with the following 3 plasmids by using the calcium phosphate method : i) a plasmid that was encoded by the hiv-1-based lentiviral sin vector segment (phser - ef1-gfp, which was described previously, a kind gift of dr. guangqian zhou, queen s university belfast, belfast, uk) ; ii) the packaging construct (pspaxi, preserved by our laboratory) ; and iii) the envelope protein - producing construct (pmgid, preserved by our laboratory). forty - eight hours after transfection, the viral supernatant was harvested, centrifuged to pellet cellular debris, and filtered through a 0.45-m filter unit. the vector titer was determined by transduction of 3.010 hacat cells with dose - dependent quantities of vector supernatant and polybrene (8 g ml). cells were collected 96 h post - transduction and analyzed by fluorescence - activated cell sorting for expression of green fluorescent protein (gfp). in these studies, 3.010 hacat cells at 6080% confluence were incubated with 1.8810 infection units per ml of the lentiviral sin vector supernatant. cells were incubated with the supernatant for 48 h in the presence of polybrene (8 g ml). transduced hacat cells were trypsinized and then seeded into a 96-well plate with defined keratinocyte - sfm culture medium (gibco, usa) containing g418 (500 g ml) by limiting dilution. after 2 weeks of continuous culture, the concentration of g418 in the medium was changed to 200 g ml. then, a typical single - cell clone appeared in 1 well after 58 weeks of continuous culture. this gfp - positive keratinocyte clone was selected and expanded in a 24-well plate with defined keratinocyte - sfm (gibco, usa), and then sub - cultured to a passage of 48 for further analysis when the cells had grown in static culture for about 6 months. proviral integration sites were cloned by ligation - mediated pcr (lm - pcr) as described previously. briefly, genomic dna was purified from 1 to 510 cells that were digested with msei and psti to prevent amplification of internal 3ltr fragments and ligated to an msei double - strand linker. lm - pcr was performed with nested primers (supplementary table 1) specific for the ltr and the linker. pcr products without purification were directly shotgun cloned by using the topo ta cloning kit for sequencing (invitrogen, usa) and transformed into top10-competent cells to form libraries of integration junctions, which were then sequenced to saturation by the gs - flx genome sequencer (roche/454 life sciences) pyrosequencing platform following the manufacturer s instructions. sequences were trimmed to remove the linker and viral dna sequences using the software program primer premier 6.0 and mapped onto the human genome (ens62, apr 2011, grch37.p3/hg19) using the online tool gtsg - quickmap, which is a system that can automatically identify genuine integration sites (target set) and calculate the frequencies of integration within or near various genomic features of interest (genes, transcription start sites, and cpg islands). in addition, gtsg - quickmap spontaneously generated a reference set of 1 million random integration sites as the matched control. spss software (version 18.0, spss, usa), and chi - square analysis was then used to compare the integration frequencies of the target versus control set. we sequenced 7017 amplified junction sequences, and only 874 of these were mapped to unique locations in the human genome. the results revealed that all integration sites were broadly distributed among autosomes and sex chromosomes (figure 1). on chromosomes 1, 11, 16, 17, and 20, a significantly higher frequency of integration was observed, whereas lower frequencies of integration than that seen in the matched control were found on chromosomes 2, 4, 5, 13, 21, and x (p0.05), which was similar to the results of a previous study. moreover, the sequences of introns do not represent junk dna, but actually play a key role in gene expression and regulation. thus, whether the expression of these genes is disturbed will form the basis of subsequent studies from our laboratory. in addition, we chose a conservative window size of 50 kb upstream and downstream of the transcription start sites for analysis. the data showed that there was enrichment for lentiviral vector integration sites within 550 kb upstream of the transcription start sites (28.95% vs. 20.98% ; p 2.5 kb from the tss, and was considered intergenic in all other cases. therefore, this distance (550 kb from the upstream region of tss) may still reside in genes or extend to intergenic regions. in any case, genes, but not transcription start sites, are favored targets for lentiviral vector integration in the human keratinocyte genome. our study also shows that the 50 kb region from the cpg islands was favored by the lentiviral vector. for cpg islands, these regions commonly correspond to gene regulatory regions containing clustered transcription factor binding sites, and many of them are within 10 kb of the gene. in other words, hiv integration extends from being disfavored at short distances (less than 1 kb) to being favored at longer distances (more than 10 kb). it is possible of course, that this distance from the cpg islands and the 550 kb upstream region of transcription start sites may have also overlapped. our integration data also suggested that lentiviral vector integration was strongly favored in sines and disfavored in lines and ltrs (gene - dense regions are rich in sines, sparse in lines and ltrs), and showed no preference for cancer - associated genes and their transcriptional start sites. taken together, these findings show some unique integration features of lentiviral vectors in the human keratinocyte genome. this means that the details of genetic integration, transcription start sites, and cpg islands are not fully parallel with those of other cell types previously reported. many host factors may influence integration site profiles, such as growth time, cell cycle, and cellular proteins. cells containing integration sites in genes become less common after prolonged growth, which suggests negative selection. fortunately, in our study, no malignant clone emerged over the extended culture period, indicating both negative selection and lack of oncogene activation, raising the question of further safety. cell cycle status can determine lentiviral integration in actively transcribed and developmentally - related genes. to date, ledgf / p75 is one of the most intensively researched cellular proteins, which can recruit the lentiviral pre - integration complex (pic) to transcriptional units, thereby promoting integration efficiency and dictating lentiviral integration site selection. ledgf / p75 can be truncated by deleting the n - terminal chromatin - reading pwwp - domain, and replacing this domain with alternative pan - chromatin binding peptides. expression of these ledgf - hybrids in ledgf - depleted cells can result in more randomly distributed lentiviral integration throughout the host - cell genome. our findings offer new data showing the pattern of lentiviral vector integration in the genome of human keratinocytes. this work will lay the foundation for further research aimed at determining and then establishing the biosafety of the lentiviral vector system and in designing a safer lentiviral vector system in the context of specific gene therapy for skin diseases. primers used for ligation - mediated pcr (lm - pcr) in this study. | backgroundlentiviral vectors have been successfully used for human skin cell gene transfer studies. defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. however, genome - wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood.material/methodsin this study, 874 unique lentiviral vector integration sites in human hacat keratinocytes after long - term culture were identified and analyzed with the online tool gtsg - quickmap and spss software.resultsthe data indicated that lentiviral vectors showed integration site preferences for genes and gene - rich regions.conclusionsthis study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases. |
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